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

PESTICIDE

BIOCHEMISTRY

Metribuzin

AND

PHYSIOLOGY

Metabolism

19,

270-281 (1983)

in Tomato: Isolation and Identification Glucoside Conjugatesll*

of N-

D. S. FREAR, E. R. MANSAGER, H. R. SWANSON, AND F. S. TANAKA Metabolism

and Radiation Research Laboratory, U. S. Department of Agriculture, Service, Fargo, North Dakota 58105

Agricultural

Research

Received September 29, 1982; accepted December 10, 1982 Metribuzin [4-amino-6-rert-butyl-3-(methylthio)-l,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 [5-14C]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 [LdC]metribuzin in 120 hr. Excised mature leaves metabolized 85-90% of the petiole-absorbed [IQmetribuzin 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 S-D-(N-glucoside) conjugate of metribuzin. The biosynthesis of (I) was demonstrated with a partially purified UDP-glucose:metribuzin Nglucosyltransferase 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 p-o-(N-glucoside) conjugate of metribuzin. INTRODUCTION

Metribuzin [4 - amino - 6 - tert - butyl - 3 (methylthio)-1,2,4-triazin-5(4H)-one] is a member of the substituted as-triazinone group of herbicides that inhibit photosynthesis (l-4). It is an effective herbicide for selective preemergence and postemergence weed control in direct-seeded or transplanted tomatoes (5) and in other tolerant crops (1, 6-8). Differences in tomato tolerance have been reported to occur during the seedling stage of growth (5, 9), under various environmental conditions (5, 9-l l), and with different cultivars (12). It has been suggested that differences in metribuzin tolerance may be related to genetic inheritance and to rates of metabolic detoxification (12, 13). Unfortunately, detailed information on the metabolism of metribuzin in

tomato and other plants is limited. The objectives of the present study were twofold: (i) to identify the major metabolites and to determine primary pathway(s) of metribuzin metabolism in the tomato plant, and (ii) to examine possible correlations between herbicide metabolism and reported differences in the tolerance of tomato plants to metribuzin treatment. METHODS

AND MATERIALS

Chemicals. Metribuzin and [5J4C]metribuzin (26.8 pCi/pmol) were provided by Mobay Chemical Corporation. Chemical and radiochemical purities were greater than 99%. Reference compounds [6-tert-butyl3(methylthio)-1,2,4-triazin-5(4I-I)-one] @A); [4 - amino - 6 - terf - butyl - 1,2,4 - triazin - 3,5 (2H,4H) - dione] (DK); and [6 - tert - butylI Presented in part at the North Central Weed Con- 1,2,4-triazin-3,5(2H,4H)-dione] (DADK) trol Conference, Des Moines, Iowa, December, 1981. were also provided by Mobay Chemical * Mention of a trademark or proprietary product does Corporation. All solvents were reagent not constitute a guarantee or warranty of the product grade except for the acetonitrile (Fisher, by the U. S. Department of Agriculture and does not HPLC grade) that was used for final HPLC imply its approval to the exclusion of other products that may also be suitable. purifications. Sephadex G-50 (fine or me270

0048-3575/83 $3.00 Copyright AII ri&ts

0 1983 by Academic F’ress, Inc. of reproductionin any form reserved.

N-CLUCOSIDE

METABOLITES

dium grade) and DEAE-Sephacel (preswollen beads) were obtained from Pharmacia. The t-butyl-dimethylchlorosilane/imidazole reagent was obtained from Applied Science Laboratories. The ultra micro Glucostat reagent was obtained from the Worthington Biochemical Corporation, I-0-Phenyl (Yand B-D-glucosides, UDP-Glc (Na salt, 98100%), and B-glucosidase were purchased from Sigma Chemical Corporation. Plant materials and treatments. Greenhouse tomato plants, Lycopersicon esculentum Mill., were germinated and grown in vermiculite with nutrient solution (14). The Sheyenne variety was used for all translocation, metabolism, and enzyme characterization studies. Other tomato cultivars, with reported differences in metribuzin tolerance (12, 15), were used for differential metabolism studies. Seeds were provided by Dr. E. A. Kerr, Horticultural Experiment Station, Simcoe, Ontario, Canada. Excised petioles from mature tomato plants were used for metabolite isolation and identification studies. Each petiole was pulse treated via the cut end with 1 ml of 1 x IO-3M [14C]metribuzin (0.46 $i/kmol). After approximately 2 hr, the absorbed [14C]metribuzin was followed by a distilled water chase. Treated tissues were maintained in distilled water under cool white fluorescent lights (50-60 PEm-2 set-1; 14hr photoperiod) at room temperature for 48 hr prior to [14C]metabolite extraction. In preliminary translocation and metabolism studies, 19-day-old tomato seedlings were transferred directly from vermiculite to test tubes (7.5 x 1.2 cm). Each seedling was pulse treated via the roots with 0.5 ml of 4.26 x 10-SM [14C]metribuzin (26.8 @i/ IJ,rnol). Absorbed [‘4C]metribuzin was chased with distilled water and seedlings were maintained, as described for excised tissues, until harvested (1-5 days). Chromatography. Silica gel HF plates (250 or 500 pm) were used for all thin-layer chromatography (TLC). TLC solvent systems were: (A) benzene-acetone (2/l) and

