Elucidation of the enantioselective enzymatic hydrolysis of chiral herbicide dichlorprop methyl by chemical modification

Up to 25% of the current pesticides are chiral, the molecules have chiral centers, but most of them are used as racemates. In most cases, enantiomers of chiral pesticides have different fates in the environment. Knowledge of the function of amino acids of enzymes involved in enantioselective behaviors contributes to the understanding of the enantioselectivity of chiral pesticides. In this work, Aspergillus niger lipase (ANL, EC3.1.1.3) was chemically modified using bromoacetic acid (BrAc), 2,3-butanedione (BD), N-bromosuccinimide (NBS), and methanal. The enantioselectivity of the enzymatic hydrolysis of 2,4-dichlorprop-methyl (DCPPM) was investigated by chiral GC. The results have suggested that histidine, arginine, and tryptophan are essential for lipase activity and might be involved in the catalytic site of ANL. In addition, histidine and lysine play an important role in determining the observed enantioselective hydrolysis of chiral herbicide dichlorprop methyl. The molecular modeling study revealed that the essential hydrogen bonds formed between DCPPM and catalytic residues of ANL might be responsible for the enantioselectivity of DCPPM. The loss of enantioselectivity can also arise from the fact that the modification of the amino acids may cause changes in both the nature of the ANL enzyme conformation and the binding pattern of DCPPM. Our study provides basic information for the exploration of the enantioselective interaction mechanism of enzymes with chiral pesticides.     

Enantioselective degradation and ecotoxicity of the chiral herbicide diclofop in three freshwater alga cultures

Aryloxyphenoxypropanoates are a class of chiral herbicides. They have a pair of enantiomers, only the R(+) form of which is herbicidally active. Diclofop, the model compound of these herbicides, is commercialized as the racemate of the ester form, diclofop-methyl, consisting of a 1:1 mixture of the enantiomers. This study evaluated the enantioselectivity in aquatic toxicity and biodegradation of diclofop and diclofop-methyl. The herbicidally inactive S(-) enantiomers of both diclofop-methyl and diclofop were similar to or higher than the corresponding R(+) forms in toxicity to algae, depending on specific species. Although no enantiomeric conversion occurred for diclofop-methyl and diclofop, the difference in the enantioselective degradation of these herbicides observed in algae cultures suggested that their application forms were an important factor determining their enantioselective environmental behavior. The cell permeability and heat treatment of algae revealed that the enantioselective degradation of diclofop in algae cultures was governed primarily by the facilitated uptake by algae, whereas the enantioselective toxicity was primarily governed by the passive uptake. These results suggested that the acute toxicity test such as the 96 h EC 50 was insufficient to assess the ecological risk of chiral pesticides because of the differential degradation as well as possibly differential action sites of enantiomers. From this study, it was concluded that the enantioselective degradation and toxicity of chiral herbicides may result in their ecotoxicological effects being difficult to predict and that specific attention should thus be paid to currently used racemic pesticides as less active or inactive enantiomers may pose higher ecological risks.     

Enantioselective environmental behavior of the chiral herbicide fenoxaprop-ethyl and its chiral metabolite fenoxaprop in soil

The enantioselective degradation behavior of fenoxaprop-ethyl (FE) and its chiral metabolite fenoxaprop (FA) in three soils under native conditions was investigated. Two pairs of enantiomers were analyzed by high-performance liquid chromatography (HPLC) with an amylose tri-(3,5-dimethylphenylcarbamate) (ADMPC) chiral column. The degradation of racemic FE in three soils showed the herbicidally inactive S-(-)-enantiomer degraded faster than the active R-(+)-enantiomer. FE was configurationally stable in soils because no interconversion to the respective antipodes was observed during incubation of the enantiopure S-(-)- or R-(+)-FE. The main metabolites of FE were confirmed as FA and 6-chloro-2,3-dihydrobenzoxazol-2-one (CDHB), and the formation of the chiral metabolite FA showed enantioselectivity in soils. The degradation of rac-FA was also enantioselective with the S-(-)-FA preferentially degraded: the half-life (t(1/2)) of the S-form in the three soils ranged from 2.03 to 5.17 days, and that of R-form ranged from 2.42 to 20.39 days. The inversion of the S-(-)-enantiomer into the R-(+)-enantiomer occurred in two of the three soils when the enantiopure S-(-)- and R-(+)-FA were incubated. The data from sterilized control experiments indicated that the enantioselectivity of FE and FA was attributed to microbially mediated processes.     

