The Chiral Herbicide Beflubutamid - American Chemical Society

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The Chiral Herbicide Beflubutamid (I): Isolation of Pure Enantiomers by HPLC, Herbicidal Activity of Enantiomers, and Analysis by Enantioselective GC-MS Ignaz J. Buerge,* Astrid Bac̈ hli, Jean-Pierre De Joffrey, Markus D. Müller, Simon Spycher, and Thomas Poiger Plant Protection Chemistry, Swiss Federal Research Station (Agroscope), CH-8820 Wädenswil, Switzerland ABSTRACT: For many chiral pesticides, little information is available on the properties and fate of individual stereoisomers. A basic data set would, first of all, include stereoisomer-specific analytical methods and data on the biological activity of stereoisomers. The herbicide beflubutamid, which acts as an inhibitor of carotenoid biosynthesis, is currently marketed as racemate against dicotyledonous weeds in cereals. Here, we present analytical methods for enantiomer separation of beflubutamid and two metabolites based on chiral HPLC. These methods were used to assign the optical rotation and to prepare milligram quantities of the pure enantiomers for further characterization with respect to herbicidal activity. In addition, sensitive analytical methods were developed for enantiomer separation and quantification of beflubutamid and its metabolites at trace level, using chiral GC-MS. In miniaturized biotests with garden cress, (−)-beflubutamid showed at least 1000× higher herbicidal activity (EC50, 0.50 μM) than (+)-beflubutamid, as determined by analysis of chlorophyll a in 5-day-old leaves. The agricultural use of enantiopure (−)-beflubutamid rather than the racemic compound may therefore be advantageous from an environmental perspective. In further biotests, the (+)-enantiomer of the phenoxybutanoic acid metabolite showed effects on root growth, possibly via an auxin-type mode of action, but at 100× higher concentrations than the structurally related herbicide (+)-mecoprop.

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INTRODUCTION In recent years, stereochemical aspects of chiral pesticides have attracted increasing attention. In many cases, the stereoisomers of pesticides differ significantly with respect to biological activity.1−3 For example, the (R)-enantiomer of the herbicide haloxyfop has at least 1000-fold higher activity than the (S)enantiomer when applied postemergent.4 Consequently, application rates may be reduced by switching from racemic to stereoisomer-pure or -enriched compounds.5 Furthermore, stereoisomers may show different metabolism in plants, in animals, in humans, and in the environment, and they may have different toxicity to humans and nontarget organisms.1,2 Possible adverse effects of “non-active” stereoisomers, i.e., stereoisomers without the desired biological activity, could therefore be avoided when using single isomer compounds. Even though the synthesis of single stereoisomers is often more complex and expensive than that of isomer mixtures, the number of such pesticides available on the market is increasing.6 They may offer opportunities for new patents and other marketing advantages, and save costs for formulation, packaging, storage, and transport.5 Further developments toward single isomer pesticides are expected from regulatory requirements. Under the new European regulation on plant protection products,7 the identity and maximum content of isomers/diastereomers in an active substance have to be © 2012 American Chemical Society

specified and active substances that contain a significant proportion of nonactive isomers are candidates for substitution. For many of the existing chiral pesticides, however, stereochemistry was not considered in the original studies submitted for registration, or such data are not available in the open literature. A basic data set for the evaluation of chiral pesticides would, first of all, include stereoisomer-specific analytical methods and data on the biological activity of the individual stereoisomers. Pure stereoisomers are thus required for efficacy tests and also for further studies on plant metabolism, toxicity, environmental fate, and ecotoxicity. For this study, we selected the herbicide beflubutamid, a chiral compound currently marketed as racemate against dicotyledonous weeds in cereals.8 Beflubutamid inhibits the enzyme phytoene-desaturase, which is involved in the biosynthesis of carotenoids. Depletion of carotenoids results in photooxidation of chlorophyll and thus bleaching/chlorosis of susceptible weeds.8 The chirality of beflubutamid is due to an Special Issue: Rene Schwarzenbach Tribute Received: Revised: Accepted: Published: 6806

