Metabolism of chlorsulfuron by plants: Biological basis for selectivity of a new herbicide for cereals

PESTICIDE

BIOCHEMISTRY

AND

Metabolism

PHYSIOLOGY

17, 18-23 (1982)

of Chlorsulfuron by Plants: Biological Basis for Selectivity of a New Herbicide for Cereals

P. B. SWEETSER,

G. S. SCHOW, AND J. M. HuTCHISON

E. I. du Pont de Nemours & Company, Inc., Biochemicals

Department,

Wilmington, Delaware 19898

Received June 4, 1981; accepted September 24, 1981 A major factor responsible for the selectivity of chlorsulfuron [2-chloro-N-[(4-methoxy-6methyl-l,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide] (formerly DPX-4189), as a postemergence herbicide for small grains is the ability of the crop plants to metabolize the herbicide. Chlorstiron is the active ingredient in Du Pont “Glean” weed killer. Tolerant plants such as wheat, oats, and barley rapidly metabolize chlorsulfuron to a polar, inactive product. This metabolite has been characterized as the O-glycoside of chlorsulfuron in which the phenyl ring has undergone hydroxylation followed by conjugation with a carbohydrate moiety. Sensitive broadleaf plants show little or no metabolism of chlorsulfuron.

respectively, by a synthesis similar to that outlined by Levitt et al. (4). The radiochemical purity of both compounds was >9% as determined by HPLC with a Zorbax-ODS Column (condition 1 of HPLC methods). The synthesis of 5-hydroxy-2-chlorobenzenesulfonamide was accomplished as follows. 6-Chloro-m-anisidine hydrochloride was reacted with 1 eq of sodium nitrite in aqueous acid solution. The resultant diazonium salt was added to 3 eq of sulfur dioxide in acetic acid with a catalytic amount of cuprous chloride according to the procedure of Yale and Sowinski (5). The sulfonyl chloride was then reacted with 3 eq of anhydrous ammonia in ether solution to afford the sulfonamide (6). 5Methoxy-2-chlorobenzenesulfonamide was converted to 5-hydroxy-2-chlorobenzenesulfonamide using sodium ethyl mercaptide in dimethylformamide, according to the method of Feutrill and Mirrington (7). The 5-hydroxy-2-chlorobenzenesulfonamide, mp 170- 172°C was characterized by NMR and IR. The synthesis of 2-chloro-5-hydroxy-N[(4-methoxy-6-methyl1,3,5-triazin-2yl)aminocarbonyl]benzenesulfonamide, compound A-l, was accomplished as follows. 2-Chloro-5-benzyloxynitrobenzene was reduced with iron powder in ethanol/

INTRODUCTION

Chlorsulfuron’ [2-chloro-N-[(Cmethoxy6-methyl- 1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide], the active ingredient in Du Pont “Glean” weed killer, selectively controls weeds in small grain crops, such as wheat, barley, oats, and rye at rates of lo-60 g ai/ha. Although chlorsulfuron is extremely active as an herbicide, it has low mammalian toxicity. The acute oral LDsO for fasted male rats is 5545 mg/kg and for fasted female rats 6293 mg/kg. Results were negative when chlorsulfuron was tested in the Ames bacterial mutagenicity assay. Mode of action studies by Ray (3) have indicated that chlorsulfuron is an inhibitor of plant growth and cell division. In general, broadleaf plants are highly sensitive, while grasses show a broad range in sensitivity to chlorsulfuron. This study was undertaken to gain insight into factors leading to selectivity. MATERIALS

AND METHODS

Synthesis of Chemicals [phenyl-14C]Chlorsulfuron and [triazine-14C]chlorsulfuron were labeled at a specific activity of 2.2 and 5.4 mCi/nmol, ’ The common name proposed Standards Institute (1). 2 Formerly DPX-4189 (2).

