ANALYTICAL

BIOCHEMISTRY

205, 100-107 (19%)

Spectroscopic Quantitation of O rganic Isothiocyanates by Cyclocondensation with Vicinal Dithiols Yuesheng Zhang,* Cheon-Gyu Cho,t Gary H. Posner,t and Paul Talalay*Tl *Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and TDepartment of Chemistry, The Johns Hopkins University School of Arts and Sciences, Baltimore, Maryland 21218

Received February

19, 1992

Organic isothiocyanates are widely distributed in plants and are responsible for a variety of beneficial and toxic biological effects. No direct and generic method for quantitating isothiocyanates has been described. Under mild conditions nearly all organic isothiocyanates (R-NCS) react quantitatively with an excess of vicinal dithiols to give rise to five-membered cyclic condensation products with release of the corresponding free amines (R-NH,). The products of the condensation of propyl-NCS with 1,2-ethanedithiol, 2,3dimercaptopropanol, and 1,2-benzenedithiol have been isolated and identified as 1,3-dithiolane-2-thione, 4-hydroxymethyl-1,3-dithiolane-2-thione, and 1,3-benzodithiole-2-thione, respectively. Since 1,3-benzodithiole-2-thione (X,,, 365 nm and a, 23,000 M-l cm-‘) can be sensitively measured spectroscopically, the reaction of organic isothiocyanates with 1,2-benzenedithiol has been developed for analytical purposes. All aliphatic and aromatic isothiocyanates tested (except tert-butyl and other tertiary isothiocyanates) reacted quantitatively with an excess of 1,2-benzenedithiol. Thiocyanates, cyanates, isocyanates, cyanides, or related compounds did not interfere with this reaction under assay conditions. The method can be used to measure 1 nmol or less of pure isothiocyanates or isothiocyanates in crude mixtures. It can also be used to measure isothiocyanates in chromatographic fractions obtained from plant extracts and for the assay of the rate of cleavage of glucosinolates by myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1). o 1992 Academic PESS, IN.

Organic isothiocyanates, also known as mustard oils, are widely distributed in plants and their seeds and are

’ To whom correspondence should be addressed at Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, M D 21205.

responsible for the pungent taste of many condiments such as mustard and horseradish. Many of these compounds occur in high concentrations in the vegetable kingdom, especially in the form of their glucosinolate precursors (1). Glucosinolates are N-hydroxysulfate and thioglucoside derivatives of isothiocyanates and usually occur together with the enzyme myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1), which is released when plant cells are injured, and cleaves glucosinolates to isothiocyanates, hydrogen sulfate, and glucose, thereby releasing the pungent taste of isothiocyanates. The most abundant glucosinolate of mustard and horseradish is sinigrin, ally1 glucosinolate, which is cleaved by myrosinase: S--&O, / CH,=CH-CH,-C

+ H,O + \\ N -

CH, = CH -

CH, -

OSO,

N = C = S + HSO;

+ D-glucose.

The reaction involves a Lossen-type rearrangement in which the ally1 group migrates from carbon to nitrogen. The quantitative determination of glucosinolates and isothiocyanates is of agricultural importance since selective breeding of vegetables is practiced to lower the content of isothiocyanates in efforts to reduce the pungency, hepatic toxicity, and goitrogenic properties of some Cruciferae. At the same time the control of the pungency of condiments such as mustard and horseradish is required to maintain the appropriate taste of these products. Although methods for the isolation and calorimetric determination of isothiocyanates by derivatization (e.g., formation of thioureas) are available, no generic analytical methods for the direct, specific, and sensitive measurement of isothiocyanates have been described [for a

100 All

Copyright 0 1992 rights of reproduction

0003-2697192 $5.00 by Academic Press, Inc. in any form reserved.