May 15, 2012 July 20, 2012 July 31, 2012 July 31, 2012 dx.doi.org/10.1021/es301876d | Environ. Sci. Technol. 2013, 47, 6806−6811

Environmental Science & Technology

Article

Figure 1. Chemical structures of the herbicide beflubutamid and two metabolites (bfl-amide and bfl-acid) that are formed in soils, plants, and animals (top), corresponding internal standards used for GC-MS analysis (middle), and the herbicide mecoprop (bottom). The asterisks indicate the asymmetrically substituted C atom of the chiral compounds.

Germany; bfl-amide (2-[4-fluoro-3-(trifluoromethyl)phenoxy]butanamide, purity unknown) and bfl-acid (2-[4-fluoro-3(trifluoromethyl)phenoxy]butanoic acid, 99.0%) were obtained from Stähler, Stade, Germany; and 2,6-dichlorobenzamide (99.9%) and mecoprop (2-(4-chloro-2-methylphenoxy)propanoic acid, >99%) were from Sigma-Aldrich, Steinheim, Germany. Enantioselective HPLC. The enantiomers of beflubutamid were separated by HPLC on a chiral Lux Cellulose 2 column (250 mm × 4.6 mm, 5 μm particle size, Phenomenex, Torrance, CA), using a Dionex P680 pump and a UVD170U UV/vis detector (Dionex, Sunnyvale, CA) or a Polar Monitor optical rotation detector (Büchi, Flawil, Switzerland). HPLC conditions were as follows: isocratic elution with hexane/ isopropanol 97/3 at a flow rate of 1 mL/min and a column temperature of 50 ± 1 °C, with detection at a wavelength of 285 nm. This method was used to manually collect fractions of the pure enantiomers. Fraction 1 was collected at a retention time of ≈13.2−13.7 min and fraction 2 at ≈14.8−15.4 min. Fractions from 20 injections of 100 μg of beflubutamid dissolved in 20 μL of eluent were combined. The pooled fractions contained ≈1 mg of each enantiomer with >99% enantiomeric purity as determined by enantioselective GC-MS (see below). Enantiomers of bfl-amide and bfl-acid were separated on a ET200/Nucleodex α-PM column (200 mm × 4 mm, 5 μm, Macherey-Nagel, Dü ren, Germany) with the following conditions: isocratic elution with methanol/water 1/1 (for bfl-acid, 50 mM NaH2PO4, pH 3 instead of water) at a flow rate of 0.7 mL/min and a column temperature of 10 ± 1 °C, detection at a wavelength of 250 nm. Fractions of the enantiomers were collected manually from 8 injections of 500 μg bfl-amide or bfl-acid dissolved in 100 μL eluent. The time intervals for sampling were selected to avoid cross-contamination with the opposite enantiomer: bfl-acid fraction 1: 20.6− 22.5 min, fraction 2: 23.3−25.3 min, bfl-amide fraction 1: 17.3− 19.0 min, fraction 2: 19.6−22.0 min. The combined fractions were then partitioned three times with 5-mL portions of dichloromethane. After evaporation of the solvent, the residues