OF

METRIBUZIN

271

(B) methylene chloride-methanol-water (651 25/4). Separated ‘4c zones were located with a Packard 7201 radiochromatogram scanner or fluorescence quenching under uv light. High performance liquid chromatography (HPLC) equipment (Waters Associates) included two 6000A pumps, a 720 system controller, an M-730 data module, a U6K injector, and an RCM-100 radial corn,pression module. HPLC chromatograms were monitored with a Vari-Chrom (Varian Instrument Division) variable wavelength detector at 254 nm, a 1055 CA1 radioactivity monitor with a 200-p,l flow cell containing glass scintillator beads, and a 440 Integral Data System printer. All HPLC separations were obtained with 10 pm Cl8 RadialPak cartridges (Waters Associates) and a solvent flow rate of 2.5 ml/min. Five different HPLC solvent systems were used: (1) 15-min linear gradient with an initial 3-min hold from 0 to 80% CH3CN in HzO; (2) isocratic CH3CN/H20 (15/85); (3) 15-min linear gradient from 30 to 80% CH&N in H,O; (4) isocratic CH3CN/H20 (85/15); and (5) ISmin linear gradient with an initial 3-min hold from 15 to 80% CH,CN in H20. A Varian 3700 gas chromatrz-?ph equipped with FID and a glass column (200 cm x 4 mm i.d.) packed with 3% SP 2340 (Supelco) on 100/200 Supelcoport was used for gas chromatographic (GLC) analysis of dimethylmalonate. Injector and detector temperatures were 180 and 260°C. respectively. The oven temperature was 80°C and the nitrogen carrier gas flow rate was 20 cm3/min. Under these conditions, the retention time for standard dimethylmalonate was 3.5 min. Instrumentation. Mass spectra [(electron impact (EI) and chemical ionization (CI)] were obtained with a Varian MAT 112s spectrometer equipped with a combination EI/CI source and a SS-200 data system. Samples were inserted directly with a solid sample probe. Ammonia was the ionizing gas for CI spectra. NMR spectra were obtained with a JEOL FX-90Q Fourier transform spectrometer

272

FREAR

ET AL.

LC Millipore) and combined for 14C determination. Methanol was removed in vacua (30°C) and the aqueous concentrate was partitioned three times with equal volumes of Et,O. The Et,0 phases were separated by centrifugation and combined for 14C analysis. Five-milliliter aliquots of the aqueous phase were applied to preconditioned Cl8 Sep-Pak cartridges (Waters Associates). Each cartridge was washed with 5 ml of HZ0 and the adsorbed [14C]metabolites were eluted with 10 ml of aqueous 80% MeOH. Eluates were combined, concentrated in vac~o at (30”(Z), streaked on preparative (500 pm) TLC plates (20 x 20 cm), and chromatographed (solvent system B). A broad r4C zone (&0.35-0.65) was removed and the silica gel was eluted with aqueous 80% CH&N. The eluate was

equipped with a microprobe and an NVVTS variable temperature controller. Tetramethylsilane was used as the internal reference and samples were analyzed in 1.8 mm o.d. tubes. Quantitative 14C measurements were made with Insta-Gel (Packard) counting cocktail and a Packard 3375 liquid scintillation spectrometer. Insoluble 14C residues were determined by combustion analysis with a Packard 306 sample oxidizer. Metabolite extraction and isolation. Procedures developed for metabolite extraction and isolation are summarized in Fig. 1. [14C]Metribuzin-treated leaves (20 g fresh weight) were ground and extracted in an Omnimixer with 200 ml of aqueous 80% MeOH (2 x ) and 100 ml of absolute MeOH (1 x). Extracts were vacuum filtered (10 pm,