QUANTITATION

review of analytical methods see Ben-Efraim (2)]. Total glucosinolate levels in plant extracts have been determined by incubation of such extracts with an excess of purified myrosinase and measurement of glucose formation by various procedures [see review by Heaney and Fenwick (3)]. Myrosinase activity has been assayed by measuring the liberation of glucose from glucosinolates (3) or by monitoring the loss of absorbance of ally1 glucosinolate (sinigrin) at 227 nm (45). These methods for measuring glucosinolates or myrosinase activity by monitoring glucose liberation are not highly specific or sensitive. Our own interest in the detection and quantitation of isothiocyanates was stimulated by the finding that these agents are inducers of enzymes that detoxify electrophilic xenobiotics and are also potent protectors against chemical carcinogenesis (6-14). Although growing evidence indicates that induction of such enzymes is a major mechanism for chemoprotection (12,15), it is unclear how much of the anticarcinogenic effects of vegetables can be attributed to their content of isothiocyanates. In related work, we have recently isolated from broccoli an unusual isothiocyanate [sulforaphane; (-)-l-isothiocyanato-R-(methylsulfinyl)butane] as a major and very potent inducer of detoxication enzymes in animal cells and tissues (16). Further progress in assessing the anticarcinogenic properties of isothiocyanates depends on developing methods for their measurement in plant products. A search of the literature did not disclose a method suitable for these purposes. We therefore took advantage of the accidental discovery of the facile cyclocondensation of isothiocyanates with vicinal dithiols to give products with strong uv absorption to develop the desired analytical method. MATERIALS

AND

METHODS

Chemicals 1,2-Ethanedithiol, (R,S)-2,3dimercaptopropanol, 1, Z-benzenedithiol, 4-methyl-1,2-benzenedithiol (3,4-dimercaptotoluene), 1,3-dithiolane-2-thione (ethylene trithiocarbonate), propyl-NCS, phenyl-NCS, cyclohexyl-NCS, tert-butyl-NCS, and sinigrin were from Aldrich (Milwaukee, WI). The commercial isothiocyanates were distilled under reduced pressure prior to use and stored at 4°C in an atmosphere of nitrogen. Sulforaphane [CH,S( 0) (CH,),NCS] and erysolin [ CH,SO,(CH,),NCS] were synthesized by methods described and referenced elsewhere (16). Several unusual isothiocyanates and oxazolidinethiones were gifts of Anders KjEr (Lyngby, Denmark). Preparation

and HPLC

101

OF ISOTHIOCYANATES

of Broccoli Extract

The procedure described previously was followed (16). Lyophilized SAGA broccoli floret powder (8 g) was

extracted three times (for 6 h each) with 280-ml portions of acetonitrile on a shaker at 4°C. The combined extracts were filtered and evaporated to dryness at reduced pressure on a rotatory evaporator (~40°C). The residue was suspended in 15 ml of methanol, filtered through a 0.45-pm porosity filter, and the insoluble fraction was discarded. The soluble fraction was evaporated in a vacuum centrifuge (Speed-Vat, Savant, Hicksville, NY) to give 120 mg of residue, which was redissolved in 1.5 ml of methanol. An aliquot (1.0 ml) was subjected to preparative reverse-phase HPLC on a Whatman Partisil 10 ODS-2M column (500 X 10 mm) with a 330-ml convex solvent gradient (Waters Gradient Program No. 5) from water/methanol (70/30, by vol) to 100% methanol. The gradient was preceded by 30 ml of the initial solvent and was followed by 90 ml of methanol. The eluting solvent was delivered at a rate of 3.0 ml per minute, and 6.0-ml fractions were collected. Aliquots of the fractions were analyzed for isothiocyanate content by reaction with 1,2-benzenedithiol under the standard assay conditions described in the text. Purification

