Study on the Correlation of Chemical Structure and Ovicidal Activities of 2-Bromoethylthiobenzenes

Formation of a sulfonium-like intermediate was assumed in the hydrolysis of the 2-bromoethylthiobenzenes. A linear free energy relationship was found between the hydrolysis rate of a certain substituted 2-bromoethylthiobenzene and the molar fraction of water in the solvent. The effect of the substituent on the rate constant was attributed not only to the activation energy but also to the entropy change of activation. The negative ρ-value in the formation of the sulfonium-like intermediate in aqueous solution was comparable with that obtained in the ρ-σ-π analysis for ovicidal activity of the compounds.

For the reaction of the substituted 2-bromoethylthiobenzenes with highly excess amount of 4-(p-nitrobenzyl)-pyridine, the ρ-value was found to be negative, which means that the formation of the sulfonium-like intermediate is a rate determining step. Whichever might be more important, the hydrolysis or alkylation, as to the ovicidal action of the compounds, the formation of the sulfonium-like intermediate, could be considered to be an essential step.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Synthesis of 18O-Labelled chlorophyll derivatives at carbonyl oxygen atoms by acidic hydrolysis of the ethylene ketal and acetal

The ethylene ketal of pyropheophorbides, chlorophylls possessing the 13-keto carbonyl group and lacking the 13(2)-methoxycarbonyl group, reacted with H(2)(18)O (ca. 95% 18O atom) by acidic hydrolysis to give efficiently and regioselectively 13(1)-18O-oxo-labelled compounds (ca. 92% 18O). The resulting 18O-labelled chlorin was modified by several chemical reactions to afford some derivatives with little loss of the 18O atom. Following the same procedures, 3(1),13(1)-doubly-18O-labelled pyrochlorophyll derivatives were also prepared. All the synthetic 18O-labelled compounds were identified by FAB-mass and vibrational spectra. Especially, in the vibrational spectroscopic results including IR and resonance Raman spectra, an about 30 cm(-1) wavenumber down-shift of the 3- and/or 13-C[double bond]O stretching vibrational bands was observed by exchanging 3(1)- or 13(1)-oxo-oxygen atom from 16O to 18O.     

Molecular requirements for the biological activity of ethylene

The molecular requirements for ethylene action were investigated using the pea straight growth test. Biological activity requires an unsaturated bond adjacent to a terminal carbon atom, is inversely related to molecular size, and is decreased by substitutions which lower the electron density in the unsaturated position. Evidence is presented that ethylene binds to a metal containing receptor site. CO₂ is a competitive inhibitor of ethylene action, and prevents high concentrations of auxin (which stimulate ethylene formation) from retarding the elongation of etiolated pea stem sections. It is suggested that CO₂ delays fruit ripening by displacing the ripening hormone, ethylene, from its receptor site. Binding of ethylene to the receptor site is also impeded when the O₂ concentration is lowered, and this may explain why fruit ripening is delayed at low O₂ tensions.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK_a of the inhibitor carboxylate group is lowered by at least 1.5 pK_a units when it binds to either enzyme. The signal at ~ 183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-¹³C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-¹³C]-L-tryptophan is not bound in the S₂′ subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~ 183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Phosphorylation of AMP by inorganic phosphorylating agents

AMP was phosphorylated by inorganic phosphorylating agents: cyclo-triphosphate and diphosphonate, in aqueous solution (70-80 degrees C, pH 6-12). The molecular structures of phosphorylated products were established by use of phosphorus-31 NMR and high-performance liquid chromatography (HPLC). The OH groups on AMP were phosphorylated by both phosphorylating agents to form 2'- or 3'-phosphate but an OH group on dAMP was not phosphorylated. Phosphorylation of OH group proceeds in two steps: formation of hydrogen bond between OH group and phosphorylating agent; subsequent nucleophilic attack of OH group on a phosphorus atom. Phosphate group on AMP was phosphorylated by diphosphonate but not by cyclo-triphosphate. The difference in the reactivities is explained in terms of charge repulsion between AMP and agents.     

Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings

Previously, it was proposed that plant growth-promoting bacteria that possess the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, can reduce the amount of ethylene produced by a plant and thereby promote root elongation. To test this model, canola seeds were imbibed in the presence of the chemical ethylene inhibitor, 2-aminoethoxyvinyl glycine (AVG), various strains of plant growth-promoting bacteria, and a psychrophilic bacterium containing an ACC deaminase gene on a broad host range plasmid. The extent of root elongation and levels of ACC, the immediate precursor of ethylene, were measured in the canola seedling roots. A modification of the Waters AccQ.Tag Amino Acid Analysis Method was used to quantify ACC in the root extracts. It was found that, in the presence of the ethylene inhibitor, AVG, or any one of several ACC deaminase-containing strains of bacteria, the growth of canola seedling roots was enhanced and the ACC levels in these roots were lowered.     

Quantum chemical study of the mechanism of ethylene elimination in silylative coupling of olefins

Silylative coupling of olefins differs from olefin metathesis. Although in both these reactions ruthenium catalysts play a crucial role and ethylene product is detected, ruthenium-carbene intermediate is formed only in the course of the metathesis reaction. In this study quantum chemical calculations based on the density functional theory (DFT) have been carried out in order to examine the mechanism of the silylative coupling of olefins leading to ethylene elimination. In the first step of the catalytic cycle, a hydrogen atom from the ruthenium catalytic center is transferred preferentially to the carbon atom bound to Si in a vinylsilane. This H transfer is coupled with the formation of Ru-C bond. Next, the rotation around the newly formed C-C single bond occurs so that silicon atom is placed in the vicinity of the ruthenium center. The following step involves the migration of a silyl moiety, and leads to Ru-Si bond formation, coupled with ethylene elimination. The next reaction, that is the insertion of ethylene (alkene) into Ru-Si bond, has an activation barrier almost as high as the reaction of ethylene elimination. However, the posibility of removing gaseous ethylene from the reactive mixture together with the entropic fators suggests that the insertion of alkene that is larger than C₂H₄ is the rate limiting step in the silylative coupling of olefins. It also suggests that the substituents attached to the silicon atom or the carbon atoms of an alkene by electronic and steric effects may significantly affect silyl migration and thus the effectiveness of the catalytic reaction. Figure Insertion of alkene into Ru-Si bond seems to be rate limiting step in the silylative coupling of olefins Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Heterogeneous ethoxylation of cellulose: influence of alkali and available-water concentrations on substituent distributions

O-(2-Hydroxyethyl)cellulose (1) as formed by the alkali-catalyzed addition of ethylene oxide to cellulose flock in a slurry process is not uniformly substituted. Most of the ethylene oxide adds to HO-6 in a chain-fashion, so that ～20% of the d-glucose residues remain totally unsubstituted at 2.0 molar substitution. Consequently, an aqueous solution thickened by 1 is highly susceptible to enzymic degradation. Stepwise decrease in concentration of alkali during etherification gives improved stability to enzymic degradation associated with a more-uniform distribution of hydroxyethyl groups between the three hydroxyl groups of the glucose residues in cellulose. The relative reactivities of hydroxyl groups and patterns of substitution were established by matching the distribution patterns from a stochastic computer-model with the distribution of substituents as determined by chemical analyses [namely hydrolysis with sulfuric acid to determine the percentage of unsubstituted glucose residues (u-2) and with periodate oxidation for determining the percentage of unsubstituted 2,3-vicinal diol groups per residue]. The reactivities of the three hydroxyl groups at various alkali concentrations in a heterogeneous, slurry-addition process approximate those observed under homogeneous conditions, wherein the reactivities have been determined by tedious chromatographic analyses. In the variable-alkali procedure for addition of ethylene oxide, the amount of water available to the cellulose matrix in the low-alkali (m) sequence is important both for the stability to enzymic degradation and for obtaining gel-free, thickened, aqueous solutions. Optimal stabilities and gel-free solutions are observed at intermediate water: cellulose ratios of 1.10–1.23. At a ratio of 1.23, the stability to enzymic degradation is less sensitive to percentage variations of u-2 than in 1 prepared at higher or lower water: cellulose ratios. Although the initial degree of degradation between 1 of high molar substitution prepared at 6.8m alkali concentration and a similar product prepared under variable alkali conditions may be related to percentage differences of u-2, the rate and final degree of degradation do not relate to percentage differences of u-2. An adequate interpretation, utilizing known cellulase turnover-rates, is found in stochastic-model projections of the distribution of consecutive 2 residues not substituted at HO-2. The results indicate that (1) more-uniform substitution through equalization of hydroxyl reactivities is achieved by lowering the alkali concentration, and (2) more-uniform substitution of 2 of the many cellulose-chains being substituted is achieved by employing an optimal amount of “available” water during etherification.     