Enantioselective separation and phytotoxicity on rice seedlings of paclobutrazol

The environmental significance of enantioselectivity in chiral insecticides and herbicides has been widely studied. However, little information is currently available on the enantioselective behavior of chiral plant growth regulators. In this study, paclobutrazol enantiomers were resolved and prepared by chiral high-performance liquid chromatography with a Sino-chiral OJ column. The relationship among absolute configuration, optical activity and circular dichroism of paclobutrazol enantiomers was established. The enantioselective behavior of paclobutrazol, including enantioselective effect of paclobutrazol on the growth of rice seedlings and cyanobacteria and enantioselective loss of paclobutrazol in rice seedling growth media, in rice culture system was studied. The (2S,3S)-(-)-enantiomer was almost 3.1 times more active than the (2R,3R)-(+)-enantiomer toward shoot growth as measured by 7 day EC50 values. Enantioselectivity could not be determined with respect to root growth of rice seedlings because a typical dosage response was not observed in the range of the concentrations studied. The dissipation of paclobutrazol in rice growth medium is not enantioselective. Enantiomers and diastereoisomer of paclobutrazol all facilitated the growth of cyanobacteria, which increase the effectiveness of rice biofertilizers. The (2S,3S)-(-)-enantiomer showed stronger stimulatory activity on Microcystis aeruginosa cyanobacteria than the (2R,3R)-(+)-enantiomer, whereas the latter was a more potent stimulator of Anabaena sp. growth. These observations indicate that application of the (2S,3S)-(-)-enantiomer of paclobutrazol and Microcystis aeruginosa in rice cultivation is a good strategy for improving rice seedling performance.     

Enantiomeric resolution and biotoxicity of methamidophos

Despite the fact that the biological processes of chiral pesticides are enantioselective, the biotoxicity of methamidophos with respect to enantioselectivity has so far received limited research. In this study, the enantiomeric separation and biotoxicity of the two enantiomers of methamidophos were investigated. Successful enantioseparation of methamidophos using high-performance liquid chromatography on a Chiralcel OD column was achieved. As indicated by the optical rotation and circular dichroism detection, (+)-methamidophos was eluted prior to (-)-methamidophos. The t test at the 95% level of confidence indicated significant differences between the enantiomers in their in vitro inhibition toward acetylcholinesterases of bovine erythrocytes and Electrophorus electricus and in vivo acute aquatic toxicity to Daphnia magna. The in vitro assays showed that (-)-methamidophos was about 8.0-12.4 times more potent to the enzymes than its (+)-form. In contrast, the (+)-enantiomer was 7.0 times more toxic to D. magna in 48 h tests. The toxicity of racemic methamidophos was intermediate in both in vitro and in vivo bioassays. These results suggest that the biotoxicity of chiral OPs to nontargeted organisms is enantioselective and therefore should be reevaluated with their pure enantiomers.     

手性异丙甲草胺在土壤中的选择性降解

Separation of chiral enantiomers and the dissipation of rac-metolachlor and S-metolachlor in soil were evaluated using achiral high-performance liquid chromatography （HPLC） and chiral gas chromatography （GC） methods. Under the experimental conditions the possible metabolite was considered to be N-（2-ethyl-6-methyl-phenyl）-2-hydroxy-acetamide. Because of the presence of two chiral elements （asymmetrically substituted carbon and chiral axis）, the baseline separation of metolachlor enantiomers was not achieved. S-metolachlor degraded faster in soil than rac-metolachlor. After a 42-day incubation, 73.4% of rac-metolachlor and 90.0% of S-metolachlor were degraded. However, due to the absence of biological processes the degradation process in sterilized soil showed no enantioselectivity. The results indicated that enantioselective degradations could greatly affect the environmental fate of metolachlor and should be considered when the environmental behavior of these compounds was assessed.     

Enantioselective residue dissipation of hexaconazole in cucumber (Cucumis sativus L.), head cabbage (Brassica oleracea L. var. caulorapa DC.), and soils

In this study, the enantioselective dissipation behavior of hexaconazole was investigated in cucumber fruit, head cabbage, and two different types of agricultural soils. The dissipation kinetics was determined by reverse-phase liquid chromatography-tandem mass spectrometry on a cellulose tris (3-chloro-4-methylphenylcarbamate) chiral column. Dissipation rates of hexaconazole enantiomers followed first-order kinetics; the residues of (+)-enantiomer decreased more rapidly than (-)-enantiomer in cucumber and head cabbage, resulting in relative enrichment of the (-)-form, while the two enantiomers showed similar degradation rates in the tested soils. These results indicate substantial enantioselectivity in the residue dissipation of hexaconazole enantiomers in cucumber and head cabbage; however, nonenantioselective dissipation was observed in the tested soils.     