of Myrosinase

Partially purified preparations of myrosinase were obtained from yellow mustard seeds (Sinapis alba, a gift of Durkee-French Foods, Inc., Wayne, NJ) according to modifications (17) of the procedure of Palmieri et al. (18). In brief, the seeds were washed and homogenized (Polytron, Brinkmann, Westbury, NY) in water. The homogenate was centrifuged and the supernatant fluid was extracted with an equal volume of hexane to remove lipids. The aqueous phase was then dialyzed first against water and then against 0.5 M NaCl-20 mM TrisCl, pH 7.4. After centrifugation the supernatant fraction was applied to a concanavalin A-Sepharose column (Pharmacia, Piscataway, NJ) which was washed extensively with the same NaCl-Tris buffer, and the myrosinase activity was then eluted with 0.25 M methyl a-~mannopyranoside (Sigma, St. Louis, MO) in the same buffer. The final product was concentrated by centrifugation (Centricon, Amicon, Danvers, MA). The protein concentration was 10.8 mg per milliliter, and the specific activity was 8.2 pmol of sinigrin hydrolyzedper minute per milligram of protein when measured at 25°C according to Palmieri and co-workers (5). Measurement

of Myrosinase

Activity

The assays were carried out at 25°C in 3.0-ml systems containing 33 mM sodium phosphate, pH 6.0, 130 pM sinigrin, and indicated amounts of partially purified myrosinase. Control vessels contained no enzyme. For spectrophotometric assays the absorbance at 227 nm was monitored with time, and the amount of sinigrin hydrolyzed was calculated (a, = 7110 M-’ cm-’ wasassumed, based on our own measurement). For measure-

102

ZHANG

ET AL.

ment of the ally1 isothiocyanate formed by reaction with 1,2-benzenedithiol, 0.3-ml aliquots were removed at indicated times and mixed rapidly with 0.7 ml of ethanol at -20°C. After storage at this temperature for 20 min, the mixtures were centrifuged, and 0.2-ml aliquots of the supernatant fluids were added rapidly to 7-ml screw-top vials containing 0.3 ml of a 13.3 m M solution of 1,2-benzenedithiol in methanol and 0.5 ml of 100 m M potassium phosphate buffer, pH 8.5. The mixtures were heated at 65°C for 1 h and cooled to 25”C, and the absorbance was determined at 365 nm. Two blanks (see Standard Assay System in text) were used as controls. The amount of isothiocyanate formed was calculated (a, = 23,000 M-’ cm-’ for 1,3-benzodithiole-2-thione).

H20 h

Ultraviolet

Spectroscopy

Preparation and Identification Trithiocarbonates

6

I

Ultraviolet absorption spectra were obtained in l.Ocm light path quartz cuvettes with a Beckman DU 7 recording spectrophotometer equipped with data storage and retrieval facilities. The molar extinction coefficients (a,) were based on concentrations determined by weight.

of Cyclic

The cyclic trithiocarbonates resulting from the reaction of propyl-NCS with 1,2-ethanedithiol, 2,3-dimercaptopropanol, and 1,2-benzenedithiol were prepared by reacting the propyl-NCS with a 5- to lo-fold molar excess of the dithiols in a mixture of equal volumes of methanol and 100 m M potassium phosphate buffer, pH 8.5, at 25°C overnight in an atmosphere of nitrogen. The reaction mixtures were extracted with chloroform and the organic solvent was evaporated at reduced pressure (~40°C). To isolate the products formed from 1,2ethanedithiol and 1,2-benzenedithiol, the residues were purified on preparative silica TLC plates developed with methylene dichloride. 1,3-Dithiolane-2-thione (ethylene trithiocarbonate) was sublimed at 0.25 mm Hg onto a cold condenser at -15°C. The 1,3-benzodithiole2-thione was crystallized from methylene dichloride. The cyclic product of the reaction of propyl-NCS with 2,3-dimercaptopropanol was purified by two preparative normal phase HPLC (Whatman Partisil M9 10150) with use first of hexane/2-propanol (65135, by vol) and then hexane/2-propanol/chloroform (60/20/20, by vol). The yellow liquid so obtained was finally purified by application to a silica dry column and elution with methylene dichloride. 1,3-Dithiolane-2-thione (ethylene trithiocarbonate; CAS 822-38-8). Mass spectra (EI) Mf 135.9474 (calcd for C3H4S3, 135.9475), m/z (rel intensity) 136 (loo), 137 (5.84; calcd 5.64), 138 (13.43; calcd 13.32), 108 (10.5), 76

