16, 141-148 (1981)
of Dibutyl-14C-Labeled Dibutylaminosulfenyl Carbofuran in the Cotton Plant’
TAKAAKI NISHIOKA,~ NORIHARU UMETSU,* AND T. ROY FUKUTO Division of Toxicology California 92521: and
and Physiology, *Agro-Chemical
Department of Entomology, Research Laboratory, Otsuka Tokushima-ken 772, Japan
of California. Riverside. Company, Ltd., Naruto,
Received April 2. 1981; accepted July 9, 1981 The fate of the di-n-butylaminosulfenyl moiety in 2,3-dihydro-2,2-dimethyl-7-benzofuranyl (din-butylaminosulfenyl)(methyl)carbamate (DBSC or Marshal) was studied in the cotton plant at 1, 3, 6. and 10 days following foliage treatment with [di-n-butylamino-W]DBSC. Dibutylamine and two major radioactive metabolites were obtained following extraction of the plant tissue with a methanol-buffer containing N-ethylmaleimide (NEM), a sulthydryl scavenger which was added to prevent the cleavage of the N-S bond during the workup procedure. The most adundant radioactive material recovered from plants was identified as a product arising from the reaction between NEM and dibutylamine. Extraction of plant tissue with straight methanol-buffer solution or with methol-buffer containing other sulthydryl scavengers resulted in 57-86% of the applied radioactivity being recovered as dibutylamine in the organosoluble fraction. When [‘4C]dibutylamine was applied to cotton leaves, most of the radioactivity, i.e., 96% of the total recovered radioactivity, was found in the organosoluble fraction as dibutylamine. Dibutylamine is the major metabolite of [di-n-butylamino-W]DBSC in the cotton plant.
ring- and carbonyl-labeled DBSC (1). The results showed that the cleavage of the NS bond in DBSC initially occurred to form carbofuran which, in turn, was oxidized at the 3-position of the ring and the N-methyl group. However, the metabolic fate of the dibutylaminosulfenyl moiety in DBSC remained unknown. This report describes the fate of DBSC in the cotton plant following leaf application of [di-n-butylamino-
The N-dibutylaminosulfenyl derivative of carbofuran [2,3-dihydro-2,2-dimethyl-7benzofuranyl (di-n-butylaminosulfenyl)(methyl)carbamate, also referred to as DBSC3 or Marshall is a highly effective insecticide which is substantially less toxic to mammals than carbofuran (1). To assess potential hazards arising from its use as an agricultural chemical, the metabolism of DBSC in cotton and corn plants was studied using
N-Arylsulfenyl and N-aminosulfenyl derivatives of carbofuran are labile to thiol residues which are present in biological tissues (2, 3). In previous investigations, Nethylmaleimide, a sulfhydryl scavenger, was routinely added to the solvent used for extraction of metabolities to prevent cleavage of the N-S bond during the workup procedure (1, 4). However, in the present study, one of the radioactive metabolities turned out to be a product resulting from the reaction between NEM and dibutylamine, the latter being a true metabolite. The effect of other sulfhydryl scavengers
’ This investigation was supported in part by federal funds from the Environmental Protection Agency under Grant R804345 and a research grant from FMC Corporation, Middleport, N.Y. The contents do not necessarily reflect the views and policies of the Environmental Protection Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. L Present address: Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan. 3 Abbreviations used: DBSC, 2,3-dihydro-2,2dimethyl-7-benzofuranyl (di-n-butylaminosulfenyl)(methyl) carbamate; DBSC-sulfone, 2,3-dihydro-2,2dimethyl-7-benzofuranyl (di-n-butylaminosulfonyl) (methyl)carbamate; TLC, thin-layer chromatography; NEM, N-ethylmaleimide. 141
0048-3575/81/050141-08$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
on the extraction studied.
