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Formation of adducts of parthenin and related sesquiterpene lactones with cysteine and glutathione
Chem.-BioL Interactions, 28 (1979) 83--89 © Elsevier/North-Holland Scientific Publishers Ltd.
83
FORMATION OF ADDUCTS OF PARTHENIN AND RELATED SESQUITERPENE LACTONES WITH CYSTEINE AND GLUTATHIONE
ANNA K. PICMANa, ELOY RODRIGUEZ b and G.H.N. TOWERSa aDepartment of Botany, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada) and bDepartment of Ecology and Evolutionary Biology, University of California, Irvine, CA (U.S.A.) (Received May 18th, 1978) (Revision received June 4th, 1979) (Accepted June 19th , 1979)
SUMMARY
Parthenin, the major sesquiterpene lactone of Parthenium hysterophorus, a weed responsible for dermatitis in man is primarily restricted to leaf and stem trichomes. Parthenin forms a m o n o a d d u c t with L-cysteine through the a-methylene group of the 7-1actone and a biadduct with the endocyclic double bond on the cyclopentenone ring. Studies with other sesquiterpene lactones support the view that the types of adducts formed are correlated with the biological activity of the sesquiterpene lactones. INTRODUCTION Parthenium hysterophorus L., a widespread composite of the Southern U.S.A., the Caribbean, Australia and parts of South America, has become a serious agricultural pest as well as a medical hazard in India where it is the cause of an epidemic of allergic contact dermatitis [1]. It is generally recognized that partbenin, the major sesquiterpene lactone of this species, is responsible for the dermatological reaction associated with this plant [1]. Sesquiterpene lactones, characteristic of the Compositae, are an important group of terpenoids which exhibit cytotoxic, antineoplastic, allergenic and other biological activities [2]. These compounds, by Michael-type addition, form adducts with amino acids containing SH groups such as cysteine [3,4] or with lysyl or histidyl residues [5]. It has been suggested that the conjugated a-methylene~/-lactone moiety of sesquiterpene lactones is responsible for cytotoxicity [6,7] as well as for allergenicity [8,9]. A structural requirem e n t for significant cytotoxic activity is the O=C--C--CH2 moiety which could be part of an ester, ketone or lactone [10]. Results of tests for aUergenicity with different sesquiterpene lactones indicated differences in response between patients [11]. The fact that
84
A
B
b H
a I HS--C½--#--COO+NH3
C
.~.~
D HOOC
°'-c°/ I s - ~
NH2
COOH
~
'-- N H2
coon Fig. 1. Parthenin, cysteine and their adducts (A-parthenin, B-cysteine, C,monoadduct, D-biadduct).
parthenin (Fig. 1A) possesses two potentially active sites (an exocyclic methylene C13 and an C2 C3 endocyclic double bond allylic to C4 carbonyl group) at which Michael-type additions could occur, might explain toxic effects on cattle and the potent allergenicity of this compound [1,12]. We have examined the reaction of parthenin and of several closely related sesquiterpene lactones w i t h cysteine and:g!utathione in order to determine whether more than one type of adduct is formed. We also have reexamined the distribution of parthenin in aerial parts of P. hysterophorus. MATERIALS AND METHODS
Plant material. P. hysterophorus plants were grown from achenes obtained from Austin, TX, U.S.A. Achenes germinated in wet sand In 1--3 weeks and when leaves had formed, seedlings were transfered to pots and maintained at 30°C during the day and 20°C at night. Light period was 16 h. Detection o f parthenin in plant material. Parthenin was isolated from Parthenium hysterophorus by the method of Rodriguez [12]. Plant material (achenes or seedlings) were extracted with chloroform for 24 hrs and the presence of parthenin checked by thin-layer chromatography (TLC) with a known sample (Eastman Si gel plates with benzene--acetone, 1 : 4, fumed with iodine vapors, Rf = 0.57). Crushed or intact plant material gave identical results. Detection o f parthenin in trichomes and stems o f P. hysterophorus. From
85 ten fresh stems 10 mg of trichomes were removed with a spatula and transfered to distilled methanol (10 ml). The stems, with all trichomes removed (1 g), were cut into small pieces and extracted with distilled methanol (25 ml) overnight. Both extracts were concentrated to minimum volumes and checked by TLC for presence of parthenin. TLC of sesquiterpene lactone-thiol adducts. Eastman cellulose plates, without fluorescent indicator, and the upper phase of n-butanol/acetic acid/ water (4 : 1 : 5) were used. Plates were sprayed with 0.25% ninhydrin (acetone), heated for several minutes at 80°C and then exposed to iodine vapors to detect sesquiterpene lactones w~hich give brown spots. The monoand biadducts of parthenin give