Subtle alteration of the active site of ribulose bisphosphate carboxylase/oxygenase by concerted site-directed mutagenesis and chemical modification

Both activities of ribulose bisphosphate carboxylase/oxygenase are dependent on carbamylation by CO2 of a specific lysyl epsilon-amino group (Lys-191 of the enzyme from Rhodospirillum rubrum). To examine the stringency of the requirement for this lysyl side chain, Lys-191 was converted to an aminoethylcysteinyl residue (net replacement of a gamma-methylene group by a sulfur atom) by a combination of site-directed mutagenesis and subsequent chemical modification. The purified Cys-191 mutant was totally devoid of both carboxylase and oxygenase activities. However, this mutant protein exhibited tight-binding of the transition-state analogue, 2-carboxyarabinitol bisphosphate, a property heretofore ascribed solely to the carbamylated form of the carboxylase. Treatment of the mutant protein with ethylene imine restored catalytic activity to 4-7% of the wild-type level. The carboxylase:oxygenase activity ratio of the aminoethylated protein was unperturbed relative to that of wild-type enzyme.     

A Curtin–Hammett mechanism for the copolymerization of ethylene and methyl acrylate monomer using a PymNox nickel catalyst as revealed by DFT computational studies

In this work, the copolymerization of ethylene and methyl acrylate (MA) as catalyzed by a new Ni-based PymNox organometallic compound was studied computationally. We recently tested the behavior of this type of catalyst in ethylene homopolymerization. Experimental results show that the unsubstituted catalyst Ni2 (aldimino PymNox catalyst) is unable to incorporate the MA monomer, whereas methyl-substituted Ni1 (acetaldimino PymNox catalyst) is able to achieve copolymerization. The reactivities of both catalysts were examined using density functional theory (DFT) models. Based on energy profiles calculated at the BP86 level, a Curtin–Hammett mechanism was proposed to explain the different reactivities of the catalysts in ethylene/MA copolymerization. Our results indicate that the methyl substituent Ni1 introduces additional steric hindrance that results in a catalyst conformation that is better suited to polar monomer incorporation. This model provides insights into the design of new catalysts to produce polar functionalized copolymers based on ethylene.     

Trichostrongylus colubriformis: isolation and characterization of ovicidal activity from Bacillus thuringiensis israelensis

Bioassay of media fractions from cultivation of Bacillus thuringiensis israelensis revealed that ovicidal activity for eggs of the ruminant nematode Trichostrongylus colubriformis was found in microbial crystals, but was not released into culture medium. The purified delta-endotoxin of B. t. israelensis, composed of two 25 kDa proteins, had no effect on nematode eggs. A fraction that had high ovicidal activity for eggs of T. colubriformis was isolated by high performance liquid chromatography from crystals of B. t. israelensis. Retention of the compound(s) on size exclusion columns indicated a mol wt of 1510 when compared to standards. The LD50 of this fraction for nematode eggs was not altered significantly by 5 mM calcium chloride with and without ethylenediaminetetraacetic acid or the enzyme inhibitor phenylmethylsulfonyl fluoride (10(-6) M). The fraction was susceptible to proteolytic hydrolysis by bacterial protease VI whereas the fungal protease XIII slightly decreased the ovicidal effects of the fraction. The ovicidal activity was stable for 2 days at ambient temperature or 2 months at 0 C, but declined after 7 days at ambient temperature. Little activity was lost after heating to 100 C for 60 min. The ovicidal effects were also pH dependent with increased toxicity at alkaline pH values. The fraction, however, had no effect on larvae of the mosquito Aedes aegypti or mice after intraperitoneal injection.     

Studies of ethylene-forming system in rat liver extract.

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of L-methionine to ehtylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule alpha-CH2-CH2- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of alpha-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that alpha-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.     

Studies of ethylene-forming system in rat liver extract

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of l-methionine to ethylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule α-CH₂-CH₂- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of α-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that α-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.     