4.45

4.40

4.15

4.10

4.00

3.90 2.00

1.50

FIG. 1. ‘H NMR spectrum of 4-hydroxymethyl-1,3-dithiolane-2thione at 600 MHz in CDCl,. The chemical shifts are given in ppm relative to TMS. Six groups of resonances, designated RI-R, are resolved. R,: 6 1.98 (broad, lH, exchangeable with D,O, OH). R, and R, overlap partially and integrate to 2H. R,, R,, and R, integrate to IH each. R,: d 3.95-3.98 (dd, H,, Jgem = 12.2 Hz, J., = 4.2 Hz). R,: 8 4.13-4.16 (dd, H,, J,,, = 12.2 Hz, Jab = 5.85 Hz). R,: 6 4.40-4.44 (m, H,). (Inset) When D,O was added, R, disappeared and the other resonances remained unchanged except R, and R,. R,: d 3.92-3.95 (dd, H,, J = 11.2 Hz, Jad = 6.2 Hz). R,: $ 3.99-4.02 (dd, H,, J,,, = 11.0 Hz, JI:m= 7.9-8.1 Hz). The sharpening and simplification (from m to dd) of R, and R, after exchange of OH with D,O indicates that the complexity of R, and R, in CDCl, arises from splitting of H, and H, by OH. The specific identification of the individual geminal protons is based on modeling and should be considered tentative.

(23.3); ‘H NMR (300 MHz; CDCl,) 6 3.95 (sharp s); uv (acetonitrile) X,,, 316, a,,, = 16,500 M-r cm-‘. These spectral findings are in agreement with literature values (19). 4-Hydroxymethyl-1,3-dithiolane-2-thione. Mass spectra (EI) M+ 165.9580 (calcd for C,H,OS,, 165.9581), m/z (rel intensity) 166 (loo), 167 (7.01; calcd 6.72), 168 (13.71; calcd 13.32), 108 (8.6), 90 (8.6), 76 (19.3), 64 (13.0), 60 (16.5), 59 (51.1), 58 (10.6), 57 (77.6). The 600-MHz (CDCl,) ‘H NMR spectrum is shown and interpreted in Fig. 1; uv (acetonitrile) X,,, 316 nm, a, = 16,400 M-’ cm-l. The product did not react with 5,5’dithiobis(2-nitrobenzoic acid), thus showing that it does not contain a free sulfhydryl group. 1,3-Benzodithiole-2-thione (CAS 934-36-l). Mass spectra (EI) M’ 183.9478 (calcd for C,H,S,, 183.9476), m/z (rel intensity) 184 (loo), 185 (10.31; calcd 9.96), 186 (13.77; calcd 13.32), 140 (75.9), 108 (15.9), 69 (36.5), 63 (12.4); H1 NMR (300 MHz; CDCl,) 6 7.43 (m, 2H), 7.48 (m, 2H); uv (acetonitrile) X,,, 363 nm, a, = 22,500 M-’ cm-‘. The mass spectra and NMR findings are in agreement with literature values (20).

QUANTITATION

103

OF ISOTHIOCYANATES

though we did not characterize this product, it seems highly likely that the expected thiocarbamate was formed: C,H,-NCS

+ HSCH,CH,OH

C,H,-NHC(S)SCH,CH,OH.

WAVELENGTH

(nm)

FIG. 2. Ultraviolet absorption spectra showing the time course of the reaction between propyl-NCS and 2,3-dimercaptopropanol. The reaction was carried out at 25°C in a 2.0.ml system consisting of 1.0 ml of 100 nIM potassium phosphate, pH 8.5, and 1.0 ml of methanol containing 100 nmol of propyl-NCS and 500 nmol of 2&dimercaptopropanol. Top, scans at 2-10 min. Bottom, scans at lo-150 min. The initial absorbances of the reactants have been subtracted. Note the early formation of products with absorbance maxima at 270 nm and with a shoulder near 250 nm and the conversion of the presumed thiocarbamate to a second product with a new absorption maximum at 316 nm. There is a well-defined isosbestic point at 287 nm.