violet colored ninhydrin spots with Rf = 0.55 and Rf = 0.19, respectively. (Rf cysteine = 0.36, Rf cystine = 0.13). Reactions o f sesquiterpene lactones with L-cysteine and reduced glutathione. To 0.01 mmol of each of the sesquiterpene lactones shown in Fig. 2 in aqueous ethanol 0.04 mmol of an aqueous solution of L~ysteine (Sigma Chemical Company) was added and the mixture allowed to stand at room temperature. The formation of adducts was checked by TLC. Parthenin and helenalin (0.01 mmol and 0.04 mmol of each of them in water) were allowed to react with 0.04 mmol of an aqueous solution of glutathione (Sigma Chemical Company) in the same way. Isolation and identification of: (a) Monoadduct -- equimolar aqueous solutions of parthenin (5.84 mg/0.58 ml) and L-cysteine (2.42 mg/0.24 ml) were mixed and allowed to stand at room temperature. After 6 h, when TLC showed only one positive spot with ninhydrin, the solution was shaken with Chloroform to remove remaining parthenin. The aqueous phase when freezedried yielded 8.2 mg of a white powder soluble in water, methanol, ethanol, n-butanol, acetone but insoluble in chloroform. It turned brown at 210--212°C and decomposed at 247°C. NMR {15.4 MHz, D20 with DSS): 8 7.67 (H-2, d), 6.17 (H-3, d), 5.06 (H-6, d), 4.03 (Hb, s), 3.10 (Ha, s), 1.27 (C5-Me, s), 1.10 (C10-Me, s). IR (KBr disc): kmax 3300--2900 (aminoacid, s), 1755 (vs), 1715 (cyclopentenone, vs), 1650--1580 (COOH, s), cm -1. (b) Biadduct-aqueous solution of parthenin (17.5 mg representing 0.06 mmol, in 1.75 ml) and L-cysteine (14.5 mg, representing 0.12 mmol, in 1.45 ml) were mixed and allowed to react at room temperature for 6 h and then freeze~lried. The residue, dissolved in water, was chromatographed on a column (25 × 2 cm) of Whatman cellulose powder which was packed and eluted with the upper phase of the solvent system n-butanol/water (4 : 5). After fast moving compounds were removed from the column, methanol/water (1 : 1) was used to elute the cysteine-parthenin biadduct. The fractions containing this product were pooled and freeze
86
CO
O..CO/---
par t h e n i n
H
hymenin
i
/
o"
o1-t
//
helenalin
damsin
H
6~ °--co cumambrin- B
tetraneurin-D
I
H
'
0
0
tenulin
H
I
.?0
,
CO
isotenulin
Fig. 2. Sesquiterpene lactones selected for the reaction with L-cysteine.
(H-3, d) 6.28 (H-13b, d), 5.63 (H-13a, d), 5.06 (H-6, d), 1.27 (C5-Me, s), 1.10 (C10-Me, s), IR (KBr disc): kmax 3420 (OH, us), 1742 (7-1actone, vs), 1718 (cyclopentenone, vs), 1662 (7-1actone, w), 1595 (cyclopentenone, w) am
-1
.
L-cysteine NMR: (15.4 MHz, D20 with DSS): 6 4.03 (Hb, s), 3.10 (Ha, s). IR (KBr disc): kmax 3300--2900 (aminoacid, s), 2560 (SH, m), 1650--1580 (COOH, s) cm -~ .
87 RESULTS
P. hysterophorus achenes and seedlings, with the first true leaf bearing trichomes, were found to contain parthenin. Younger seedlings with cotyledons only and lacking trichomes did n o t contain detectable amounts of parthenin. A comparison of methanolic extracts of trichomes which had been carefully excised from the stems with extracts of stems that had been depilated confirmed that parthenin is restricted to trichomes [13]. TLC of a mixture of aqueous solutions of parthenin and L~cysteine yielded t w o additional ninhydrin positive spots. One of them, identified as a monoadduct (Fig. l b ) , was obtained directly from an equimolar mixture o f both reactants. The second one, a biadduct (Fig. lc), was isolated b y column chromatography from a reaction mixture containing an excess of L-cysteine. The NMR spectrum of the m o n o a d d u c t did not show the characteristic signals for = CH2 of parthenin. Instead new signals due to a cysteine residue (Ha and Hb) were apparent. Proton signals corresponding to H2 and H3 in the cyclopentenone ring remained. The IR spectrum exhibited the cyclopentenone chromophore of parthenin with the additional absorption of the cysteinyl carboxyl group. This adduct therefore contains one molecule of cysteine attached through a sulfhydryl link to parthenin on the = CH2 carbon of the 7-1actone ring. The NMR spectrum of the second adduct showed the disappearance of the signals for = CH2 as well as for the protons on carbons of the cyclopentenone ring and in addition two new signals for L~cysteine (Ha and Hb). This c o m p o u n d was identified as a biadduct containing one molecule of cysteine at each active site. Rapid decomposition of the biadduct to m o n o a d d u c t and to cysteine, cystine and parthenin was detected by TLC in the first hour after isolation. There was also a slower transforma-
TABLE
I
POTENTIAL ACTIVE SITES THEIR ADDUCT FORMATION Sesquiterpene
OF SELECTED SESQUITERPENE WITH L-CYSTEINE
a-CH2-7-1actone
Cyclopentenone
LACTONES
AND
Adducts of sesquiterpene lactones with L-cysteine
Parthenin