were prepared from dibutylamine and Nbutyl- and N-methylaminosulfonyl chloride, respectively (6). 1-Ethyl-3-(di-n-butylamiMATERIALS AND METHODS no)succinimide was prepared by heating 2 g Chemicals. [di-n-butyfamino-‘4C]DBSC, N-ethylmaleimide (NEM) with 3 g di-nhereinafter referred to as [‘“C]DBSC, spe- butylamine at 100°C for 1 hr. The product cific activity 19.8 mCi/mmol, was available was purified by column chromatography from a previous study (5) and was purified and distillation, bp 143°C (0.8 mm), n23D by column chromatography. A sample of 1.4690. Analysis calculated for C,,H,,N,O,: 32 mg [l”C]DBSC was applied to a florisil C, 66.10; H, 10.30; N, 11.02. Found: C. column (20 g, 60/100 mesh) and eluted step- 66.44; H, 10.43; N; 11.35. N-Ethyl-3-(di-nwise with 30 ml n -hexane, 30 ml rz-hexane butylamino)succinamic acid was prepared ethyl ether (19:1), 110 ml n-hexane-ethyl by alkaline hydrolysis of 1-ethyl-3-(di-nether (9: l), and 70 ml n-hexane-ethyl ether butylamino)succinimide in ethanol. NMR (4: 1). A total of 44 fractions (fractions l-2, and mass spectral data of the synthesized 20 ml; 3-6, 10 ml; 7-42, 5 ml: 43-44, 1.5 compounds were consistent with the assigned ml) were collected; purified DBSC was structures. Other chemicals were analytical present in fractions 27-29. The solvent was reagent grade and all solvents were redisremoved from the combined fractions and tilled before use. [ 14C]DBSC of >99% radiochemical purity Foliage treatment with [‘“Cl DBSC and was dissolved in absolute ethanol for storage. extraction of metabolites. The two bifoliate Authentic standards of anticipated leaves of cotton plants (Dehapine 61) in the metabolities are listed in Table 1 along with early trifoliate stage were painted with 10 or their thin-layer chromatographic (TLC) 20 ~1 each of an ethanol-acetone-water properties (I$ values) in different solvent (1:3:6) solution containing 4.1 pg (0.21 &i) systems. DBSC-sulfone was available from or 13.3 pg (0.17 &I) [‘“CIDBSC, respecan earlier study (1) and the bis(di-ntively. A set of three plants was analyzed butylamino) sulfide and bis(di-n-butylfor metabolites at 1, 3, 6, and 10 days folamino) disulfide were provided by FMC lowing treatment. The leaves were excised Corporation, Middleport, New York. N,- at the base of the stem and rinsed with 40 ml N,N’,N’-tetra-n-Butylsulfuric diamide and benzene-dichloromethane (1:l) to remove N,N-di-n-butyl-N’-methylsulfuric diamide surface residue. The wash, designated as TABLE Thin-Layer
Compound DBSC DBSC-sulfone Bis(di-n-butylamino)suKde Bis(di-n-butylamino)disultide N,N,N’,N’-tetra-n-Butylsulfuric N,N-di-n-Butyl-N’-methylsulfuric n-Butyric acid di-n-Butylamine
0.61 0.61 0.73 0.73 0.62 0.41 0.39 0
0.66 0.66 0.72 0.72 0.21 0
0.64 0.66 0.66 -
0.52 0.52 0 0
0.57 0.53 0
’ Solvent system: (A) hexanes-ethyl acetate (7:3); (B) dichloromethane-acetonitrile (9:l); (C) benzenemethanol (19:l); (D) ethyl ether-hexanes (3:2): (E) ethyl ether-hexanes (I:l): (F) ethyl acetate-methanolammonium hydroxide (18: 1: 1).