asymmetrically substituted C atom at position 2 of the phenoxybutanamide moiety (Figure 1). Two metabolites were found to be formed in soils, plants, and animals: a primary phenoxybutanamide (a minor metabolite, in the following denoted as bfl-amide) and a phenoxybutanoic acid (the major metabolite, bfl-acid).9 These metabolites still contain the stereogenic C atom and are thus chiral as well (Figure 1). An analytical method for determination of the isomeric ratio of beflubutamid was developed for European registration,9 but is confidential information. In addition, the herbicidal activity of beflubutamid enantiomers against selected weeds was tested.10 The efficacy tests showed that only (S)-beflubutamid is herbicidally active, but detailed information on how the enantiomers were prepared and characterized is missing in the summary report.10 In this study, we first developed analytical methods for the separation of the enantiomers of beflubutamid, bfl-amide, and bfl-acid based on chiral high-performance liquid chromatography (HPLC). These methods were used to assign the optical rotation of the enantiomers and to prepare milligram quantities of the pure enantiomers for further characterization with respect to their herbicidal activity and for a soil incubation study (see accompanying paper, ref 11). Garden cress was selected to test the herbicidal activity of beflubutamid and bflacid, since germination and growth are reproducible and fast. As only small quantities of the enantiomers were available, biotests were miniaturized. Finally, sensitive analytical methods were developed for enantiomer separation and quantification of beflubutamid and its metabolites at trace level, using chiral gas chromatography−mass spectrometry (GC-MS). These methods were also employed in a study on the enantioselective degradation of these compounds in soils.11

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EXPERIMENTAL SECTION Chemicals. Beflubutamid (2-[4-fluoro-3-(trifluoromethyl)phenoxy]-N-(phenylmethyl)butanamide, purity, 99.5%), flutolanil (N-[3-(1-methylethoxy)phenyl]-2-(trifluoromethyl)benzamide, 99.5%), and cloprop (2-(3-chlorophenoxy)propanoic acid, 99.0%) were from Ehrenstorfer, Augsburg, 6807

dx.doi.org/10.1021/es301876d | Environ. Sci. Technol. 2013, 47, 6806−6811


Article

medium containing different concentrations (up to 100 μM) of rac-, (+)-, or (−)-beflubutamid. Deionized water and potato dextrose agar (Difco, BD, Franklin Lakes, NJ) were autoclaved at 121 °C during 10 min. For each concentration level of beflubutamid, 10 mL of an aqueous solution was prepared from an organic stock solution and mixed with 10 mL of 7.8% agar (at 60 °C). Note that stock solutions had been prepared in ethanol (racemate) or hexane/ isopropanol 97/3 (pure enantiomers), but for the biotests, the organic solvent was either completely evaporated (hexane/ isopropanol) or reduced to a final concentration of ≤0.01% ethanol in agar after dilution. In separate biotests with the same content of ethanol, but without beflubutamid, no herbicidal effect was observed. Aliquots of 200 μL of agar gel were drawn into the pipet tips (10−20 replicates), and the ends were sealed with modeling clay (Staedtler Mars, Nürnberg, Germany). Thereafter, one cress seed was added per pipet tip onto the gel. The pipet tips were placed in a rack and covered with a transparent PET lid. The bottom of the rack was filled with ≈2 mm of water (not in contact with the tips) to maintain moist conditions for germination of the seeds. The rack was then exposed to a diurnal cycle of 16 h of light (using four Philips TLD 36W/86 incandescent lamps) at 20 °C and 8 h of dark at 18 °C in a climate chamber with 70% relative humidity. After 2 days, the lid was removed from the rack. After 5 days, the two cotyledons, which were fully developed at this point, were sampled from 10 pipet tips, weighed, and extracted for analysis of chlorophyll a. Further biotests with bfl-acid and, for comparison, with the herbicide mecoprop were performed in the same way, except that the root length of 3-day-old cress seedlings was measured instead of chlorophyll a. Analysis of Chlorophyll a. The cress leaves (10 replicates) were extracted with 5 mL of methanol. After 5 min of sonication and subsequent filtration (0.45 μm cellulose acetate filters, VWR, Radnor, PA), the solutions were transferred into a 1-cm quartz cuvette. Within 30 min, the absorbance was recorded at wavelengths of 665, 652, and 750 nm using a Cary 100 Bio UV/vis spectrophotometer (Varian, Mulgrave, Australia). Concentrations of chlorophyll a were calculated using the following equation:15