by the American

18 0048-3575/82/010018-06$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

METABOLISM

OF

CHLORSULFURON

acetic acid solution (8). The resultant aniline was carried on to the corresponding benezenesulfonamide as described in the preceding paragraph. 2-Chloro-5-benzyloxybenzenesulfonylisocyanate, prepared according to the method of Uhich and Sayigh (9), was added to a suspension of 2-amino4-methoxy-6-methyl-1,3,5triazine in acetonitrile as described by Levitt et al. (4). The benzyl protecting group was removed by heating the sulfonylurea in trifluoroacetic acid for 1 hr. The resultant 2-chloro-5-hydroxy-IV-[ (Cmethyld-methoxy-1,3,5-u-u&n2-yl)aminocarbonyl]benzenesulfonamide , mp 184-186°C was characterized by NMR, IR, and mass spectral data. Plant Material All plant materials were grown in controlled-environment growth rooms in plastic pots containing the potting media, Terra-Lite Metro-Mix-350.3 The growth conditions were: 16-hr photoperiod at -3000 fc, 22-23°C day and 19-21°C night temperature. The pots were watered with tap water twice a day, and after the first week, were fertilized with Peters 2020-20 general purpose fertilizer. Metabolism studies with chlorsulfuron were made on l- to 3-week-old plants. Fifteen to twenty microliters of 50 to 400 ppm solutions of either Cphenyl-14C] or [triazineY]-chlorsulfuron (in lo-20% acetone, 0.2% Tween-20) was applied to the upper leaf surface and the plants returned to the growth room. This amount of chlorsulfuron was in the same range as that which might be expected in field applications (i.e., lo-60 g/ha). After 24 hr, the treated leaves were removed from the plants and the leaf surface washed three times with acetone. The washed, treated leaves were frozen in liquid nitrogen and stored at -20°C until the extraction step. Preparation of Plant Extracts All reagents and solvents were ACS grade. One to five grams of frozen plant 3 Terra-Lite Metro Mix growing tured by Grace & Co., Cambridge,

medium manufacMassachusetts.

19

BY PLANTS

tissue were extracted in a blender containing 50-75 ml of cold 80% acetone. The extract was centrifuged 75OOg at 4°C for 10 min. The excess acetone was removed from the supernatant with a rotating evaporator. The solution was titrated to pH 3.0 with H,SO, and extracted twice with equal volumes of ether. The ether layers were combined and taken to dryness under nitrogen. The remaining aqueous layer was extracted twice with equal volumes of n-butyl alcohol and the combined n-butyl alcohol layers taken to dryness with a rotating evaporator. High-Performance Chromatography Extracts

Liquid (HPLC)

of Plant

HPLC separations of the chlorsulfuron metabolites, acid hydrolysis products, and unreacted chlorsulfuron were made on a Du Pont Model 850 liquid chromatograph using three column-mobile phase combinations. 1. Du Pont Zorbax-ODS column (6.2 x 25 mm) with a mobile phase of A = H,O + 0.1% formic acid; B = acetonitrile + O.l$G formic acid, and a nonlinear concave No. 2 gradient of 5% B to 100% B in 30 min; flow, 2.7 mllmin; temperature, 35°C. 2. Du Pont Zorbax-ODS column (6.2 mm x 25 cm) with a mobile phase of A = Hz0 + 0.1% formic acid; B = MeOH + 0.1% formic acid, and a nonlinear concave No. 2 gradient of 5% B to 100% B in 30 min; flow, 2.7 ml/min; temperature, 35°C. 3. Whatman Partisil M-9 ODS column (9.4 mm x 50 cm) with a mobile phase of 15% acetonitrile, 85% H,O, 0.1% formic acid; flow, 10 ml/min; temperature, 25°C. The HPLC separations were made on the ether and n-butyl alcohol extracts after dissolving the dried extracts in 5 to 20% acetonitrile. Twenty to four hundred microliters of the extracts were injected onto the column and the distribution of the radioactivity was followed by collecting 0.5- to l.O-min fractions of the eluant directly into scintillation vials with a Model 568 ISCO fraction collector. The 14C activity was determined by scintillation counting of the fractions.

20

SWEETSER,

SCHOW,

Semipreparative amounts of metabolite A were collected with the Partisil M-9 ODS column from 1.O-ml sample injections of the n-BuOH wheat leaf extracts of plants which had been treated for 24 to 48 hr with [phenyl-14C]chlorsulfuron. PGlucosidase Hydrolysis of Metabolite A

Samples of metabolite A from wheat extracts were purified by semipreparative HPLC and the dried fraction made up in 0.1 M sodium acetate (pH 5.0). To this solution was added an equal volume of pglucosidase (EC 3.2.1.21), 1 mg/ml (1000 units/mg) in 0.1 M sodium acetate (pH 5.0). The reaction mixture was incubated at 30-32°C for 4-6 hr, the pH adjusted to 3.5, and extracted twice with equal volumes of ether. The combined ether extracts, containing the p-glucosidase hydrolysis product of metabolite A, were taken to dryness under nitrogen, giving A-l. RESULTS

AND

DISCUSSION

A number of factors can be responsible for the selectivity of an herbicide. These include differences in penetration, translocation, or rate of metabolism.