the leaf rinse, was analyzed by TLC for alteration products. The rinsed leaves, stems, and roots were cut into small pieces and ground thoroughly with a mortar and pestle in 20 ml (9:l) methanol-buffer (10 mM phosphate, pH 7.5) containing 9 mM NEM. In the experiments concerning the effect of sulfhydryl scavengers on the extraction of metabolities, a methanol-buffer solution containg the sulfhydryl reagent at a concentration of 9 mM was used. After filtration, the residue was extracted twice with 20 ml each of the same methanol-buffer solution. The residue was designated as the unextractable fraction. The filtrate and the extract were combined and concentrated to remove most of the methanol. The concentrate was added to 5. ml phosphate buffer (pH 7.5) and extracted three times with a total of 40 ml dichloromethane. The combined extract Was dried over anhydrous sodium sulfate and made up to a total of 50 ml. This was designated as the organosoluble fraction. The residual aqueous phase was made up to a total of 20 ml and designated as the water-soluble fraction. Metabolism of [l”C]di-n-butylamine. Tenmicroliter samples of an ethanol-acetonewater (1: 3 :6) solution containing [ 14C]di-nbutylamine (7.73 pg, 0.399 &i) were painted on the two foliate leaves of cotton plants. After 3 days, the distribution of radioactive metabolites was determined by the same procedure as described for the [‘“C]DBSC metabolism. The methanol-buffer solution used for metabolite extraction, however, did not contain any sulthydryl reagent. Analysis. Organic extracts were concentrated and analyzed by TLC using precoated silica gel GHLF plates (0.25 mm, Analtech Inc.). Location of radioactive spots on the TLC plates was accomplished by means of a Berthold (Varian-Aerograph) thin-layer radioscanner (Model LB 2723) equipped with a dot printer and was confirmed by autoradiography using Kodak X-ray film (BB5) exposed for 7-15 days. Radioactivity was quantitated with a
Beckman Model LS-3 145T liquid scintillation counter using 10 ml of a scintillation cocktail consisting of 6 g PPO, 0.2 g POPOP, 333 ml Triton X-100, and 666 ml toluene. Liquid samples were determined by counting 0.2-ml aliquots. Radioactivity in each spot on TLC plates was determined by scraping the spot from the plate and placing the silica gel in counting vials with scintillation cocktail. The radioactivity in the unextractable fraction was determined after oxygen combustion of 50- to 90-mg samples of the dried plant materials using a Model 306 Packard sample oxidizer. NMR spectra of model compounds and metabolites isolated from the extract were determined in a Varian EM-390 spectrometer using deuterochloroform as solvent and tetramethylsilane as lock signal. Mass spectra were recorded by direct insertion probe in a Finnigan Model 1015 mass spectrometer. Isolation c?f’M-l. A sample of M-l large enough for spectroscopic analysis was collected as follows. An ethanol-acetone solution containing [14C]DBSC was applied by brush on the leaves of 250 cotton plants in the early trifoliate stage. Approximately 170 pg DBSC was applied per leaf and the total applied radioactivity was 39.1 &i. Six days following treatment, the leaves were collected, homogenized in a blender with 9:l methanol-buffer solution (50 mM phosphate, pH 7.5) containing 9 mM NEM at the rate of 200 ml solvent per 30 plants. Of the applied radioactivity, 58% was organosoluble and of this 75.6% was dibutylamine, 10.7% was DBSC, 8.1% was M-l, and 5.5% were other unknown metabolites, including M-l 1. M-l was purified by repeated preparative TLC (silica gel, 60 PF-254, EM Laboratories) using four different solvent systems: hexanes-ethyl acetate (7:3), dichloromethane-acetonitrile (9:1), ethyl ether-hexanes (3:1), and chloroform-acetonitrile (4: 1). The amount of M- 1 collected after purification was 0.4% of the applied radioactivity.
(M-l), and metabolite 11 (M-11) were the major products at each time interval, representing 52-76% of the recovered radioactivity. Rf values for M-l using solvent systems A-D (Table 1) were 0.48, 0.54, 0.41, and 0.44, respectively. Rf values for M-11 with solvent systems A, B, and D were 0.17, 0.30, and 0.14. None of the available standards showed TLC properties corresponding to either M-l or M-l 1. M-l was stable in acidic and alkaline methanol and inert to L-cysteine and p-nitrobenzenethiol, i.e., reagents which readily react with compounds containing a sulfenyl-nitrogen linkage (2).