were dissolved in 10 mL of methanol. This yielded amounts of ≈1 mg of each enantiomer with >99% enantiomeric purity (determined by enantioselective GC-MS; bfl-acid, after methylation, see below). The method for bfl-acid was also used for enantiomer separation of the herbicide mecoprop (application note no. 106080 in the Macherey-Nagel database under www.mn-net. com) and to collect fractions of the enantiomers. Mecoprop served as a reference compound in biotests with bfl-acid (see later). Enantioselective GC-MS. For GC-MS analysis, bfl-acid was first methylated to the corresponding methyl ester.12,13 Methanolic solutions (0.1−0.5 mL) were transferred to 2-mL glass vials, carefully evaporated to a volume of ≈50 μL, and acidified with 2−3 drops of ≈0.5% trifluoroacetic acid in methanol. Bfl-acid was then derivatized using diazomethane in diethyl ether until the yellow color of diazomethane persisted. After derivatization and evaporation of the solvents, the residues were dissolved in an appropriate volume of ethyl acetate (0.2−1 mL). To compensate for potentially incomplete methylation and for other losses during sample preparation and analysis, the following internal standards were used: cloprop for bfl-acid, 2,6dichlorobenzamide for bfl-amide, and flutolanil for beflubutamid (chemical structures; see Figure 1). They were added prior to extraction (for soil samples, see ref 11) and methylation. The enantiomers of beflubutamid and flutolanil were separated on a chiral GC column made in-house, coated with a mixture of 2 mg of octakis(bis-tert-butyldimethylsilyl)-γcyclodextrin and 6 mg of PS086 (≈86% dimethyl-(14%)diphenylsiloxane copolymer; 20 m, 0.25 mm i.d., 0.05 μm film14). GC conditions were as follows: 1 μL split/splitless injection (240 °C, initial 48 s splitless); temperature program: 70 °C, 1 min isothermal, 25 °C/min to 100 °C, 2 °C/min to 190 °C, isothermal hold at 220 °C; constant flow, 1 mL/min helium. The GC was coupled to a Quattro Micro triple quadrupole MS (Micromass, Manchester, U.K.), operated under electron impact ionization (EI, 70 eV, 180 °C) and selected-ion-monitoring (SIM) conditions. Beflubutamid and flutolanil were quantified using the ions m/z 176 (221 for confirmatory purposes) and 173 (281), respectively. Enantiomers of bfl-amide and bfl-acid-methyl, 2,6-dichlorobenzamide, and cloprop-methyl were separated on a column coated with permethyl-β-cyclodextrin (15%) in OV1701 (BGB 171, 20 m, 0.25 mm i.d., 0.15 μm film, BGB, Boeckten, Switzerland), under the following conditions: 1 μL split/ splitless injection (250 °C, 48 s splitless); temperature program: 70 °C, 1 min isothermal, 10 °C/min to 100 °C, 2 °C/min to 190 °C, isothermal hold at 220 °C; constant flow, 1 mL/min helium. These compounds were analyzed with a Voyager single quadrupole MS (Finnigan, Manchester, U.K.), operated under EI (70 eV, 180 °C) and SIM conditions. Bflamide, bfl-acid-methyl, 2,6-dichlorobenzamide, and clopropmethyl were quantified using the ions m/z 221 (265), 221 (280), 173 (189), and 155 (214), respectively. Quantification was based on peak area ratios relative to the internal standards and in reference to suitable standard solutions of the racemic compounds. Biotests. The herbicidal activity of beflubutamid was investigated with garden cress (Lepidium sativum, from fenaco/UFA, Winterthur, Switzerland). Biotests were performed in 200 μL polypropylene pipet tips filled with agar

Chl a [μg/mL] = 16.29Abs(665 nm) − 8.54Abs(652 nm)

where Abs(λ) is the absorbance at the indicated wavelength minus the absorbance at 750 nm.