II14C 1Mebibuzin-treated

Leaf Tissue 80%

MeOH

I Insoluble

I Soluble I Aqueous

C’s

Sep I TLC

I Et,0

Pak 80%

MeOH

Solvent C’B

S

HPLC System

1

I

I

Metabolite C’*

HPLC I

Acetylate

Metabolite

(1)

C’s

System

2 Methylate

or Silylate (Separately) C’*

(II:) I HPLC System 2 I and Acetylate I

HPLC System

3

I Purified FIG.

1. Scheme

Metabollte for

the isolation

(I)

and

(H

and derivation

1 Derivatives of metribuzin

metabolites.

N-GLUCOSIDE

METABOLITES

taken to dryness in vucuu (3O”C), dissolved in a small volume of aqueous 50% CH,CN, and chromatographed (HPLC system 1). Two 14C peaks were eluted (metabolite I; 10.8 min, and metabolite II; 9 min). Peak fractions were collected separately, taken to dryness in vucuo (3o”C), dissolved in a small volume of aqueous 50% CH,CN, and rechromatographed (HPLC system 2). Purified metabolites I and II eluted at 6.8 and 3.2 min, respectively.

OF

METRIBUZIN

273

ment at m/z 605, and a base peak protonated aglycone ion fragment at m/z 200. Silylated glucose ions resulting from fragmentation on either side of the metribuzin amino group were observed at m/z 520,406, and 391. NMR spectroscopy. Proton NMR spectra of metribuzin in acetone-d, gave peaks at 1.38 (t-Bu), 2.51 (S-Me), and 5.54 ppm (NH,). Spectra in Me2SO-d6 gave peaks at 1.33 (t-B@, 2.44 (S-Me), and 5.88 (NH?). Metabolite derivatization and purificaAU peaks were observed as singlets. Extion. Isolated [14C]metabolites (I and II) cept for the amino group, all peaks that were were derivatized and purified as summarelated to the metribuzin moiety in NMR rized in Fig. 1. Metabolite I was acetylated spectra of underivatized and silylated meat 40°C for 3 hr with an excess of acetic tabolite I were observed at approximately anhydridejpyridine (9: I). Excess reagents the same location as with the metribuzin were removed with a stream of nitrogen and standard. Resonance of the conjugated by TLC separation with Et,O. Acetylated amino group was shifted downfield to the products (Z$ 0.15) were eluted from the sil- spectral region ranging from 6.0 to 8.0 ppm. ica gel and chromatographed (HPLC sysProton NMR spectra of underivatized tem 3). Two acetylated metabolite I prod- metabolite I in Me$O-ds showed the anoucts were separated with retention times of merit proton as a doublet centered at 4.92 8.12 and 8.94 min, respectively. Metabolite ppm with a coupling constant of 7.2 Hz. II was methylated with diazomethane at 4°C Addition of D,O caused peaks in the 4.4 to for 1.5 min (16). Excess diazomethane was 6.0 ppm region to disappear. The peak at removed with a stream of nitrogen and the 4.92 ppm remained, but a satisfactory resmethylated product was acetylated and olution of the doublet was not possible. purified as described above for metabolite The NMR spectra of both acetylated meI. Two methyl/acetyl derivatives of metabtabolite I derivatives were identical. In olite II were separated (HPLC system 3). DCC& or acetone-d,, the nonanomeric proRetention times were 8.2 and 9.0 min, re- tons of the glucose moiety were observed spectively. as multiplets ranging from 3.9 to 5.5 ppm. Silyl derivatives of metabolite I and I-OProton NMR spectra of the silylated mephenyl CX-and P-D-glucoside reference stan- tabolite I in DCC13 showed the anomeric dards were prepared for NMR analysis by proton as two doublets centered at 4.34 and reaction with excess t-butyldimethylsilane/ 4.50 ppm with a coupling constant of .I = imidazole reagent at 80°C for 20 min. Reac- 7.0 Hz. Integration of the two doublets intions were stopped by the addition of H,O. dicated the presence of one proton. Spectra Silylated products were extracted with Et,0 taken in acetone-d, showed the anomeric and chromatographed (HPLC system 4). A proton as two doublets at 4.32 and 4.46 ppm with a coupling constant of .I = 9.2 Hz. major (>85%) silylated derivative of metabolite I eluted as a sharp 14C peak with a Addition of DzO caused the NH doublet retention time of 5.8 min. Two minor 14C (6.44 ppm; J = 6.56 Hz) to disappear, but peaks with retention times of 7.4 and 11 .O the two doublets for the anomeric proton min were also observed. The CI mass spec- remained. tra of the major HPLC peak fraction showed Proton NMR spectra of silylated I-Oa weak protonated molecular ion (M+ 1) at phenyl cx- and p-D-glucosides in DCCl, m/z 719, a protonated (M- 115) ion frag- showed the anomeric proton of the (Y an-