TABLE Penetration

and

Translocation

of [WlChlorsulfuron

AND

HUTCHISON

Studies on the amount of penetration and translocation of [14C]chlorsulfuron after 24 hr in sensitive and tolerant plants show no correlations to account for the wide degree of selectivity seen with these plants (see Table 1). There is a slightly greater amount of penetration of chlorsulfuron into the leaves of some sensitive plants vs tolerant plants, however, this difference would not account for the up to 4000-fold difference in tolerance to chlorsulfuron found between sensitive and tolerant plants. In contrast, a good correlation has been found between sensitivity of a plant to chlorsulfuron and the rate of metabolism of the herbicide. It should be noted that the terms tolerant and sensitive plant are subjective terms. The sensitivity of a plant to chlorsulfuron may vary with growth condition and age. In these studies, plants are classified as tolerant if they show only slight to moderate temporary growth inhibition by a foliar spray (to near runoff) of a 50-400 ppm solution of chlorsulfuron. Sensitive plants show severe growth inhibition from a 0.1 to 0.5 ppm foliar spray. Tolerant plants under the conditions of these studies include wheat, barley, wild oats, annual bluegrass,

1 in Sensitive

and Tolerant

Plants

(24-hr

Study)

Percentage 14C translocated out of treated leaf*

Plant” Sensitive plants Sugar beet Soybean (primary) Soybean (trifoliate) Mustard Cotton Tolerant plants Wheat Barley Wild oats

Percentage penetration into plant

To leaves and stems

To roots

91.2 82.0 93.3 97.7 56.0

2.6 12.3 1.9 17.6 4.3

0.1 4.0 0.9 0.8 0.5

68.7 66.3 72.9

5.4 1.1 2.7

3.3 1.4 0.6

a [phenyl-L4C]Chlorsulfuron applied to the first two true leaves of sugar beet and mustard, the primary and first trifoliate leaves of soybeans, cotyledon leaves of cotton, and the first true leaf of wheat, barley, and wild oats. b Values represent percentage of total added l*C activity which translocated out of the treated leaves to roots or leaves and stems in 24 hr.

METABOLISM

OF

CHLORSULFURON

johnsongrass, and giant foxtail. The sensitive plants were sugar beet, mustard, soybean, rape, cotton, and Galium aparine L. When a leaf of a sensitive plant (sugar beet) and a leaf of a tolerant plant (wheat) were treated with [phenyl-14C]chlorsulfuron, nearly 97% of the radioactivity in the treated leaf of sugar beet was recovered as chlorsulfuron after 24 hr, while with wheat only about 5% of the 14C in the leaf was chlorsulfuron. This same leaf residue pattern was observed in a variety of other tolerant and sensitive plants (Fig. 1). Thus, after 24 hr, less than 10% of the 14C activity in the treated leaves of tolerant plants was present as chlorsulfuron. With sensitive plants 80 to 97% of the 14C was unmetabolized chlorsulfuron. HPLC Characterization of Chlorsulfuron Metabolites in Leaf Extracts High-performance liquid chromatography has been an important tool in studying the metabolism of chlorsulfuron by plant tissue. Excellent separations of chlorsulfuron from its metabolites and possible acid breakdown products can be achieved with a Zorbax-ODS column using the water-acetonitrile gradient (see column-mobile phase 1 under Materials and Methods). The de-

TOLERANT

PLANTS

SENSITIVE

BY

7or t

WHEAT

21

PLANTS

EXTRACT

,i

5ot

%

1OOr

9 E I

80:

f” $

R

i

SUGAR

BEET

EXTRACT

60i do:

IO TIME

(MINUTES)

I2

14 -

16 HPLC

18

20

22

FIG. 2. The 14C activity in metabolite A and c,hlorsulfuron in wheat and sugar beet leaf extracts. Thr percentage WI? is related to the total 14C in the treutetl leaf after the surface wash (24-hr study).

