Eugene R. Mansager

Acifluorfen metabolism in soybean: Diphenylether bond cleavage and the formation of homoglutathione, cysteine, and glucose conjugates

Abstract Metabolism of the substituted diphenylether herbicide, acifluorfen [sodium 5-(2-chloro-4-trifluoromethylphenoxy)-2-nitrobenzoate], was studied in excised leaf tissues of soybean [ Glycine max (L.) Merr. ‘Evans’]. Studies with [ chlorophenyl - 14 C]- and [ nitrophenyl - 14 C]acifluorfen showed that the diphenylether bond was rapidly cleaved. From 85 to 95% of the absorbed [ 14 C]acifluorfen was metabolized in less than 24 hr. Major polar metabolites were isolated and purified by solvent partitioning, adsorption, thin layer, and high-performance liquid chromatography. The major [ chlorophenyl - 14 C]-labeled metabolite was identified as a malonyl-β- d -glucoside (I) of 2-chloro-4-trifluoromethylphenol. Major [ nitrophenyl - 14 C]-labeled metabolites were identified as a homoglutathione conjugate [ S -(3-carboxy-4-nitrophenyl) γ-glutamyl-cysteinyl-β-alanine] (II), and a cysteine conjugate [ S -(3-carboxy-4-nitrophenyl)cysteine] (III).

Aryl hydroxylation of diclofop by a cytochrome P450 dependent monooxygenase from wheat

Abstract In tolerant wheat, diclofop metabolism in vivo was rapid (33% in 6 hr) and was sensitive to cytochrome P450 inhibitors (CO, tetcyclasis, and 1-aminobenzotriazole). Microsomal fractions from etiolated wheat seedling shoots were shown to support the in vitro hydroxylation of diclofop in the presence of molecular oxygen and NADPH. Enzyme activity was strongly inhibited by tetcyclasis, but showed less sensitivity to 1-aminobenzotriazole and CO. The major enzymatic reaction product in vitro was isolated and identified by HPLC and electron impact mass spectrometry as the NIH shift product, 2-[4-(2,5-dichloro-4-hydroxyphenoxy)phenoxy]propanoic acid. The enzyme had a pH optimum of 7.4, a Vmax of 5.7 nmol/mg protein · h, and an apparent Km of 41.6 μM.

Metribuzin metabolism in tomato: Isolation and identification of N-glucoside conjugates☆☆☆

Abstract Metribuzin [4-amino-6- tert -butyl-3-(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in tomato ( Lycopersicon esculentum Mill. “Sheyenne”). Pulse-treatment studies with seedlings and excised leaves showed that [5- 14 C]metribuzin was rapidly absorbed, translocated (acropetal), and metabolized to more polar products. Foliar tissues of 19-day-old seedlings metabolized 96% of the root-absorbed [ 14 C]metribuzin in 120 hr. Excised mature leaves metabolized 85–90% of the petiole-absorbed [ 14 C]metrubuzin in 48 hr. Polar metabolites were isolated by solvent partitioning, and purified by adsorption, thin-layer, and high-performance liquid chromatography. A minor intermediate metabolite (I) was identified as the polar β- d -( N -glucoside) conjugate of metribuzin. The biosynthesis of (I) was demonstrated with a partially purified UDP-glucose: metribuzin N -glucosyltransferase from tomato leaves. A possible correlation between foliar UDP-glucose: metribuzin N -glucosyltransferase activity levels and differences in the tolerance of selected tomato seedling cultivars to metribuzin was suggested. The major polar metabolite (II) was identified as the malonyl β- d -( N -glucoside) conjugate of metribuzin.

Alternate pathways of metribuzin metabolism in soybean: Formation of N-glucoside and homoglutathione conjugates☆☆☆