274

FREAR

omer as two doublets centered at 5.30 and 5.39 ppm with a coupling constant of J = 3.5 Hz. Spectra of the l3 anomer showed the anomeric proton as two doublets at 4.84 and 4.98 ppm with a coupling constant of J = 7.0 Hz. Heating the (Yanomer at 60 and 80°C in the spectrometer did not cause any merging of the two doublets. Glucose determinations. Purified [ 14C]metabolites I and II were hydrolyzed with 0.5 ml of 1 N HCl at 80°C for 3 hr. Hydrolysates were analyzed qualitatively (TLC) and quantitatively (glucose oxidase) for glucose as reported previously (17). Malonate Determinations. Purified [Wlmetabolite II (30-40 pg) was dissolved in 0.1 ml MeOH and reacted at room temperature for 3 hr with an excess of diazomethane dissolved in Et,0 (16). At the end of the reaction, excess diazomethane and Et,0 were removed with a gentle stream of nitrogen. Aliquots of the MeOH-soluble products were analyzed quantitatively for dimethylmalonate (GLC) and 14C. Enzyme

extraction

and purification.

ET AL.

The standard reaction mixture contained 100 pmol of K phosphate buffer, pH 8.0, 0.9 pmol of UDP-Glc, 0.026 pmol of the [i4C]metribuzin substrate (3.06 pXi/p,mol), enzyme, and distilled water to give a final volume of 1 .O ml. The reaction was initiated by the addition of [14C]metribuzin and incubated at 25°C for 1 hr. Controls were run either with boiled enzyme or without UDPGlc. The reaction was stopped by rapid freezing in a dry ice-acetone bath. Frozen reaction mixtures were lyophilized and dissolved in 0.5 ml of aqueous 80% MeOH. Aliquots (50 or 100 ~1) were analyzed quantitatively for unreacted substrate and the Nglucoside reaction product by HPLC (system 5) using the radioactivity flow monitor coupled with the integral data system. Retention times for metribuzin and the ZV-glucoside were 7.0 and 3.4 min, respectively. Under the assay conditions, enzyme activity was proportional to protein concentration and linear with time. The enzyme unit (U) was defined as that amount of enzyme required for the biosynthesis of 1 nmol of N-glucosidelhr under the conditions of the assay. Protein was determined by the method of Bradford (18) with crystalline bovine serum albumin as the standard.

Leaves (3WO g fresh wt) from 4- to 5-weekold tomato plants were ground to a fine powder in liquid nitrogen and homogenized in a mortar with 2 vol of 0.1 M K phosphate buffer (pH 7.5) containing 0.01 M cysteine. The homogenate was strained through Enzyme activity determinations in tocheesecloth and centrifuged at 15OOg for 10 mato seedlings. Leaves (~1 g fresh wt) from min. The crude cell-free supernatant was tomato cultivar seedlings were harvested at centrifuged at 70,OOOg for 30 min. The the same stage of development (6-8 days 70,OOOg supernatant was precipitated with after emergence) and ground in a glass tis(NH4)#04 (30-60% saturation), dissolved sue homogenizer with 3 ml of 0.1 M K in a small volume (5-10 ml) of 0.05 M K phosphate buffer (pH 8.0) containing 0.01 phosphate buffer (pH 7.5) and placed on a M cysteine. Homogenates were centrifuged Sephadex G-50 (tine) column (2.5 x 25 cm) at 20,OOOg for 1 hr and supernatant fracequilibrated with the same buffer. The en- tions were placed on Sephadex G-50 colzyme was eluted with two void volumes of umns (1 x 20 cm) equilibrated with 0.05 M the equilibrating buffer at a flow rate of 1 K phosphate buffer (pH 7.5). Single sharp ml/min. Peak fractions were combined, ly- protein peaks (~2 void volumes) were eluted ophilized, and stored at -15°C. All steps in with equilibrating buffer at 1 ml/min. Peak the extraction and purification of the en- fractions were combined, lyophilized, and zyme were carried out at WC. stored at - 15°C. Lyophilized preparations Enzyme assay. The UDP-glucose: metriwere dissolved in 2 ml of distilled water and buzin N-glucosyltransferase assay was assayed for enzyme activity. Enzymatic synthesis and isolation of mebased on the rate of N-glucoside formation.