RESULTS

Principle

AND of

DISCUSSION

the Analytical

Method

Organic isothiocyanates (R-N = C = S) are highly reactive compounds in which the central carbon atom is strongly electrophilic and is attacked with great facility by nucleophiles such as amino groups or mercaptans to furnish thioureas or thiocarbamates, respectively. Thus the reaction of phenyl-NCS with the amino groups of amino acids is the basis for the widely used Edman degradation for the sequencing of peptides (21). Thiourea derivatives have been especially valuable in the characterization of isothiocyanates. Nevertheless, we were unable to find a convenient, sensitive, and generic method for measuring isothiocyanates directly. Spectroscopic studies of the nonenzymatic reaction of isothiocyanates with 2-mercaptoethanol in aqueous or aqueous-methanol solutions under mildly basic conditions revealed the formation of a product(s) with high ultraviolet absorption. Thus, propyl-NCS (a, = 1000 -1 cm-l M at 240 nm) gave a product(s) with a broad ultraviolet absorption band at 270 nm (a, = 10,000 M-’ cm-‘) and a broad shoulder centered near 250 nm. Al-

The absorption spectra of known thiocarbamates are consistent with this interpretation (22). Reaction of isothiocyanates under similarly mild conditions with compounds containing two thiol groups on adjacent carbon atoms, such as 1,2-ethanedithiol or 2,3dimercaptopropanol, also resulted in the rapid formation of products absorbing maximally near 270 nm with shoulders at 250 nm. Upon longer incubation, however, this primary product(s) underwent modification to a secondary product(s) (as judged by a well-defined single isosbestic point), which absorbed light maximally at even longer wavelengths and with greater intensity. The time course of the reaction between propyl-NCS and 2,3-dimercaptopropanol is shown in Fig. 2. Since other isothiocyanates behaved similarly, these observations suggested the potential usefulness of the reaction of isothiocyanates with vicinal dithiols for analytical purposes. Further investigation of this reaction indicated that the major product of the reaction of propyl-NCS with 1,2-ethanedithiol was the known 1,3-dithiolane-2thione (ethylene trithiocarbonate) (1) (19). This product probably results from an attack of the second sulfhydryl of the reagent on the thiocarbamate (formed by addition of the first sulfhydryl group) to give rise to a cyclic product and release of a primary amine, as shown in Scheme 1. The analogous formation of 4-hydroxymethyl-1,3-dithiolane-2-thione (2) from the reaction of propyl-NCS with (R,S)-2,3-dimercaptopropanol was demonstrated by isolation and spectroscopic structure determination (see Materials and Methods). This com-

HE ‘\

H R-;-&S

R-N=C=S B:d

H. 7 S

S’kS/H

-

S’”

r:B

i( R’

H I R;+%7Cq-f-f

s A R-NH2

+

R’ I

S

J

t-

S

R’ R’

Scheme 1.

1, R’ = H; 2, R’ = CH,OH.

104

ZHANG

ET AL.

pound, which is racemic, has not been previously described. These findings suggested that a hitherto undescribed reaction of isothiocyanates had occurred. Although intramolecular cyclizations between isothiocyanates and vicinal amino groups or vicinal amino and thiol groups have been reported (23), a cyclocondensation with vicinal dithiols has not been described. A survey of the reactivity of various dithiols with isothiocyanates disclosed that only reagents with vicinal thiol groups participated in the reaction. A favorable reagent for analysis of isothiocyanates was found to be 1,2-benzenedithiol, which upon reaction with isothiocyanates gave rise to 1,3-benzodithiole-2-thione [3; Ref. (20)].

300

250

WAVELENGTH

350

4 0

(nm)

FIG. 3. Ultraviolet absorption spectrum of synthetic 1,3-benzodithiole-2-thione (35 pM) in a mixture containing 1.0 ml of methanol and 1.0 ml of 100 mM potassium phosphate, pH 8.5.

.m=s 3, R = H; 4, R = CH,.