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RESULTS AND DISCUSSION Separation and Characterization of Enantiomers with Chiral HPLC. Baseline-separation of the enantiomers of beflubutamid was achieved using a Lux Cellulose 2 HPLC column and normal-phase chromatography (Figure 2). The enantiomer resolution was clearly better at 50 °C than at room temperature. The stationary phase in this column contains cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector. The column has also been used for enantiomer separation of other chiral amides, such as the anesthetic drug bupivacaine (application note no. 18187 in the Phenomenex database under www.phenomenex.com). The elution order of the beflubutamid enantiomers, determined with the optical rotation detector, was (+) prior to (−) (Figure 2). Enantiomers of bfl-acid and bfl-amide were separated reasonably well using an ET200/Nucleodex α-PM column and reversed-phase chromatography (Figure 2). In this case, a 6808



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The enantiomers of methylated bfl-acid and bfl-amide were separated on a permethylated β-cyclodextrin column with a resolution of ≈1.3 and 1.4, respectively (Figure 3). The smaller metabolites apparently interacted more strongly with a βcyclodextrin-derivative (a cyclic chiral selector made up of seven glucose molecules) whereas beflubutamid was better resolved with a γ-cyclodextrin-derivative (eight glucose units). The elution order of the bfl-amide enantiomers was the same under GC conditions as with chiral HPLC, but reversed for bflacid (Figures 2 and 3; note that, with GC, bfl-acid was analyzed as the methyl ester). The internal standards eluted after 21.7 min (first enantiomer of cloprop-methyl) and 46.1 min (2,6dichlorobenzamide). The two enantioselective GC-MS methods were used for characterization of content and purity of the isolated enantiomers and, in a separate study, to quantify the three analytes in extracts from soil incubation experiments.11 Detailed information on extraction procedure and recoveries from the respective soils and on limits of detection are given in ref 11. Biotests with rac-Beflubutamid and Pure Enantiomers. To characterize the isolated enantiomers of beflubutamid regarding their herbicidal activity, we performed several small-scale biotests with garden cress. For range finding, we first tested rac-beflubutamid at concentration levels of between 0.1 and 100 μM (definitive test at eleven concentrations). Five days after sowing on herbicide-treated agar, the roots had grown through the agar down to a length of ≈4−5 cm, whereas the shoots had reached a length of ≈1−2 cm and the two trilobate cotyledons were fully developed. Photos of leaves grown with and without herbicide addition are shown in Figure 4. Between the concentration levels, no obvious differences could be recognized with regard to plant size and morphology, but certainly with regard to color. With increasing concentration of rac-beflubutamid, the color of the cotyledons visibly changed from green to white. This bleaching effect is consistent with the mode of action of the herbicide, which acts by inhibition of carotenoid biosynthesis and eventually causes photooxidation of chlorophyll. The symptoms were most pronounced at the leaf edges (Figure 4). The chlorophyll a content in the 5-day-old cotyledons decreased steadily from ≈1.4 mg/g (wet weight) in control samples to 0.8 mg/g at 1 μM and 0.07 mg/g at 100 μM racbeflubutamid. The corresponding dose−response curve is depicted in Figure 4 and illustrates that chlorophyll a is a good measure of the herbicidal effect of beflubutamid. Further biotests were carried out with the pure enantiomers of beflubutamid at concentrations of 1−100 μM (+)-beflubutamid (3 concentration levels) and 0.1−100 μM (−)-beflubutamid (10 concentrations). In tests with (+)-beflubutamid, no bleaching symptoms were observed at any dose level. Even at the highest dose, the chlorophyll a content was not different from that in untreated control plants. In contrast, in the presence of (−)-beflubutamid, the cotyledons were clearly bleached. Chlorophyll a decreased from ≈1.3 mg/g in the controls, to 0.6 mg/g at 0.4 μM and 0.04 mg/g at 100 μM (−)-beflubutamid. The data in Figure 4 were then fitted with a log−logistic dose−response model, implemented in the dose response curve (drc) package 16 of the statistics program R:17