N-GLUCOSIDE

METABOLITES

tabolite I. The reaction mixture contained 500 p,mol of K phosphate buffer (pH 7.5), 88 pmol of UDP-Glc, 7.1 pmol [14C]metribuzin (0.32 $Yp,mol), lyophilized Sephadex G-50 enzyme (150 mg protein), and distilled water to a final volume of 50 ml. The reaction was initiated by the addition of [14C]metribuzin and incubated at 25°C for 12 hr. [14C]Metabolite I yields were greater than 80% of theoretical (TLC solvent system B). Ten-milliliter aliquots of the reaction mixture were placed directly on preconditioned Cl8 Sep-Pak cartridges. Each cartridge was washed with 5 ml of distilled water and eluted with 10 ml of aqueous 80% MeOH. Eluate fractions were combined and concentrated in vucuo (30°C). [14C]Metabolite I was isolated and derivatized as indicated in Fig. I. RESULTS

olized by the tomato seedlings (Table 1). The presence of deaminated and diketo products (DA, DK, and DADK) was questionable because only trace amounts of the methanol-soluble 14C cochromatographed with corresponding reference compounds (TLC solvent A). Metribuzin metabolism studies in soybean (19-22), sugarcane (23), potato (24, 25), and several weed species (21,26) have reported the presence of small amounts of DA, DK, and DADK as plant metabolites. It should be noted, however, that these products are also formed as degradation products in the soil (22, 27) and by photochemical reaction on surfaces and in solution (28-30). Presumably, these degradation products would also be subject to root or foliar absorption by plants. The significance of these products as in vivo plant metabolites should be examined further. Metabolite isolation and identification. Since the bulk of the root-absorbed [‘“Clmetribuzin was rapidly translocated to the photosynthetic tissues and metabolized, excised petioles from mature tomato plants were pulse treated with higher concentm tions of [14C]metribuzin and used for preparative-scale metabolite isolation and identification. Under the experimental conditions used, excised tissues exhibited little, if any, apparent injury and metabolized 85-90% of the absorbed [14C]metribuzin within 48 hr. Polar metabolites I and II were separated from unreacted metribuzin and other pos-

AND DISCUSSION

Initial metabolism studies with tomato seedlings showed that [14C]metribuzin was rapidly absorbed by the roots and translocated to the leaves. Within 24 hr, 96% of the absorbed 14C was present in the foliar tissues. Aqueous 80% MeOH extracts of seedling tissues, 24 and 120 hr after [14C]metribuzin treatment, showed that less than 0.1 and 7%. respectively, of the absorbed 14C was associated with methanolinsoluble tissue residues. Chromatographic analysis (TLC) of the methanol-soluble extracts showed that absorbed [14C]metribuzin was rapidly metabTABLE [VJMetribuzin

Metabolism

1 in Tomato

Seed/inns1

TLC solvent system (R,) Compound Metribuzin DA DADK DK Metabolite Metabolite

I II

(A)

(B)

0.48 0.33 0.24 0.22

1.00 1.00

0.00 0.00

275

OF METRIBUZIN

1.00

14CDistribution 24

hr

(‘%)h 120 hr

18.4

?.I

-

0.6

0.9

{ -

81.0

1.oo 0.58 0.43

y Four 19-day-old seedlings pulse treated via roots (1.27 x lo6 dpm/plant; sp act 26.8 pXi/~mol). Ir Values represent percentage of total MeOH-soluble j4C activity in seedling tissues.