The identity of the cyclic reaction product was established by spectroscopic methods (see Materials and Methods). 1,3-Benzenedithiol does not react with isothiocyanates under these conditions. The use of this reaction for the synthesis of amines from various isothiocyanates has been reported recently (24). The advantages of the use of 1,2-benzenedithiol as the dithiol reagent for analytical purposes are: (a) the product has an absorption maximum at 363 nm in acetonitrile and at a slightly higher wavelength (365 nm) in a mixture of equal volumes of methanol and 100 mM potassium phosphate buffer, pH 8.5 (Fig. 3), and the product can therefore be measured with less interference from common contaminants; (b) the extinction coefficient of the product is very high (a, = 23,000 M-l cm-’ at 365 nm); (c) disposable plastic rather than quartz cuvettes can be used; and (d) the product appears to form rapidly and to be very stable under a variety of conditions, including heating for 18 h at 65°C in the reaction mixture at pH 8.5 (see Fig. 5). One minor disadvantage in the use of 1,2-benzenedithiol as a reagent is that it is a low melting (mp 27-28°C) solid and has an offensive odor. 3,4-Dimercaptotoluene which is commercially available is somewhat easier to handle (mp 30-33°C) and has a less offensive odor. The cyclocondensation product of 3,4-dimercaptotoluene with isothiocyanates (4) has identical uv spectroscopic properties to 3. When the reaction of 1,2-benzenedithiol with propylNCS was studied with time, no spectroscopic evidence for the formation of a transient intermediate (such as occurred in the reaction with 2,3-dimercaptopropanol; Fig. 2) was obtained. Several reasons for the failure to observe a reaction intermediate(s) may be advanced, including differences in the acidities of the sulfhydryl groups and steric effects that could affect rates and equi-

libria. The most plausible explanation is that the conformational rigidity imposed by the aromatic ring on the sulfhydryl groups of 1,2-benzenedithiol favors more rapid cyclization than that observed with aliphatic vicinal dithiols that are conformationally mobile. Standard

Assay System

Based on these observations, standard conditions for the measurement of isothiocyanates by reaction with 1,2-benzenedithiol were developed, and all experiments were carried out under these conditions unless otherwise indicated. The reaction system contained in a final volume of 2.0 ml 900 ~1 of 100 m M potassium phosphate buffer (pH 8.5), 900 ~1 of methanol, 100 ~1 of a solution of the isothiocyanate to be determined (l-100 nmol) diluted in water, methanol, or other water-miscible organic solvents that do not absorb light at 365 nm, and in methanol. The 100 ~1 of 80 m M 1,2-benzenedithiol 1,2-benzenedithiol was added last to initiate the reaction. The reactions were carried out in 7-ml screw-top glass vials equipped with plastic caps fitted with tightfitting Poly-Seal cones (Aldrich Z-15152-1). The vials were heated at 65°C for 60 min and cooled to room temperature, their contents decanted into l.O-cm light-path polystyrene cuvettes, and the absorbances at 365 nm determined against a solvent blank. Each sample to be analyzed was accompanied by a paired blank containing all ingredients, except 1,2-benzenedithiol. Each set of determinations also included a control vessel that contained the 1,2-benzenedithiol reagent but no isothiocyanate. The absorbances were corrected for those of 4 m M 1,2-benzenedithiol (A,,, = 0.050) and when necessary (i.e., in crude extracts) for the initial absorbance at 365 nm of the sample to be analyzed. A typical calibration curve for 1.25-40 PM propylNCS and phenyl-NCS obtained under these conditions

105

OF [SOTHIOCYANATES

QUANTITATION

nm was observed. Other likewise unreactive. w

/

I

~J/, 0

10

ISOTHIOCYANATE

20

I

, ,) 30

CONCENTRATION

40

MM)