Distribution and Relative Amounts of Radioactive Metabolites Extracted by Methanol-Buffer Solution Containing NEM Analysis of radioactive materials following extraction of cotton leaves with methanol-buffer solution containing NEM was made 1, 3, 6, and 10 days after treatment with [‘“CIDBSC (Table 2). Total recovery of radioactivity at each time interval ranged from 88 to 101% of the applied amount. Virtually all of the recovered radioactivity was soluble in organic solvent, i.e., benzene-dichloromethane for the leaf rinse and dichloromethane for the internal extract. Radioactivity in the water-soluble and unextractable fractions was less than 1.5%. TLC separation of the various metabolites in the organosoluble fraction (internal extract) is shown in the two-dimensional chromatogram presented in Fig. 1. Dibutylamine (origin spot), metabolite 1
Metabolites Extracted Solution following
Extraction of Metabolites in the Presence of Other Suljhydryl Reagents Because of the possibility that M-l was a compound resulting from the reaction between NEM and a DBSC metabolite, experiments also were conducted in which other sulfhydryl scavengers such as iodoacetamide, mercuric chloride, 4,4’-dipyridyl
TABLE 2 from Cotton Leaves with NEM-Methanol -Buffer Treatment of Cotton Plants with [‘“C]DBSC Percentage
External extract DBSC M-2 to 20 Dibutylamine Subtotal
9.5 1.1 3.0 13.6
3.0 1.4 3.0 7.4
1.1 1.7 1.5 4.3
0.6 0.7 1.9 3.2
1.1 34.1 13.8 7.1 18.5 11.1 85.7 0.4
1.1 30.2 12.0 6.7 13.8 27.9 91.7 0.5
0.4 41.5 15.7 7.9 18.3 10.8 94.6 0.8
23.8 9.1 16.6 10.5 35.8 95.8 0.6
Internal extract Organo-soluble DBSC M-l M-2 to 10 M-11 M-12 to 20 Dibutylamine Subtotal Water-soluble conjugate Unextractable Total
‘r &+2o 2nd
FIG. 1. Twso-dimensional thin-layer chromatogram of the organosoluble fraction obtained following extraction of cotton plants by NEM containing methanol-buffer solution. First development: dichloromethane-acetonitrile (9:I); second development: ether-hexanes (1:l).
disulfide, and 5,5’-dithiobis(2-nitrobenzoic acid) were added to the methanol-buffer extracting solvent. Analyses of leaf extracts containing these reagents revealed dibutylamine as the predominating metabolite and, further, evidence was not found for the presence of M-l (see Fig. 2). TLC analysis of radioactivity following leaf extraction
FIG. 2. Thin-layer chromatogram of the organosoluble fractions obtained following extraction of cotton plants by methanol-buffer containing different sulfhydryl scavengers. (A) iodoacetamide; (B) mercuric chloride, (C) 4,4’-dipyridyl disulfide; (D) 5.5’dithiobis(2-nitrobenzoic acid); (E) N-ethylmalemide; (F) no scavenger. Solvent: dichloromethane-acetonitrile (9:l).