Figure 2. Enantioselective HPLC separation of beflubutamid (column, Lux Cellulose 2, 50 °C; 100 μg injected), bfl-acid, and bfl-amide (ET200/Nucleodex α-PM, 10 °C; 500 μg injected). Superimposed chromatograms with UV detection (black lines) and optical rotation detection (red lines, arbitrary scales).

better resolution was obtained at a low column temperature of 10 °C. On this column with permethylated α-cyclodextrin as chiral selector, baseline separation is also achieved for the enantiomers of the herbicide mecoprop, a compound with structural similarities to bfl-acid (see later). The elution order of bfl-acid was (−) prior to (+) (Figure 2). The optical rotation of the enantiomers of bfl-amide was low and could not be assigned unambiguously, possibly also due to interfering optically active impurities (Figure 2). Note that the purity of the bfl-amide reference material was not known. Separation of Enantiomers with Chiral GC-MS. Several chiral GC columns were tested for separation of the enantiomers of beflubutamid, bfl-amide, and bfl-acid. However, it was not possible to obtain satisfactory separation for all compounds on a single chiral column so that two different columns were finally used. For beflubutamid, an enantiomer resolution of ≈1.2 was achieved on a column with a γ-cyclodextrin-derivative as chiral selector (the enantiomer resolution is defined by R = 2 (tr2 − tr1)/(w1 + w2), where tri is the retention time of enantiomer i and wi is the width at the base of peak i). A representative chromatogram from a soil incubation experiment with beflubutamid11 is shown in Figure 3. The elution order was the same as in chiral HPLC with the (+)-enantiomer eluting first (retention times, 35.0 min for (+)-beflubutamid, 35.3 min for (−)-beflubutamid, and 37.3 min for the internal standard flutolanil).

Figure 3. Enantioselective GC separation of beflubutamid (column, PS086/BDMS-γ-CD; concentration, 0.74 μg/g soil), bfl-acid-methyl, and bfl-amide (OV1701/PM-β-CD; 0.50 and 1.5 μg/g, respectively) in soil extracts with MS detection (arbitrary scales).

Chl a = 6809

1 1 + 10

b(log c − log EC50)



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mecoprop or cloprop (Figure 1). These compounds have an auxin-type mode of action and affect plant growth, in particular stem and root elongation.18 Bfl-acid may therefore show some herbicidal activity as well, but presumably not on the same target enzyme as the parent compound beflubutamid (phytoene-desaturase). In further biotests with bfl-acid, the root length of 3-day-old cress seedlings was used as a measure of the herbicidal effect. For comparison, additional biotests were performed with mecoprop as a reference compound. Bfl-acid and mecoprop are both chiral at position 2 of the alkanoic acid. It is well established that (+)-mecoprop is clearly more herbicidally active than (−)-mecoprop,6 and for this reason, the biotests were performed with the pure enantiomers of mecoprop and bfl-acid. Range finding tests with (+)- and (−)-mecoprop at concentrations of 0.001−10 μM confirmed the higher herbicidal activity of the (+)-enantiomer. A significant influence on root length was observed at concentrations of ≥0.1 μM (+)-mecoprop, whereas, with (−)-mecoprop, ≈100× higher concentrations were needed for a comparable inhibition of root growth (Figure 5). This test also confirmed that root length is a reasonable measure of the activity of auxin-type herbicides.