Chemical influences on the specificity of tyrosine phosphorylation

Biological tyrosine phosphorylation has become an extensively studied reaction. Little, however, is known of the chemistry involved. The acetylation of the tyrosyl phenolic hydroxyl group by N-acetylimidazole was studied as a model acylation reaction over the pH range 7.5-9.5. The reactivities of tyrosine and 3-fluorotyrosine were compared. The ratio of reactivities, kappa F-Tyr/kappa Tyr, decreases with increasing pH. Extrapolation to the state in which equivalent concentrations of the two derivatives exist indicates that, consistent with Br?nsted theory, tyrosine is 17 times more reactive than fluorotyrosine. No reactivity was observed with tetrafluorotyrosine, 3-nitrotyrosine, or 3,5-dinitrotyrosine. A peptide containing fluorotyrosine was synthesized and compared with the tyrosine-containing peptide as a substrate for the insulin receptor/tyrosine kinase. In both the presence and absence of insulin, the tyrosine peptide was phosphorylated with higher Vm and Km values than the fluorotyrosine peptide was. These results suggest that ionization of the tyrosyl hydroxyl group has an effect on both the chemical and enzymatic reactivities of the tyrosyl residue in acylation reactions. A model is suggested in which deprotonation of the acceptor occurs upon binding of the substrate to the kinase and implicates a role for the substrate site microenvironment in defining substrate specificity.     

Estimation of the hydrophobicity of 2,4-diphenyl-1,3-oxazoline analogs and QSAR analysis of their ovicidal activity against Tetranychus [corrected] urticae

Partition coefficients of six 2-phenyl-1,3-oxazoline congeners containing 2-I, 2-NO2, 2-CF3, 2,6-(CH3)2, 2,6-F2, and 2-F-6-Cl substitutions on the phenyl moiety were measured in a 1-octanol/water system using the flask-shaking method. The effect on the hydrophobicity (LogP) of substituents on the phenyl moiety of 2-phenyl-1,3-oxazolines linearly correlated with that of benzamide congeners. logP values of other 2-(substituted phenyl)-1,3-oxazoline analogs were empirically estimated from the corresponding substituted benzamides. The ovicidal activity of 2-(substituted phenyl)-4-phenyl-1,3-oxazoline analogs against the two-spotted spider mite Tetranychus [corrected] urticae was quantitatively analyzed using the classical QSAR (Hansch-Fujita) method. Results showed that ovicidal activity increases with hydrophobicity. The introduction of inductive electron-withdrawing groups at ortho-positions increased ovicidal activity, but addition of steric bulk was unfavorable. Substitution at either the meta- or para-position was detrimental to the acaricidal activity.     

Analysis of defensive responses activated by volatile allo-ocimene treatment in Arabidopsis thaliana

Since volatile allo-ocimene enhances resistance of Arabidopsis thaliana against Botrytis cinerea, we attempted to dissect the factors involved in this induced resistance. The penetration of B. cinerea hyphae into Arabidopsis epidermis and the growth of hyphae after penetration were suppressed on allo-ocimene-treated leaves. allo-Ocimene also induced lignification on cell walls and veins of the leaves. The treatment induced accumulation of antifungal substances including the Arabidopsis phytoalexin, camalexin. Induction of lignification and accumulation of camalexin elicited by B. cinerea infection on Arabidopsis leaves after treating with allo-ocimene was faster and more intense than that observed with the leaves that had not been treated with this volatile. This suggested that allo-ocimene could prime defensive responses in Arabidopsis. allo-Ocimene enhanced resistance against B. cinerea in an ethylene resistant mutant (etr1-1), a jasmonic acid resistant mutant (jar1-1) and a salicylic acid resistant mutant (npr1-1). Thus, it is suggested that a signaling pathway independent for ETR1, JAR1 and NPR1 was operative to induce the resistance. The series of responses observed after allo-ocimene-treatment was mostly similar to that observed after C6-aldehyde-treatment. The effect of C6-aldehyde-treatment has been largely accounted to the chemical reactivities of the compounds; however, from this result it can be suggested that resistance responses of Arabidopsis could be induced by the volatiles mostly independent on their reactivities and that a common signaling pathway unaffected by the reactivities of compound was activated by the volatiles.     