with straight methanol-buffer (no sulfhydry1 scavenger) gave results similar to that observed following extraction with methanol-buffer solvent containing iodoacetamide. Figure 3 shows a two-dimensional thin-layer chromatogram of radioactive products present in a methanol-buffer extract. Dibutylamine again was the predominating metabolite but small amounts of several unknown metabolites (I-l to I-6) also were observed. None of them corresponded to M-l or M-l 1. Because of the small amounts in which they were present, identification of these unknown metabolites was not pursued. Table 3 summarizes data for the relative amounts of the various metabolites extracted from cotton plants using methanolbuffer solution containing iodoacetamide or straight methanol-buffer solution after application of [i4C]DBSC. Of the recovered radioactivity, 57-86% was dibutylamine and 5--26% was DBSC. These two compounds accounted for 82-91% of the total recovered radioactivity. Identification
The mass spectrum of M-l, recorded by direct insertion probe at 70 eV, gave the following ions; mle 254 (4.4%), 225 (2.8%), 211 (74.8%), 197 (2.1%), 183 (2.0%) 169 (53.2%), 155 (17.1%). 149 (11.8%), 128
FIG. 3. Two-dimensional thin-layer chromatogram of the organosoluble fraction obtained following extraction of cotton plants by methanol-buffer solution. First development: dichloromethanr-acetonitrile (9:l); second de~~elopment: ether-hexanes (3:2).
of [ “C]DBSC Containing
Extracted from Co!ron Plants by Methanol-Buffer Iodoacetamide or by Methanol-Buffer Solution
Percentage of total DBSC applied Extraction by iodoacetamide methanol-buffer -
Extraction by methanol-buffer
External extract (leaf rinse) DBSC Unknowns Dibutylamine Subtotal
23.3 3.0 2.2 28.5
9.7 0.9 5.1 15.7
5.3 1.0 3.9 10.2
25.8 3.6 2.2 31.6
9.7 0.8 4.5 15.0
5.0 0.3 3.2 8.5
Internal extract Organosoluble DBSC I-l I-2 to 6 Dibutylamine Subtotal Water-soluble conjugate
1.5 2.1 6.8 60.6 71.0 0.5
1.7 5.8 5.9 70.2 83.6 0.7
1.3 7.7 7.5 72.2 88.7 1.2
1.9 4.4 7.4 54.5 68.2 0.2
2.0 6.4 8.8 67.3 84.5 0.5
0.4 3.1 4.8 82.7 91.0 0.3
co. 1 100.0
co. 1 100.0
co. 1 100.0
Unextractable (radioactivity Total
(30.6%), 126 (26.1%), 113 (14.8%), 112 and NMR spectra of the synthesized mate(14.0%). 98 (18.7%), 84 (26.2%), 70 (36.00/o), rial were identical to those of M-l. There57 (66.6%), 56 (56.0%), 55 (76.4%), 43 fore, unknown metabolite M-l was the prod(64.7%), 41 (lOO%), 30 (17.2%). Ion m/e 128 uct resulting from the reaction between NEM and dibutylamine. suggested the presence of the dibutylamino moiety. Fragment ions, m/e 254, 211, 169, Concerning the structure of the other 155, and 57 corresponded to a fragmentaprincipal unknown metabolite (M-l l), cotion pattern observed with di-n-butylamine, chromatography of M-l 1 and its alkaline dei.e., m/e 129 (M+), 86, 44, 30, and 57 (7). composition product with authentic stanThe adduct of NEM and dibutylamine has dards showed that it did not correspond to the empirical formula C,,H,,N,O, and a any of the standard compounds listed in mass of 254. M-l was assigned the structure Table 1 nor with methyl di-n-butylsulfa1-ethyl-3-(di-n-butylamino)succinimide (I). mate. A number of unsuccessful attempts This structure reasonably explains the MS were made to obtain M- 11 from DBSC, fragmentation of M- 1. Proton magnetic reso- DBSC-sulfoxide, and M-l by different reactions. Oxidation of [l”C]DBSC with m-chlonance (NMR) spectrum of M-l, recorded on a Bruker WH-90 with D lock and 5.9 roperoxybenzoic acid or with chromium tripsec pulse, also supported structure I; 6 oxide - pyridine did not result in any M- 11. 3.95 (H,, dd, JAX = 5.5, JBX = 7.8 Hz), 2.48 Acid-catalyzed alteration of [‘“CIDBSC (HA, dd, JAB = 17.0 Hz), 2.82 (HB, dd), 3.57 using acetic acid in dichloromethane (8) also did not produce any M- 11. Reaction of (>N-CH,-CH,, q, J = 7.9), 1.18 (>NNEM with DBSC-sulfoxide, a possible inCH2-CH,, t), 2.48 [N(CH,CH,CH,CH,),, m], 1.2-1.4 [N(CH.$ZH&H,CH&, m], termediate in the metabolic pathway of 0.90 [N(CH,CH,CH,CH,),, ml. The mass DBSC, did not yield any M-l 1. The sulfox-