Figure 4. Chlorophyll a content in 5-day-old cress cotyledons at different concentrations of rac-, (+)-, or (−)-beflubutamid, normalized to content in control samples without beflubutamid (1.2−1.4 mg/g wet weight). Error bars indicate standard deviations of n = 10 samples. Solid lines show fits according to a sigmoidal dose−response model (see text). The photos illustrate the bleaching effect of racbeflubutamid at concentrations of 0 and 2 μM.

where Chl a is the chlorophyll a content in the actual sample, normalized to the content in control samples without beflubutamid, c is the herbicide concentration, EC50 is the concentration that results in a 50% decrease in chlorophyll a, and b is a parameter that describes the slope of the dose− response curve. In this equation it is assumed that the lower level of the dose−response curve tends to a value of 0, i.e., that no chlorophyll a can be extracted from the leaves at high herbicide concentration. The experimental data were fitted reasonably well with this dose−response model (Figure 4). Estimated EC50 values were 1.06 ± 0.14 and 0.50 ± 0.10 μM for rac- and (−)-beflubutamid, respectively (errors signify 95% confidence intervals). The (−)-enantiomer was thus about twice as active as the racemate, which is reasonable in view of the fact that (+)-beflubutamid did not show any activity at all. The best estimates of the slope b were 1.24 and 1.01 for rac- and (−)-beflubutamid, respectively. The chlorophyll a content was somewhat underestimated at high herbicide concentrations (Figure 4). This may be due to the fact that photooxidation of chlorophyll a is a secondary effect of the herbicide beflubutamid, caused by an increased formation of singlet oxygen due to the missing protection by carotenoids.18 Apparently, chlorophyll a was not completely photooxidized after 5 days. Biotests with Pure Enantiomers of Bfl-acid. The chemical structure of bfl-acid, a phenoxybutanoic acid, resembles those of phenoxypropionic acid herbicides such as

Figure 5. Root length of 3-day-old cress seedlings at different concentrations of (+)- or (−)-bfl-acid (metabolite of beflubutamid) in comparison to (+)- or (−)-mecoprop (an auxin-type herbicide). Error bars indicate standard deviations of n = 30 samples. Root growth is clearly more inhibited by the (+)-enantiomers, but (+)-mecoprop is ≈100× more active than (+)-bfl-acid.

Root growth was also affected by bfl-acid, and as for mecoprop, the (+)-enantiomer was more active (≈10×) than the (−)-enantiomer (tested concentration levels, 1−100 μM, Figure 5). Bleaching symptoms were not observed. These findings and the structural similarity to mecoprop point to an auxin-type mode of action. However, bfl-acid was clearly less active than mecoprop and would certainly not be a promising herbicide. Further tests for an accurate determination of EC50 values were therefore not performed. A weaker activity of bflacid is consistent with the finding that phenoxyalkanoic acids with a Cl- or methyl-substituent in the ortho position of the phenoxy-ring tend to show higher herbicidal activity than corresponding compounds without ortho substitution.18 The (+)-enantiomer of mecoprop has (R)-configuration.6 The similar structure of mecoprop and bfl-acid (Figure 1) and the observed activities of the enantiomers in the biotests (Figure 5) are therefore indirect evidence that (+)-bfl-acid may 6810



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InformationActiveSubstances/InformationActiveSubstances_ documents/PlantProtectionProducts_InformationActiveSubstances_ docs_node.html. (11) Buerge, I. J.; Müller, M. D.; Poiger, T. The chiral herbicide beflubutamid (II): Enantioselective degradation and enantiomerization in soil, and formation/degradation of chiral metabolites. Environ. Sci. Technol. 2013, DOI: 10.1021/es301877n. (12) Baumgarten, H. E. Organic Syntheses; Wiley: New York, 1973. (13) Vogel, A. I., Furniss, B. S., Eds. Vogel’s Textbook of Practical Organic Chemistry, 5th ed.; Harlow Longman: London, 1989. (14) Buerge, I. J.; Poiger, T.; Müller, M. D.; Buser, H.-R. Influence of pH on the stereoselective degradation of the fungicides epoxiconazole and cyproconazole in soils. Environ. Sci. Technol. 2006, 40, 5443−5450. (15) Porra, R. J.; Thompson, W. A.; Kriedemann, P. E. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 1989, 975, 384−394. (16) Ritz, C.; Streibig, J. C. Bioassay analysis using R. J. Stat. Software 2005, 12, 1−22. (17) R Foundation for Statistical Computing. R: A language and environment for statistical computing; Vienna, Austria, 2012. (18) Cobb, A. H., Reade, J. P. H., Eds. Herbicides and Plant Physiology, 2nd ed.; Wiley-Blackwell: Chichester, U.K., 2010.