Abstract Metribuzin [4-amino-6- tert -butyl-3(methylthio)-1,2,4-triazin-5(4H)-one] metabolism was studied in soybean [ Glycine max (L.) Merr. Tracy]. Pulse treatment studies with seedlings and excised mature leaves showed that [5- 14 C]metribuzin was absorbed rapidly and translocated acropetally. In seedlings, >97% of the root-absorbed 14 C was present in foliar tissues after 24 hr. In excised leaves, 50–60% of the absorbed 14 C remained as metribuzin 48 hr after pulse treatment, 12–20% was present as polar metabolites, and 20–30% was present as an insoluble residue. Metabolites were isolated by solvent partitioning, and were purified by adsorption, ion-exchange, thin-layer, and high-performance liquid chromatography. The major metabolite (I) was identified as a homoglutathione conjugate, 4-amino-6- tert -butyl-3- S -(γ-glutamyl-cysteinyl-β-alanine)-1,2,4-triazin-5(4H)-one. Metabolite identification was confirmed by qualitative analysis of amino acid hydrolysis products, fast atom bombardment (FAB), and chemical ionization (CI) mass spectrometry, and by comparison with a reference glutathione conjugate synthesized in vitro with a hepatic microsomal oxidase system from rat. Minor metabolites were identified as an intermediate N -glucoside conjugate (II), a malonyl N -glucoside conjugate (III), and 4-malonylamido-6- tert -butyl-1,2,4-triazin-3,5(2H,4H)-dione ( N -malonyl DK, IV) by CI and FAB mass spectrometry. Alternative pathways of metribuzin metabolism are proposed.

Soybean shoot metabolism of isopropyl-3-chlorocarbanilate: Ortho and para aryl hydroxylation

Abstract This laboratory reported that isopropyl-3-chlorocarbanilate-phenyl-U- 14 C (chlorpropham-phenyl- 14 C) was absorbed, translocated, and metabolized by soybean plants. Both polar metabolites and insoluble residues were found in roots, whereas only polar metabolites were found in shoot tissues. In both roots and shoots the polar metabolites were shown to be the O -glucoside of isopropyl-2-hydroxy-5-chlorocarbanilate (2-hydroxy-chlorpropham). In shoot tissue there were other polar metabolites that were not identified. The experiments with soybeans have been repeated, but with new isolation and purification procedures. The plants were root treated with both chlorpropham-phenyl- 14 C and isopropyl-3-chlorocarbanilate-2-isopropyl- 14 C. The roots and shoots were extracted and separated into the polar, nonpolar, and insoluble metabolic components, using the Bligh-Dyer extraction method. The polar metabolites were separated by gel permeation chromatography. Further purification was accomplished on Amberlite XAD-2. The polar metabolites from the shoot and root tissues were hydrolyzed either by β-glucosidase or hesperidinase. The enzyme liberated aglycones were derivatized and separated by gas-liquid chromatography, and the components were characterized by mass spectrometry or NMR. The results of this study showed that the polar metabolites of soybean shoots were 2-hydroxy-chlorpropham and isopropyl-4-hydroxy-3-chlorocarbanilate (4-hydroxy-chlorpropham). These two hydroxy-chlorpropham metabolites were found in soybean shoots at a ratio of approximately 1:1. The only aglycone found in root tissue was 2-hydroxy-chlorpropham. Using the new procedures, no evidence was obtained for the presence of the unidentified polar metabolites that were previously observed in shoot tissues.

Aryl hydroxylation of isopropyl-3-chlorocarbanilate by soybean plants

Abstract Isopropyl-3-chlorocarbanilate-phenyl UL- 14 C (CIPC- 14 C) is absorbed, translocated and metabolized by soybean plants. Water-soluble metabolites in root and shoot were purified and the root major metabolite characterized. The acetylated aglucones from the β-glucosidase hydrolysis and the esters from the direct acetylation of CIPC- 14 C polar metabolites were purified by GLC and analysed by mass spectrometry. The data showed that the phenyl riong of CIPC- 14 C was hydroxylated by both root and shoot tissues. Isopropyl-5-chloro-2-hydroxycarbanilate (hydroxy-CIPC) was the predominant aglucone liberated by β-glucosidase from polar metabolites in root and shoot. The o -glucoside of hydroxy-CIPC was shown to be present, by direct acetylation and characterization. In shoot tissue the major metabolites were dechlorinated hydroxy-CIPC and were not hydrolysed by β-glucosidase. These data show that soybean root or shoot tissues hydroxylate the phenyl ring of CIPC- 14 C but do not alter either the isopropyl alcohol moiety or the e arbamate bond.