Enantiomeric resolution and growth-retardant activity in rice seedlings of uniconazole

The increasing application of chiral pesticides has enhanced interest in their enantioselectivity. However, little relevant information is currently available for enantioselective activity of chiral plant growth regulators. In an attempt to screen active enantiomers of uniconazole, this work investigated enantiomeric separation and the enantioselective effect of uniconazole on the growth of rice seedlings and cyanobacteria. Baseline resolution of uniconazole enantiomers was achieved on a Chiralpak AD column by chiral high-performance liquid chromatography (HPLC). The relationship among circular dichroism (CD), optical rotation (OR), and absolute configuration was successfully established by coupling of CD and OR detection. The t test at the 95% level of confidence indicated significant differences between the enantiomers in their retardant activity toward growth of rice seedlings and stimulation effect on growth of cyanobacteria, the natural biofertilizers in rice paddy fields. The S-(+)-enantiomer was more active than the R-(-)-enantiomer in retarding growth of rice seedlings and stimulating growth of Microcystis aeruginosa . This special enantiomeric selectivity was further elucidated by probing the binding mode of enantiomers to gibberellin (GA) 20-oxidase by molecular docking. The S-(+)-enantiomer was found to bind tightly with GA 20-oxidase. The results suggested that the S-(+)-enantiomer instead of a racemate of uniconazole should be used to improve rice seedling quality.     

Antioxidant phytochemicals in hazelnut kernel (Corylus avellana L.) and hazelnut byproducts

Antioxidant efficacies of ethanol extracts of defatted raw hazelnut kernel and hazelnut byproducts (skin, hard shell, green leafy cover, and tree leaf) were evaluated by monitoring total antioxidant activity (TAA) and free-radical scavenging activity tests [hydrogen peroxide, superoxide radical, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical], together with antioxidant activity in a beta-carotene-linoleate model system, inhibition of oxidation of human low-density lipoprotein (LDL) cholesterol, and inhibition of strand breaking of supercoiled deoxyribonucleic acid (DNA). In addition, yield, content of phenolics, and phenolic acid profiles (free and esterified fractions) were also examined. Generally, extracts of hazelnut byproducts (skin, hard shell, green leafy cover, and tree leaf) exhibited stronger activities than hazelnut kernel at all concentrations tested. Hazelnut extracts examined showed different antioxidative efficacies, expected to be related to the presence of phenolic compounds. Among samples, extracts of hazelnut skin, in general, showed superior antioxidative efficacy and higher phenolic content as compared to other extracts. Five phenolic acids (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid) were tentatively identified and quantified (both free and esterified forms). Extracts contained different levels of phenolic acids. These results suggest that hazelnut byproducts could potentially be considered as an excellent and readily available source of natural antioxidants.     

Contribution of malolactic fermentation by Oenococcus oeni and Lactobacillus plantarum to the changes in the nonanthocyanin polyphenolic composition of red wine

The changes in the nonanthocyanin phenolic composition during red wine malolactic fermentation carried out spontaneously and by four different starter cultures of the species Oenococcus oeni and Lactobacillus plantarum were examined to determine whether differences in nonanthocyanin polyphenolic compounds could be attributed to the lactic acid bacteria (LAB) strain that performs this important step of the wine-making process. The polyphenolic compounds were analyzed by high-performance liquid chromatography with photodiode array detection and HPLC with electrospray ionization-mass spectrometry detection. The malolactic cultures selected for this study were indigenous wine LAB strains from the A.O.C. Rioja (Spain). Results showed different malolactic behaviors in relation to wine phenolic compositions for O. oeni and L. plantarum, and also, a diversity was found within each group. The hydroxycinnamic acids and their derivatives, the flavonols and their glycosides, the flavanol monomers and oligomers, and trans-resveratrol and its glucoside were the main compounds modified by the different LAB. The wild LAB population exerted a greater impact in the wine content of some of these phenolic compounds than the inoculated selected monocultures of this study.     

Enantioselective analysis of triazole fungicide myclobutanil in cucumber and soil under different application modes by chiral liquid chromatography/tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of myclobutanil enantiomers by chiral liquid chromatography coupled with tandem mass spectrometry. The separation and determination were performed using reversed-phase chromatography on a Chiralcel OD-RH column, with ACN-water (70/30, v/v) as the mobile phase under isocratic conditions at 0.5 mL/min flow rate. The matrix effect, linearity, precision, accuracy, and stability were evaluated. The proposed method then was successfully applied to the study of enantioselective degradation of rac-myclobutanil in cucumber and soil under different application modes. The results showed that the preferential degradation of (+)-myclobutanil resulted in an enrichment of the (-)-myclobutanil residue in plant and soil. Moreover, in cucumber, the stereoselective intensity of myclobutanil under root douche treatment was stronger than that under foliar spraying treatment, whereas in soil, the intensity was exactly opposite. The probable reasons underlying these enantioselective effects were also discussed. This study highlighted the importance of examining the fate of both enantiomers in the greenhouse system for the correct use of chiral pesticides.     

Stereoselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol by lipase-catalyzed hydrolysis of 3-acetylthioesters

The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetylthioesters was investigated. The stereochemical course of the kinetic resolutions was followed by capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E value (E = 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E = 85) and the use of tert-butyl alcohol as cosolvent (E = 49) improved the enantioselectivity. Modification of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-containing compounds investigated possess attractive odor properties, and only one of the enantiomers exhibits the pleasant citrus type note.     

硅藻土吸附增强的(RS)-2,4-DP对映体选择性酶促水解

利用手性气相色谱技术研究了硅藻土吸附作用对(RS) 2 ,4 二氯苯氧丙酸甲酯(2 ,4 DP)酶促水解对映体选择性的影响。实验结果表明,硅藻土吸附作用显著增强了酶促反应的对映体选择性(ER值由1.5 8增加到5 .31)。进一步研究表明,硅藻土对脂肪酶的吸附,引起酶构象变化,影响农药底物与酶结合的微环境,是酶促反应的对映体选择性增强的主要原因。吸附在硅藻土上的脂肪酶,与R 2 ,4 DP结合更为困难,反应速率下降;而S 2 ,4 DP接近酶反应中心更加容易,反应速率上升。此外,硅藻土吸附作用对农药底物的束缚引起处于“自由状态”的底物减少,也使酶促反应的对映体选择性略有增强     

Relationship between antibacterial activity of (+)-catechin derivatives and their interaction with a model membrane

(+)-Catechin derivatives with different alkyl chain lengths were synthesized from (+)-catechin and various straight chain alkylaldehydes in the presence of methyl mercaptan, and their antibacterial activities against Gram-positive bacteria were evaluated. The antibacterial activity increased markedly with elongation of the alkyl chain lengths of the derivatives and reached a maximum at a chain of four to seven carbons. Subsequently, interaction of the (+)-catechin derivatives with a model membrane using liposome was investigated. The derivatives with a chain of three carbons or more were found to have very strong affinity for the membrane. The injury action of the derivatives against the membrane was examined with liposome in which calcein was enclosed as a fluorescent indicator. The leakage was observed in the derivatives with chain lengths of four carbons or more. Particularly the derivatives with chains longer than five carbons are considered to destroy the liposome membrane judging from the degree of the fluorescent leakage. These results implied that the lipophilicity and disrupting ability of the (+)-catechin derivatives to the liposome membrane participate in their antibacterial activity.     

Composition, enantiomeric distribution, and antibacterial activity of the essential oil of Achillea ligustica All. from Corsica

The essential oil of Achillea ligustica from Corsica was investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). A total of 82 compounds representing 94.0% of the oil were tentatively identified. The main constituents were the camphane derivatives, representing >30% (camphor, 21.3%; borneol, 6.2%; bornyl acetate, 3.5%) of the whole oil, and santolina alcohol (19.3%). The enantiomeric distribution of 8 chiral constituents was determined by GC-MS using two enantioselective stationary phases (DIME-beta-CD and Lipodex-E). Racemic santolina alcohol, required for optimization of the enantioselective GC conditions, was prepared by an original two-step synthesis from 2,5-dimethylhexa-2,4-diene. The whole essential oil was tested for its antibacterial activity against a wide range of bacteria using a paper disk method. The results show a promising activity against Streptomyces species.     

Enantioselective degradation and chiral stability of pyrethroids in soil and sediment

Synthetic pyrethroids contain two or three chiral centers, making them a family of chiral pesticides with a large number of stereoisomers. Recent studies showed significant differences in aquatic toxicity between enantiomers from the same diastereomers of pyrethroids. To better understand the ecotoxicological effect and fate of pyrethroid insecticides, chirality in biodegradation must also be considered. In this study, we examined enantiomer compositions of selected pyrethroids in field sediment samples taken from various locations in southern California. Enantioselective degradation was frequently observed for cis-bifenthrin, permethrin, and cyfluthrin under field conditions. We further conducted long incubation experiments under laboratory-controlled conditions using single enantiomers of cis-bifenthrin, cis-permethrin, and cypermethrin. The half-lives for individual enantiomers were calculated to be 277-770 days for cis-bifenthrin enantiomers, 99-141 days for cis-permethrin enantiomers, and 52-135 days for cypermethrin enantiomers, respectively. The direction and degree of enantioselectivity in degradation were found to closely depend on the specific compound as well as experimental conditions. Because no significant difference in degradation was observed after samples were sterilized, the observed enantioselectivity may be attributed to preferential biological transformations.     