tection limit for chlorsulfuron with the 254nm wavelength uv detector was 5 to 20 ng. A slightly lower detection limit was possible with [14C]chlorsulfuron and scintillation counting of OS- to 1-min chromatographic fractions. Liquid chromatographic examination of leaf extracts from [phenyl- 14C]chlorsulfuron-treated wheat leaves shows the presence of a metabolite (metabolite A) which elutes at 14-15 min under conditions in which chlorsulfuron would elute at 22-23 min. Similar sugar beet leaf extracts show no metabolite A formation. The percentages of 14C activity in metabolite A and chlorsulfuron in wheat and sugar beet leaf extracts, which have been treated with [14C]chlorsulfuron for 24 hr, are given in

PLANTS

1. The percentage of unmetabolized chlorsuljitron in the treated leaves of sensitive and tolerant plants 24 hr after a leaf application of [W]chlorsulfk ron. The percentage chlorsulfuron is related to the total 14C in the leaf after the surface wash. FIG.

FIG. 3. HPLC characteristics glucosidase hydrolysis product Chromatographic conditions similar

of A-l, the pof‘ metabolite A. to those in Fig. 2.

22

FlG.

sulfuron

SWEETSER,

4. Proposed by wheat.

scheme

for

metabolism

SCHOW,

of chlor-

Fig. 2. Leaf extracts of other tolerant grasses, 24 hr after treatment with [phenylshow similar HPLC 14C]chlorsulfuron, patterns of 14C activity. Metabolite A is the major metabolite of chlorsulfuron in the tolerant grasses. No metabolite A was found in heat-killed tissue. Characterization and A-l

of Metabolites

A

Metabolite A reacts rapidly with the enzyme /3-glucosidase to yield a less polar product (A-l). This hydrolysis product extracts from aqueous solutions (pH 3.0) with ether whereas metabolite A extracts from similar aqueous solutions with n-butyl alcohol but not with ether. The less polar nature of A-l is also demonstrated by its HPLC elution (Fig. 3). Metabolite A was isolated from wheat leaves by HPLC and hydrolyzed to A-l by the enzyme P-glucosidase. Small quantities of A-l were then isolated by HPLC and characterized as follows: 1. Results from the use of chlorsulfuron, which had the 14C label either in the phenyl or triazine ring, indicated that both rings are still present in metabolite A and A-l. 2. Acid hydrolysis of chlorsulfuron resulted in the formation of 2-chlorobenzenesulfonamide and the corresponding aminotriazine. Acid hydrolysis of A-l gave the aminotriazine but no compound identical to 2-chlorobenzenesulfonamide. 3. Mass spectrographic analysis of A-l demonstrated the presence of a hydroxyl

AND

HUTCHISON

group on the phenyl ring by the presence of fragment ions at 127, 191, and 233 m/e, corresponding to C,H,Cl(OH), C,H,Cl(OH)SOP, and CsHSCl(OH)S02N=C=0 ions, respectively. 4. Independent synthesis of 2-chloro-5hydroxybenzenesulfonamide and 5-hydroxychlorsulfuron. Comparison of HPLC retention times (in two solvent systems), mass spectra, NMR, and IR show the above compounds in 4 to be identical to the acid hydrolysis product of A-l and A- 1 itself, respectively. The 220-MHz NMR spectrum of A-l provides further evidence that hydroxylation occurs in the 5 position. The protons on the aromatic ring appear as follows: 7.7 6, doublet, J = 3.7 Hz (para coupling); 7.15 6, doublet, J = 8.5 Hz (ortho coupling); 6.8 6, doublet of doublets, J = 3.7, 8.6 Hz (para and ortho coupling). A proposed scheme for the metabolism of chlorsulfuron in wheat and other tolerant cereals is shown in Fig. 4. It is proposed that chlorsulfuron is first metabolized to the 5-OH derivative (II) and then rapidly conjugated to the 5-glycoside of chlorsulfuron (III). No free A-l has been detected in the leaf extracts. This would indicate that either the 5-hydroxy group is rapidly conjugated after its formation, or that the 5glycoside is formed directly from the chlorsulfuron without release of the 5-hydroxy derivative. Although no positive characterization of the carbohydrate moiety has been made, the most probable structure, based on the rapid hydrolysis by pglucosidase, would be that of an Oglucoside. No significant herbicidal activity has been seen with metabolite A or A-l on sensitive seedlings of corn, soybean, and lettuce. Metabolite A represents the major metabolite in tolerant grasses. The amounts of metabolite A formed by a number of plants 24 hr after foliar application of chlorsulfuron are given in Fig. 5. Tolerant grass plants show a rapid rate of formation of metabolite A, while sensitive broadleaf plants form