-

276

FREAR

sible less polar metabolites by solvent partitioning and purified by chromatography (Fig. 1). All steps in the isolation scheme resulted in >90% recoveries of metabolites. Metabolite I was initially present as a minor constituent (~10% of metabolite II). During metabolite workup and isolation, however, it was noted that metabolite II degraded slowly to metabolite I. Purified metabolite I was not hydrolyzed by B-glucosidase, but qualitative TLC analysis of acid hydrolysis products showed the presence of glucose and the [i4C]metribuzin diketo degradation product (DK). Mass spectra (EI) of the [‘4C]product isolated from acid hydrolysates of either [14C]metabolite I or [14C]metribuzin (1 N HCl; 80°C; 3 hr) were identical to DK reference spectra. Characteristic molecular (M3+ and base peak (M-NH?)+ ions were observed at m/z 184 [184.094784 (C7H12N402)] and m/z 168 [168.077519 (C,HION302)], respectively. Quantitative determinations of glucose and 14C also showed that metabolite I hydrolysates contained 1 mol of glucose for each mole equivalent of [14C]metribuzin. These data suggest that metabolite I was the Nglucoside of metribuzin. A similar metabolite has been suggested to occur in soybean (20). Anion-exchange chromatography (DEAESephacel) of purified metabolite II showed that it was acidic. This information, together with the observed degradation of metabolite II to metabolite I during its isolation, suggested that metabolite II was possibly an acylated N-glucoside of metribuzin. Indeed, when purified metabolite II was reacted with excess diazomethane at room temperature (16), equal molar quantities of [14C]metabolite I and dimethylmalonate were formed. Confirmation of metabolite I and II hydrolysis data was obtained by mass spectral analysis of purified methylated and/or acetylated derivatives (Fig. 1). Electron impact (EI) and chemical ionization (CI) mass spectra of acetylated metabolite I are shown in Fig. 2. Two acety-

ET AL.

lated derivatives were isolated (HPLC system 3), but their mass spectra were identical. EI spectra showed the presence of a tetraacetate derivative with a weak molecular ion at m/z 544 and a strong aglycone ion fragment at m/z 198. Acetylated glucose ions, resulting from fragmentation on either side of the metribuzin amino group, were observed at mlz 346,331,286,271, 169, and 109. CI spectra showed a base peak protonated molecular ion (M+ 1) at m/z 545 and strong protonated aglycone ions at m/z 199 and 200. Similar protonated ion species were observed in CI spectra of metribuzin and have been reported for substituted anilines (31, 32). Weaker acetylated glucose ion fragments identical to those present in EI spectra were also observed. Both spectra confirmed the identity of metabolite I as the N-glucoside of metribuzin. Mass spectra (EI and CI) of methylated and acetylated metabolite II are shown in Fig, 3. Again, two derivatives were isolated (HPLC system 3) that gave the same mass spectra. EI spectra showed the presence of a methylated tetraacetate derivative with a weak molecular ion at m/z 602 and a (MOCH3) ion fragment at m/z 571. A strong aglycone ion fragment was observed at ml z 198. Fragmentation on either side of the metribuzin amino group yielded methylated malonyl glucose acetate ions at m/z 404, 389, and 227 and acetylated glucose ions at mlz 346, 331, 169, and 109. Characteristic methyl malonate and tert-butyl ion fragments were observed at m/z 101 and 57, respectively. CI spectra showed a significant protonated molecular ion (M-t 1) at m/z 603 together with a protonated (M- 59) ion fragment at mlz 545 and a base peak protonated aglycone ion fragment at m/z 200. Weak methylated malonyl glucose acetate and glucose acetate ion fragments were also present. Both spectra confirmed the identity of metabolite II as the malonated N-glucoside of metribuzin. Enzymatic synthesis of metabolite I. Additional support for the rapid in vivo biosynthesis of a N-glucoside metabolite (I) was

N-GLUCOSIDE

METABOLITES

OF

277

METRIBUZIN

120-

+x25

El/MS

above

m/z

45O-

169

100: 109

80-I

60: 40-I

0

100

346 286

331,271, 169,109

198

200

300

400

500

700

600

160,

;::IC”MS oA&HgL% OAc)

100-j

(

331.2*. 169,109

80

-

N

545(M

35 266

+

1)

I

200(199+1)

60

331 20:

109

0

169

100

425

200 FIG.

2. Mass

400

300

spectra

of metabolite

obtained by the isolation and partial purification of a soluble UDP-glucose:metribuzin N-glucosyltransferase from tomato leaves (Table 2). A 2%fold purification was obtained by differential centrifugation, ammonium sulfate precipitation, and gel filtration. Ammonium sulfate was inhibitory as shown by the sharp decreases in enzyme specific activity and recovery. Activity was restored, however, by gel filtration (Sephadex G-50) or dialysis. The enzyme was stable for 3 weeks when lyophilized and stored at - 15°C. In the development of a standard assay for enzyme activity, a broad pH optimum (pH 7.6-8.5) was determined with K phosphate and N-[tris-(hydroxymethyl)-methyllglycine (tricine) buffers. A direct comparison of the isolated enzyme reaction product with the isolated plant metabolite I

485 Irk+4,“‘,‘~~“““1 500 600

700

I derivative.

by chromatography (TLC and HPLC) and mass spectral analysis showed that both products were identical. Proton NMR characterization of metabelite I. The observation that the acetylation of the N-glucoside (metabolite I) resulted in the formation of a mixture of two isomerit tetracetate derivatives with the same mass spectra suggested the possibility of acetyl migration (33). Indeed, both derivatives appeared to be in equilibrium since rechromatography (HPLC system 3) of each derivative resulted in the appearance of the same two HPLC peaks in approximately the same ratio. NMR analysis of the acetylated metabolite I derivatives showed that the nonanomeric proton multiplets of glucose were in the same spectral region as the anomeric proton (34, 35); consequently, anomerit proton data were not obtained.

278

FREAR ET AL.

31-l

El/MS 109

0

kx20

1017

above

mlz

450--+

169

200

100

300

400

500

800

700

I20:

CVMS

200( IQ9 + 1)

100:

801

40:

545

20:

603(M+l) ,~~“~~“~,“~~‘~“~I I 1 0

100

200

300

400

500

I, , , , , , , , ‘, 600

700

FIG. 3. Mass spectra of metabolite II derivative.

In order to determine the anomeric configuration of the N-glucoside metabolite (I), a preparative scale enzyme reaction system was developed to rapidly generate sufficient quantities of the N-glucoside metabolite (I) for NMR studies. Also, to avoid acetyl migration, the N-glucoside was prepared as a silylated derivative. The t-butyldimethylsilane/imidazole reagent reacts with hydroxyl groups, but not with amino groups, in the same manner as the more commonly used trimethylsilane/imidazole

Partial Purification

reagent (36). In addition, the bulky t-butyldimethylsilyl groups are resistant to hydrolysis in aqueous media (37). The anomeric proton doublet observed in the NMR spectrum of the underivatized metabolite I suggested a p configuration (34, 35). Unfortunately, poor resolution of this doublet upon the addition of D20 led to some uncertainty in such an interpretation. Proton NMR spectra of the silylated metabolite I also supported a l3 configuration (38), but the presence of two doublets with

TABLE 2 of Tomato UDPGlucose: Metribuzin

IV-Glucosyltransferase

Fraction

Volume (ml)

Total protein Om)

Total units0

Specific activity (units/mg protein)

Recovery (%I

Purification (%I

1SOOgSupernatant 70,OOOg Supematant 3O-60% (NH&SO, Sephadex G-50

70 60 13 28

252 192 169 151

105 162 47 176

0.42 0.84 0.28 1.17

100 154 45 168

2.00 2.79

u 1 unit = 1 nmole of metribuzin

N-glucosidelhr.