Pochonia chlamydosporia fungal activity in a solid medium and its crude extract against eggs of Ascaridia galli

The present study aimed to evaluate the ovicidal activity (type 3 effect) of VC1 and VC4 isolates of Pochonia chlamydosporia in a solid medium and the action of a crude extract of P. chlamydosporia against eggs of Ascaridia galli. To evaluate ovicidal activity in culture medium, 1000 A. galli eggs were plated on Petri dishes containing 2% water-agar with grown fungal isolates (VC1 or VC4) and without fungus (control group) and were examined at 1, 3 and 5 days post-inoculation (assay A). Then, to test the action of crude extracts of P. chlamydosporia (VC1 or VC4), 500 eggs of A. galli were plated on Petri dishes of 4.5 cm diameter with 5 ml of fungal filtrate from each tested isolate. The control group consisted of 500 eggs of A. galli with 10 ml of distilled water on each Petri dish (assay B). Fungal isolates were effective (P < 0.01) at destroying these eggs, showing a type 3 effect at the studied intervals. On the other hand, the crude extract of isolates (VC1 or VC4) reduced the number of A. galli eggs in the treated group compared with the control group by 64.1% and 56.5%, respectively. The results of the present study show that P. chlamydosporia is effective at destroying eggs of A. galli and could therefore be used in the biological control of nematodes.     

Self-/conspecific discrimination and superparasitism strategy in the ovicidal parasitoid Echthrodelphax fairchildii （Hymenoptera： Dryinidae）

Superparasitism in solitary parasitoids results in fatal competition between the immature parasitoids, and consequently only one individual can emerge. In the semisoli- tary ovicidal parasitoid Echthrodelphaxfairchildii （Hymenoptera： Dryinidae）, 2 adults can emerge under superparasitism with a short interval （〈24 h） between the first and second ovipositions. We determined the female parasitoid＇s behavioral responses under self- and conspecific superparasitism bouts with first-to-second oviposition intervals of 〈2 h. The self- and conspecific superparasitizing frequencies increased up to an oviposition interval of 0.75 h, with the former remaining lower than the latter, particularly for oviposition intervals of _〈0.25 h, suggesting the existence of self-/conspecific discrimination. The superparasitizing frequency plateaued for oviposition intervals of _〉0.75 h, with no dif- ference between self- and conspecific superparasitism. The ovicidal-probing frequency did not differ under self- and conspecific superparasitism, and was usually 〈20%. The females exhibited no preference for the oviposition side （i.e., ovipositing on the side with or without the first progeny） and almost always laid female eggs for any oviposition in- terval under self- and conspecific superparasitism. The sex ratio was not affected by the type of superparasitism, oviposition sides, or the occurrence of ovicidal probing. These observed results about the oviposition side, ovicidal probing, and sex ratios differed from the predictions obtained assuming that the females behave optimally. Possible reasons for the discrepancies are discussed： likely candidates include the high cost of selecting oviposition sides and ovicidal probing, and, for the sex ratio, the low frequency of encountering suitable hosts before superparasitism bouts.     

Uptake and polar transport of indoleacetic acid in moss rhizoids

In the cucumber ( Cucumis sativus L. cv. Straight Eight) cotyledon expansion assay, cytokinin-stimulated ethylene production was separated from cytokinin-stimulated growth through the use of potassium and calcium salts. Low concentrations of KC1, which dramatically promoted growth induced by cytokinin, inhibited ethylene evolution, while CaCl₂ at a concentration that had no effect on growth, strongly promoted the cytokinin-induced ethylene evolution. In contrast to the growth response, stimulation of ethylene production was not directly related to the presence of potassium or calcium but to their relative concentrations. Concentrations of KCl and CaCl₂ which promoted ethylene evolution singly, strongly inhibited it when mixed together. Low rates of exogenous ethylene had no effect on the growth response. Both the growth and ethylene responses were found to be general cytokinin phenomena. Cotyledon respiration was promoted by KC1, CaCl₂ and cytokinin, but its stimulation was not correlated with either growth or ethylene production. In the presence of KClm cytokinin-induced respiration sharply lowered the content of certain sugars during the large growth response and followed KCl uptake. Analysis of KCl uptake showed that its growth promoting synergism with cytokinin was not due to osmotic effects.