also have (R)-configuration. It may further be speculated that (+)-beflubutamid has (R)-configuration as well and, therefore, that (S)-beflubutamid is the herbicidally active enantiomer, which would be in line with the results reported in ref 10. Follow-up Studies. In our biotests, (−)-beflubutamid showed at least 1000× higher herbicidal activity than (+)-beflubutamid. Consequently, the agricultural use of enantiopure (−)-beflubutamid rather than the racemic compound may be advantageous from an environmental perspective. Beflubutamid is applied to soil, where it is subject to biotransformation processes before the herbicide is taken up by the roots of the weeds. The transformation reactions in soils may include enantioselective degradation as well as interconversion of the active enantiomer to the inactive enantiomer and vice versa, which may be mediated by soil microorganisms. In the accompanying paper,11 we therefore investigated the behavior of the enantiomers of beflubutamid (and its metabolites) in soil. From this study, sufficient amounts of enantiopure material were available to conduct several incubation experiments, and the enantioselective analytical methods were successfully employed to quantify traces of the compounds in soil extracts.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +41 44 783 6383. Fax: +41 44 780 6341. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Stähler International for providing the reference substances and Nina Spiess, Irene Hanke, and Michael Stüssi (Agroscope) for their assistance with the biotests.

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REFERENCES

(1) Ariëns, E. J., van Rensen, J. J. S., Welling, W., Eds.; Stereoselectivity of Pesticides, Biological and Chemical Problems; Elsevier: Amsterdam, 1988. (2) Kurihara, N., Miyamoto, J., Eds. Chirality in Agrochemicals; John Wiley & Sons Ltd.: Chichester, U.K., 1998. (3) Ulrich, E. M.; Morrison, C. N.; Goldsmith, M. R.; Foreman, W. T. Chiral pesticides: Identification, description, and environmental implications. Rev. Environ. Contam. Toxicol. 2012, 217, 1−74. (4) Gerwick, B. C.; Jackson, L. A.; Handly, J.; Gray, N. R.; Russell, J. W. Preemergence and postemergence activities of the (R) and (S) enantiomers of haloxyfop. Weed Sci. 1988, 36, 453−456. (5) Williams, A. Opportunities for chiral agrochemicals. Pestic. Sci. 1996, 46, 3−9. (6) Tomlin, C. D. S., Ed. The Pesticide Manual, 14th ed.; The British Crop Protection Council: Alton, U.K., 2006. (7) European Union. Regulation (EC) No 1107/2009 of the European Parliament and of the Council. Off. J. Eur. Union 2009, 11, L 309/50. (8) Takamura, S.; Okada, T.; Fukuda, S.; Akiyoshi, Y.; Hoshide, F.; Funaki, E.; Sakai, S. UBH-820: a new selective herbicide for weed control in cereals. Proc. Br. Crop Prot. Conf. Weeds 1999, 1, 41−46. (9) Rapporteur Member State Germany. Monograph on beflubutamid, 2002. http://www.bvl.bund.de/EN/04_PlantProtectionProducts/04_ EUReviewActiveSubstances/02_InformationActiveSubstances/ InformationActiveSubstances_documents/PlantProtectionProducts_ InformationActiveSubstances_docs_node.html (10) Rapporteur Member State Germany. Addendum 3 to the draft assessment report of beflubutamid, 2006. http://www.bvl.bund.de/EN/ 04_PlantProtectionProducts/04_EUReviewActiveSubstances/02_ 6811


The Chiral Herbicide Beflubutamid - American Chemical Society

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