METABOLISM K METABOLITE PLANTS

1

5

IO

15

20

25

10

OF CHLORSULFURON

A S.5

40

45

SO

55

GO

. WHEAT =

Wll.0

2 y e

A. BLUEGRASS JOHNSONGRAss GIANT FOXTAIL

OATS

SOYBEAN RAPE GALIVM

$1

MUSTARD I SUGAR

23

(J. C.-Y. Han, DuPont, unpublished data). No intact chlorsulfuron or identifiable degradation products were present in grain and straw extracts. ACKNOWLEDGMENTS

COTTON w z 5

BY PLANTS

BEET

FIG. 5. The percentuge of metabolite A in the treated leataes ofplants 24 hr after a leaf application of [phenyl-L4C]chlorsulfuron. The percentage A is related to the total [“C in the treated leaf. Chromatographic conditions similar to those in Fig. 2.

The authors gratefully acknowledge the work of Robert W. Reiser in obtaining and interpreting the mass spectra of A-l; J. J. Reap and M. P. Rorer for the synthesis of [“Clchlorsulfuron; J. C.-Y. Han for the residue studies on field grown wheat; Gade S. Reddy. Central Research and Development Department, for interpreting the NMR data: and Ivan M. Turner for hih technical assistance. REFERENCES

only trace amounts of the metabolite, as in cotton, to no detectable metabolite in sugar beet and soybean. Kinetic studies on the rate of chlorsulfuron metabolism indicate a half-life of only 2-3 hr in wheat leaves. This rate of metabolism is sufficient to account for the strong tolerance of wheat to the herbicide. In contrast to the above short-term studies, 3- to 4-month residue studies with [phenyl-‘*Cland [triazine-14C]chlorsulfuron on field-grown wheat indicate that the 14C-residue levels in the mature straw and grain were extremely low (Table 2) TABLE 2 Metabolism-Mature Field-Grown (Treatment. 70 g/ha)

Wheat

Total 14C residueb (ppm calculated as chlorsulfuron)

Position of label

Wheat grain

Straw

Phenyl Triazine

0.02 0.01

0.05 0.04

” Postemergent treatment at fourth-leaf stage. b Dry-weight basis.

1. G. Levitt, C. W. Bingeman, and G. E. Barrier, A new herbicide for cereals, Weed Sci.. 26, (1980). 2. “DPX-4189 Experimental Herbicide,” Du Pont Product Information Bulletin, E. I. du Pont de Nemours & Co., Inc., Biochemicals Department, Wilmington, Del. 3. Thomas B. Ray, Studies on the mode of action of chlorsulfuron: A new herbicide for cereals. Pestic. Biochem. Physiol., 17, 10 (1982). 4. G. Levitt, H. L. Ploeg, R. C. Weigel, Jr., and D. J. Fitzgerald, 2-Chloro-N[(4-methoxy-6methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide, a new herbicide. J. Agr. Food Chem. 29, 418 (1981). 5. H. L. Yale and F. Sowinski, 1-Alkyl-3-(u,u,cutrifluorotolylsulfonyl)ureas, J. Or~q Chem. 25. 1824 (1960). 6. M. L. Crossley, E. H. Northey, and M. E. Hultquist, Sulfanilamide derivatives 1, J. Aftrer. Chem. Sot. 60, 2217 (1938). 7. G. I. Feutrill and R. N. Mirrington, Demethyla.tion of aryl methyl ethers with thioethoxide ion in dimethyl formamide, Tetrahedron Lett.. 1327 (1970).

8. D. C. Owsley and J. J. Bloomfield, The reduction of nitroarenes with iron/acetic acid, Synthesis, 118 (1977).

9. H. Ulrich and A. A. R. Sayigh, Synthesis of isocyanates by fragmentation of sulfonylureas, Angel{,. Chem. Int. Ed. Engl. 8, 724 (1966).