Effect of different washing procedures on phenolic metabolism of shredded, packaged iceberg lettuce during storage

Different washing treatments applying chlorinated, ozonated, and tap water were examined for their effect on the phenolic metabolism of minimally processed iceberg lettuce (Lactuca sativa L.) during storage in consumer-sized bags at 4 degrees C for up to 9 days. To eliminate problems associated with raw material inhomogeneity, processing was conducted on a pilot-plant scale under operating conditions of industrial practice. Inherent product heterogeneity caused by diverse lettuce leaf tissues was compensated for by pooling large-sized samples, and frequent sampling ensured significant data about the activities of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD), as well as the contents of caffeic acid derivatives over storage time. In the homogeneous lettuce samples, specific responses caused by different washing procedures were detectable. PAL activity in the samples increased for up to 5-8 days of storage. Compared to tap and ozonated water, the use of chlorinated water (100-200 mg/L free chlorine) for washing trimmed heads or shredded lettuce significantly reduced PAL activity and the concomitant rise of 3,5-di-O-caffeoylquinic acid (isochlorogenic acid isomer) concentrations. The phenolic acids O-caffeoyltartaric (caftaric acid), di-O-caffeoyltartaric (chicoric acid), 5-O-caffeoylquinic (chlorogenic acid isomer), and O-caffeoylmalic were less influenced by different washing treatments. Individual contents either were constant or decreased during storage. Additionally, the novel finding of a further caffeic acid isomer, tentatively identified as meso-di-O-caffeoyltartaric acid, is reported. PPO and POD activities were less affected by different washing treatments and thus were less suitable physiological indicators of stress reactions triggered by alternative processing.     

Biogenetic studies in Mentha x piperita. 1. deuterium-labeled monoterpene ketones: synthesis and stereoselective analysis.

Pulegone, menthone, and isomenthone isotopomers are synthesized as regioselectively deuterated d(5)- and d(8)-stereoisomers. Deuterium-labeled menthone and isomenthone enantiomers are analyzed using enantioselective multidimensional gas chromatography/mass spectrometry. The deuterated stereoisomers of menthone and isomenthone are separated from the unlabeled menthone and isomenthone on a glass capillary column, coated with 50% octakis(2, 3-di-O-butyryl-6-O-tert- butyldimethylsilyl)-gamma-cyclodextrin in OV 1701vi as the chiral stationary phase. These deuterium-labeled monoterpene ketones are proved to be highly valuable substrates in biosynthetic studies of terpenoid compounds.     

Development of pyrethroid substrates for esterases associated with pyrethroid resistance in the tobacco budworm, Heliothis virescens (F.)

Assays to detect esterases associated with resistance to organophosphorus and pyrethroid insecticides in larvae of H. virescens were developed and evaluated. Cross-resistance to a variety of insecticides was measured in strains resulting from selection with either profenofos (OP-R) or cypermethrin (PYR-R), and resistance in both strains appeared to have a metabolic component. Esters were synthesized that coupled 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate, the acid moiety of some pyrethroid insecticides, with groups (e.g., p-nitrophenyl-) that could be detected spectrophotometrically following hydrolysis of the resulting esters. Activities toward these pyrethroid esters were significantly higher in both resistant strains than those in a susceptible reference strain. In addition, all pyrethroid esters significantly increased the toxicity of cypermethrin in bioassays with larvae from both PYR-R and OP-R strains. The biological and biochemical activities of these compounds are compared with those with more conventional esterase substrates and insecticide synergists, and the utility of pyrethroid esters as components of rapid assays for detecting esterases associated with insecticide resistance is discussed.     

Some chemical composition and biological activity of northern Argentine propolis

Twenty-five samples of propolis were collected from seven different regions in northern Argentina; ethanolic extracts of propolis were prepared from all samples, and the respective samples were examined for UV absorption spectra, RPHPTLC, RPHPLC, antimicrobial activity, antiradical activity, and total phenolic content. It was found that 16 of the 25 samples showed a phenolic profile similar to that found in samples from southern Brazil and corresponding to poplar-based propolis and that the rest of the samples showed a different profile and higher antimicrobial and antiradical activities.