FIG. 4. Spectrophotometric determination ofpropyl-NCS (0) and phenyl-NCS (0) by reaction with 1,2-benzenedithiol. The reactions were carried out in 2.0.ml systems containing 0.9 ml of 100 mM potassium phosphate buffer, pH 8.5, and 1.0 ml of a 8 mM solution of l&benzenedithiol in methanol. The isothiocyanates (1.25-40 nmoll were added in small volumes of methanol. The reaction vessels were heated at 65°C for 60 min and cooled to room temperature and the ahsorhances determined at 365 nm against a blank treated identically but containing no isothiocyanate. The initial absorbance of the highest concentrations of isothiocyanate used at 365 nm is negligible. The line represents the expected amount of l$benzodithiole-2-thione formed (a, = 23,000 MA’ cm-i at 365 nml if the isothiocyanates had reacted quantitatively.

is shown in Fig. 4. As expected, the two compounds gave identical calorimetric responses. Under these conditions 2 nmol(0.2 pg) of propyl-NCS gave an absorbance change of 0.023 at 365 nm in a 2.0-ml system. The sensitivity can be increased by a factor of five or more by using smaller reaction volumes.

Specificity

isothiocyanates

were

Rates of Reaction of Isothiocyanates Although considerable differences in the rates of reaction of various isothiocyanates with 1,2-benzenedithiol were observed under standard reaction conditions, completion of the reaction was attained in less than 60 min at 65°C with all of the compounds examined. As shown in Fig. 5, phenyl-NCS reacted with 1,2-benzenedithiol much more rapidly than propyl-NCS, which in turn reacted more rapidly than cyclohexyl-NCS. The product of the reactions, 1,3-benzodithiole-2-thione, was stable for many hours under the reaction conditions (pH 8.5; 65”C), as judged from its absorption at 365 nm. Measurement of Isothiocyanate Content of Chromatographic Fractions of Extracts of Broccoli When fractions obtained by reverse-phase HPLC of extracts of lyophilized broccoli were analyzed for isothiocyanate content by reaction with 1,2-benzenedithiol, most of the isothiocyanate was detected in a single peak that was eluted at 65% methanol: 35% water (Fig. 6). This is the position of elution of sulforaphane, which is

A 0.6

25’C

PHENYL-NCS /

1

CYCLOHEXYL-NCS

of Reaction

Among compounds potentially related to isothiocyanates, none of the following (tested at 10 PM concentrations in the standard assay system) gave increases in absorbance at 365 nm: KCN, KOCN, KSCN, C,H,COCN, CGH,CH,CN, C,H,CH,NCO, or C,H,CH,SCN. Furthermore oxazolidinethione, EGvinyloxazolidinethione (goitrin), and 5,5-dimethyloxazolidinethione did not produce any changes in absorption at 365 nm under these conditions. The glucosinolate sinigrin (ally1 glucosinolate) was also totally unreactive. A wide variety of isothiocyanates were found to be fully reactive in the assay system with 1,2-benzenedithiol, including CH,(CH,),-NCS, where n = 2, 3, or 4; cyclohexyl-NCS; C&H,-(CH,),-NCS, where n = O-6; and CH, = CH(CH,),-NCS. Various aliphatic isothiocyanates containing sulfur in the alkyl side chain were also tested, including CH,S(CH,),-NCS where n = 2,4,8,11 or 15; CH,S(O)(CH,),-NCS (sulforaphane); and CH,SO,(CH,),-NCS (erysolin), and all were fully reactive in the assay system. tert-Butyl-NCS was inert under standard reaction conditions, and even after heating for 18 h at 65”C, no change in absorbance at 365

tertiary

a z 2

B

65’C

0.6-

2

(min)

TIME

PW

FIG. 5. Time course of the reaction of propyl-NCS (01, cyclohexylNCS, (0) and phenyl-NCS (0) with 1,2-benzenedithiol at 25°C (A) and 65°C (Bl. The reactions were carried out in 2.0-ml systems consisting of 1.0 ml of 100 mM potassium phosphate buffer, pH 8.5, and 1.0 ml of methanol containing 4.0 mM 1,2-benzenedithiol and 30 fiM propyl-NCS, cyclohexyl-NCS, or phenyl-NCS. The absorbances at 365 nm are shown as a function of time. The expected change in absorbance for complete reaction of the isothiocyanates is 0.690 at 365 nm.