Fate of [14C]diflubenzuron on cotton and in soil

Abstract Aqueous suspensions and oil emulsions of a commercial [14C]diflubenzuron (N-[[(4-chlorophenyl)amino]carbonyl]-2,6-difluorobenzamide) formulation (Dimilin W-25) remained on the leaf surface of greenhouse-treated plant tissues. Absorption, translocation, and metabolism of the [14C]diflubenzuron were not significant. Less than 0.05% of the applied 14C was found in newly developed plant tissues 28 days after spray treatment. [14C]Diflubenzuron was degraded in soil. After 91 days, biometer flask studies showed that 28% of the 14C incorporated into the soil as [14C]diflubenzuron was recovered as 14CO2. Major dichloromethane-soluble soil residues were identified as unreacted [14C]diflubenzuron and [14C]4-chlorophenylurea. A minor unknown degradation product cochromatographed with 2,6-difluorobenzoic acid. Insoluble 14C-residues increased with time and represented 67.8% of the residual 14C in the soil 89 days after treatment. Cotton plants grown for 89 days in [14C]diflubenzuron-treated soil contained only 3% of the 14C applied to the soil. Small quantities of acetonitrile-soluble [14C]4-chlorophenylurea were isolated from the foliar tissues. Root tissues contained small amounts of [14C]diflubenzuron and trace quantities of a minor 14C-product that chromotographed similarly to 2,6-difluorobenzoic acid. Most of the 14C in the plant tissues (84–93%) was associated with an insoluble residue fraction 89 days after treatment.

Alfalfa metabolism of propham

Abstract Root-treated alfalfa absorbs, translocates, and metabolizes [phenyl- 14 C]isopropyl carbanilate ([ 14 C]propham). After 7 days of root treatment, the distribution of radiolabel was 73% for shoots and 27% for roots. Shoots and roots were extracted and separated into the polar, nonpolar, and solid residual components using a mixture of chloroform, methanol and water. The insoluble residues accounted for approximately 40% of the 14 C found in shoots and roots. The nonpolar fraction (6.1% of the radiolabel in shoots and roots) was not characterized, but was shown to be some component other than parent propham. Propham was not found in either shoots or roots. The polar metabolites were partly purified on Amberlite XAD-2. Cellulase-liberated aglycones were derivatized and separated by high-performance liquid and gas-liquid chromatography. The infrared, nuclear magnetic resonance, and mass spectral data showed that the polar metabolites of alfalfa shoots and roots were glycoside conjugates of isopropyl 2-hydroxycarbanilate (2-hydroxypropham) and isopropyl 4-hydroxycarbanilate (4-hydroxypropham). Conjugated 4-hydroxypropham accounted for 45.9% of the 14 C in the shoots and 3.4% of the 14 C in the roots. Conjugated 2-hydroxypropham accounted for 3.4% of the 14 C in the shoots and 1.4% of the 14 C in the roots.

Metabolism of isopropyl carbanilate by soybean plants

Abstract Root-treated soybean plants absorb, translocate, and metabolize isopropyl carbanilatephenyl- 14 C (propham- 14 C). After a 3-day treatment period and removal of the exogenous 14 C treating solution, only small concentrations of 14 C-labeled materials were found in newly emerging tissues. A measurable concentration of radiocarbon was found in the seed pods, but the fruit tissues were shown to be free of any dectable 14 C-labeling. Three days after removal of the exogenous propham- 14 C, the parent herbicide was completely metabolized by all tissues. Polar products and nonextractable residues were found in roots, stems, and leaves after a 3-day treatment period. The polar metabolites were not translocated once they were formed in either the roots or shoots. Conjugated polar metabolites were isolated, partially purified, and the prophamphenyl- 14 C moiety characterized. The aglycone moiety of the polar metabolites was liberated either by methanol-HCl solvolysis or by enzyme hydrolysis with β-glucosidase or hesperidinase. The aglycone from all three procedures was derivatized, purified and characterized by NMR, ir, and mass spectral analysis. The only aglycone was the derivative of isopropyl-2-hydroxycarbanilate which was at least in part conjugated as a glycoside.

The pyrolysis of isopropyl 5-chloro-2-hydroxycarbanilate during gas chromatography☆

Abstract Isopropyl 5-chloro-2-hydroxycarbanilate has been shown to thermally degrade to 5-chloro-2-benzoxazolinone and unidentified polymeric products. Isopropyl 2-acetoxy-5-chlorocarbanilate was thermally degraded to N-acetyl 5-chloro-2-benzonazolinone and polymeric products. When isopropyl 2-acetoxy-5-chlorocarbanilate was pyrolyzed in the presence of dimethylamine isopropyl 2-hydroxy-5-chlorocarbanilate 5-chloro-2-benzoxazolinone and 3-(2-hydroxy-5-chlorophenyl)-1,1-dimethylurea were isolated. The acetylated carbamate was shown to be thermally more stable than the hydroxy analog.