ide, an unstable compound, was prepared by the reaction between the N-chlorosultinyl derivative of carbofuran and dibutylamine (9). M-l 1 did not cochromatograph with Nethyl-3-(di-n-butylamino)succinamic acid nor with any other alkaline hydrolysis products of M-l. Hydrolysis of M-l in the presence of NEM did not result in any M-l 1. Thus, the structure of M-11 remains unknown. However, since M-l 1, as in the case of M- 1, was observed as a metabolite of [‘“C]DBSC in the cotton plant only when NEM was added to the extracting methanol-buffer solvent, M-11 is believed to be an artifact involving NEM. Distribution of [‘“Cldi-n-butylamine Cotton Plant following Foliage Application
The above results showed that the dibutylaminosulfenyl moiety of DBSC was metabolized mainly to dibutylamine in the cotton plant. Since some of the unknown metabolites indicated in Table 3 might be derived from the further metabolism of dibutylamine, the metabolism of [‘“Cldi-nbutylamine in the cotton plant was examined 3 days after the foliage application. Total radioactivity recovered was 38.6% of the applied radioactivity. Most of the radioactivity, i.e., 96.3% of the total recovery, was recovered in the organosoluble fraction as dibutylamine. Traces of five other radioactive spots were detected upon TLC analysis of the organosoluble fraction. Their& values (solvent B) were 0.43, 0.25, 0.09, 0.07, and 0.04. However, these metabolites were too low in quantity to identify. The radioactivity in the water-soluble fraction was 1.6% of the total recovery. Overall, this experiment showed that dibutylamine applied externally was metabolized only to a minor extent in the cotton plant.
dure to minimize cleavage of the N-S bond by sulfhydryl groups present in plant and animal tissue. In the cases of [ring-14C]- or [carbonyl-‘4C]DBSC, NEM played a useful role in preventing anomalous formation of carbofuran during the plant extraction and workup process (1). However, in the present study with [dibutylamino-‘4C]DBSC, use of NEM gave rise to an unexpected product (M-l) which made the study more complex. Equations depicting the breakdown of [dibutylamino-14C]DBSC and subsequent formation of M-l are indicated in Fig. 4. Dibutylamine is the predominating radioactive metabolite recovered from cotton plants after foliage application of [dibutylarnino-14CIDBSC. This is evident from the data in Table 2 by summing the amount of radioactivity attributable to dibutylamine and M-l, and from the recovered amounts of dibutylamine indicated in Table 3. Dibutylamine is surprisingly stable in the cotton plant, constituting almost 60% of the recovered radioactivity 10 days following treatment (see Table 2). This is in marked contrast to the behavior of DBSC in soil where more than 50% of the radioactivity recovered 30 days following incorporation of [dibutylamino-‘“CIDBSC was carbon dioxide (5). No dibutylamine was recovered from soil following treatment with dibutylamino-labeled material. Besides M-l and M-l 1, a number of other minor metabolites were observed in methanol-buffer extracts of cotton plants treated with [‘“CIDBSC. As in the case of M-l 1, it was not possible to isolate sufficient quanti-
In previous studies on the metabolism of N-arylsulfenyl and N-aminosulfenyl derivatives of methylcarbamate insecticides, NEM was added during the workup proce-
FIG. 4. Equations (dibutylamino-14CjDBSC mation of M-l.
showing the breakdown of in the cotton plant and for-
ties of these materials analysis.
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