106

ZHANG

a major inducer of detoxication enzymes in broccoli (16). Minor quantities of isothiocyanates were also eluted at earlier (Fractions 3-5) and at later (Fractions 60-61 and 64) points in the solvent gradient. The isothiocyanate analyses were carried out on aliquots of each fraction. The validity of these measurements is supported by the finding that the product measured at 365 nm was strictly proportional to the volume of fraction analyzed, and that when a standard amount (10 nmol) of phenyl-NCS was added as an “internal standard” to 20 ~1 of a fraction containing 13.4 nmol isothiocyanate, the standard was recovered quantitatively (104%) in the presence of this HPLC fraction. Measurement

of Myrosinase

Activity

Although several methods for assaying myrosinase activity have been described, the simplest is the direct measurement of the loss of absorption of sinigrin (ally1 glucosinolate) at 227 nm (a, = 7110 M-’ cm-‘). However, this method is not suitable for the measurement of low levels of myrosinase activity in crude plant tissue extracts. The reaction of the isothiocyanate, liberated by the action of myrosinase, with 1,2-benzenedithiol is useful for measuring low levels of myrosinase activity in crude extracts since the wavelength of measurement (365 nm) is much more favorable, and the extinction coefficient of 1,3-benzodithiole-2-thione (a, = 23,000 M-’ cm-‘) is more than three times higher than that of sinigrin. The application of the method developed in

ET AL.

TIME (min) FIG. 7. Time course of formation of isothiocyanate from sinigrin by partially purified preparations of myrosinase from yellow mustard seeds. The reactions were carried out as described under Materials and Methods. The upper (0) and lower (0) curves were obtained with 10.8 and 5.4 I.rg of partially purified myrosinase, respectively.

this paper to the measurement of purified myrosinase activity is shown in Fig. 7. Comparison of the spectrophotometric method that measures the hydrolysis of sinigrin (at 227 nm) directly with the measurement of isothiocyanate formed showed that there was reasonably good agreement at all time points, although the amount of isothiocyanate formed was usually about 12% higher than the amount of sinigrin consumed (results not shown). We attribute this difference to the difficulty of arresting the myrosinase reaction without destroying the isothiocyanate formed, and that cooling of the mixture probably does not completely arrest the reaction.

ACKNOWLEDGMENTS

.10

20

30

40

50

60

70

80

FRACTION

FIG. 6. Measurement of isothiocyanate content of reverse-phase HPLC fractions of an acetonitrile extract of lyophilized SAGA broccoli. The method of preparing the extract and the conditions of chromatography are described under Materials and Methods. About 80 mg of extract was applied to a 1.0 X 50.cm column and eluted with a gradient from methanol/water (30/70, by vol) to 100% methanol at a flow rate of 3 ml/min. The absorbance at 280 nm was monitored and 6.0-ml fractions were collected. Suitable aliquots of the fractions were analyzed for isothiocyanate content by reaction with 1,2-benzenedithiol in the standard assay system described in the text. Note that the scale for the isothiocyanate measurements is logarithmic. A total of 11.0 pmol of isothiocyanate was recovered in four regions of the chromatogram: Fractions 3-5, 535 nmol; Fractions 15-21,10.4 pmol; Fractions 60-61, 72.4 nmol; and Fraction 64, 38.3 nmol.

These studies were supported by a U.S. Public Health Service Grant (PO1 CA 44530) awarded by the National Cancer Institute, Department of Health and Human Services. We thank Anders Kjzr, Technical University of Denmark, Lyngby, Denmark, for gifts of numerous isothiocyanates identified in the text; J. L. Kachinsky, The Johns Hopkins University, for carrying out the mass spectra; L.-S. Kan, Division of Biophysics, Johns Hopkins School of Hygiene, for the 300 MHz NMR spectra; and C. Abeygunawardana and A. S. Mildvan for the 600 MHz NMR spectra and their interpretation. We have also received valuable advice from C. H. Robinson and T. Prestera, Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, and from L. Xue, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The University of Illinois at Chicago. We thank Gale Doremus for preparing the manuscript and the illustrations.

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