N-GLUCOSIDE

METABOLITES

the same coupling constant for the anomerit proton was unexpected. To determine if silylation was the factor that caused the anomeric proton to be observed as two doublets, the NMR spectra of silylated l0-phenyl IX- and P-D-glucosides were examined. Both spectra showed the anomeric proton as two doublets with identical coupling constants (Jo = 3.5 Hz; Jp = 7.0 Hz). The pair of doublets observed for the anomeric proton suggested that bulky t-butyldimethylsilyl groups might be sterically effecting two different conformations. However, no coalescence of the anomeric proton doublet pair was observed when the silylated (Y anomer of l-0-phenyl D-ghCOside was heated at 80°C in the spectrometer. This result suggested that two conformers were not present, but rather a mixture of two isomeric structures was indicated. Mass spectra (CI) of the major silylated metabolite I product showed a protonated molecular ion (M+ 1) at m/z 719 and confirmed the presence of only three t-butyldimethylsilyl groups. Apparently only trisubstitution of the glucose was atforded with the bulky t-butyldimethylsilyl reagent. The lack of tetrasubstitution might be due either to steric hinderance of formation or to steric relief via hydrolysis during the isolation of reaction products. Trisubstitution should leave one of the three glucose secondary hydroxyl groups underivatized and result in a mixture of isomers. According to the NMR spectra, only two isomeric t-butyldimethylsilyl derivatized structures were favored. Therefore, a pair of inseparable isomers would then account for the two sets of doublets with the same coupling constant that were observed for the anomeric proton. Thus, the proton NMR data for both the underivatized metabolite I (4.92 ppm; J = 7.1 Hz) and the t-butyldimethylsilylated metabolite I (4.32 and 4.46 ppm; J = 9.2 Hz) strongly support a l3 configuration for the anomeric proton. Metribuzin tolerance in tomato seedlings. The differential tolerance of tomato seedling cultivars to metribuzin has been

OF

279

METRIBUZIN

reported to be related primarily to the seedling age (5,9) and to differential metabolism rather than differential uptake or translocation of the herbicide (12). Differences in UDP-glucose:metribuzinN-glucosyltransferase activity levels were determined in leaf tissues of tolerant and sensitive tomato seedling cultivars. In three separate experiments, specific activities (Ulmg protein) of enzyme preparations from tolerant cultivars (Fireball, Harvestvee, and Vision) were consistantly greater (1 S-fold) than those from sensitive cultivars (Ont. 771, Heinz 1706, and Trimson). Studies with leaf tissues from older seedlings of the same cultivars failed to show such differences. Therefore, it would appear that the early development of enzyme activity in young leaves of sensitive cultivars may be limited in terms of metribuzin metabolism and detoxification. More fully developed, older leaves, however, appear to have more than enough enzymatic capacity to provide ad.. equate metribuzin tolerance at recommended application rates.

I

UPDG:

N-glucosyl-

transferase

(I) ‘Malonyl

-&A

tranSferem*

FH200C-CH2COOH

(HI

FIG. 4. tribuzin

Proposed by tomato.

scheme,for

the

metabolism

qfmr-

280

FREAR ET AL.

A reduced tolerance of tomato plants to metribuzin has been observed with plants maintained under low light conditions prior to herbicide treatment (9, 10). Such conditions might be expected to reduce carbohydrate reserves and/or UDP-Glc substrate levels for N-glucoside biosynthesis and metribuzin detoxification. This might be particularly significant in the case of young tomato seedlings. A situation of this nature has been reported for N-glucoside formation with chloramben (39) and pyrazon (40). Although the present studies were short term and designed primarily to determine initial metabolic reactions in the plant, it should be noted that N-glucosides appear to be resistant to enzymatic hydrolysis and may persist as “terminal residues” in those tissues where they are formed (39, 41). In conclusion, the major pathway of metribuzin metabolism and detoxification in tomato plants (Fig. 4) involves an initial enzymatic synthesis of a @NV-glucoside) conjugate (I) followed by a rapid acylation to form the malonyl p-D-(N-glucoside) conjugate (II). This metabolic pathway appears to be correlated with metribuzin tolerance in tomato plants. ACKNOWLEDGMENTS The authors thank Carol Jean Lamoureux for her assistance in the mass spectrometric measurements and the Mobay Chemical Corporation for providing radioactive metribuzin and necessary reference compounds. REFERENCES 1. L. Eue, Sencor(R), a herbicide of the triazinone group, Pflanzenschutz Nachr. 25, 175 (1972). 2. W. Draber, Neuere ergebnisse zur strukur-aktivitats-beziehung, 2. Naturforsch. 34, 973 (1979). 3. 0. T. DeViiers, M. J. Van Der Merwe, and H. M. Koch, Comparative effects of methabenzthiazuron and metribuzin on photosystem II and ATPase activity of chloroplasts of Phase&s vulgaris, Soufh African J. Sci. 75, 315 (1979). 4. A. Trebst and H. Wietoska, Hemmung des photosynthetischen elektronentransports von chloroplasten durch metribuzin, Z. Nuru&rsch. 30, 499(1975).

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N-GLUCOSIDE

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