Hymenin Helenalin Damsin Cumambrin-B Tetraneurin-D Tenulin Isotenulin
+ + + + + + ---
+ + + ---+ +
monoadduct
biadduct
+ + + + + + + +
+ + ÷ ------
88
tion of the isolated monoadduct to biadduct and also to parthenin, cysteine and cystine. These transformations were detectable by TLC 24 h after isolation and indicated a higher stability of the monoadduct. TLC of reaction mixtures of selected pseudoguaianolides (Fig. 2) with L-cysteine indicated the formation of monoadducts with iactones containing only one potential active site, i.e., either the exomethylene function on the lactone ring or the cyclopentenone ring. Biadducts were obtained with parthenin, hymenin and helenalin, compounds which possess both potentially active sites. This is shown in Table I. Equimolar quantities of parthenin or helenalin with reduced glutathione gave only monoadducts. When 4-fold quantities of glutathione were used, helenalin formed a biadduct. Parthenin, however, formed only a monoadduct even after standing for six days. DISCUSSION Parthenin was found only i n seedlings older than 10 days and its presence was associated with the development and differentiation of trichomes. The restriction of parthenin to the trichomes of P. hysterophorus was confirmed [13]. The higher concentration of this lactone in leaves [17] may be explained, in part, by the fact that leaves elaborate more trichomes than do other parts of the plant. In the present study we have demonstrated that parthenin forms two unstable adducts with L-cysteine. The monoadduct contains a cysteinyl moiety attached to parthenin through the C13 and the biadduct possesses an additional molecule of cysteine on the C2 position. The formation of the biadduct was obtained with higher concentrations of cysteine in the reaction mixture. The amounts of thiols, including proteins with sulphydryl groups, in living cells are probably high enough so that both active sites of parthenin or of certain other sesquiterpene lactones become involved. The results suggest that the presence of two active sites in parthenin is related to the fact that it is a strong sensitizer in man. A similar case may be made for helenalin which has antibiotic properties [15] and which is also reported to be a good sensitizer in delayed hypersentitivity [9]. However, hymenin, a diastereoisomer of parthenin, which also forms two adducts, is only a weak sensitizer in some cases [16] suggesting that additional factor(s) influence allergenicity. ACKNOWLEDGEMENTS We thank Dr. C.-K. Wat for the helpful discussions and Pat Jamieson and the Department of Chemistry, U.B.C. for measurements and help with inter. pretation of the NMR spectra. This study was supported by the National Research Council of Canada.
89 REFERENCES 1 G.H.N. Towers, J.C. Mitchell, E. Rodriguez, P.V. Subba Rao and F.D. Bennett, Biology and chemistry of Parthenium hysterophorus L., a problem weed in India, J. Sci. Ind. Res., 36 (1977) 672. 2 E. Rodriguez, G.H.N. Towers and J.C. Mitchell, Biological activities of sesquiterpene lactones -- a review, Phytochemistry, 15 (1976) 1573. 3 S.M. Kupchan, T.J. Giacobbe, I.S. Krull, A.M. Thomas, M.A. Eakin and D.C. Fessler, Reaction of endocyclic ~, t-unsaturated 7-lactones with thiols, J. Org. Chem., 35 (1970) 3539. 4 I.H. Hall, K.-H. Lee, E.C. Mar, C.O. Starnes and T.G. Waddell, Antitumor agents. 21. A proposed mechanism for inhibition of cancer growth by tenulln and helenalin and related cyclopentcnones, J. Med. Chem., 20 (1977) 333. 5 G. Dupuis, J.C. Mitchell and G.H.N. Towers, Reaction of alantolactone, an allergenic sesquiterpene lactone, with some amino acids, Can. J. Biochem., 52 (1974) 575. 6 S.M. Kupchan, Recent advances in the chemistry of tumor inhibitors of plant origin, Trans. N.Y. Acad. Sci., 32 (1970) 85. 7 S.M. Kupchan, M.A. Eakin and A.M. Thomas, Tumor inhibitors. 69. Structure-cytotoxicity relationship among the sesquiterpene lactones, J. Med. Chem., 14 (1971) 1147. 8 J.C. Mitchell, B. Fritig, B. Singh and G.H.N. Towers, Allergic contact dermatitis from FruUania and Compositae, J. Invest. Dermatol., 54 (1970) 233. 9 J.C. Mitchell and G. Dupuis, Allergic contact dermatitis from sesquiterpenoids of the Compositae family of plants, Br. J. Dermatol., 84 (1971) 139. 10 K.-H. Lee, E.S. Huang, C. Piandosi, J. Pagano and T.A. Geissman, Cytotoxicity of sesquiterpene lactones, Cancer Res., 31 (1971) 1649. 11 J.C. Mitchell, T.A. Geissman, G. Dupuis and G.H.N. Towers, Allergic contact dermatitis caused by Artemisia and Chrysanthemum species, J. Invest. Dermatol., 56 (1971) 98. 12 E. Rodriguez, Sesquitcrpene lactones: distribution, biological activity and isolation, Phytochem. Bull., 8 (1975) 7. 13 E. Rodriguez, M. Dillon, T.J. Mabry, J.C. Mitchell and G.H.N. Towers, Dermatologically active sesquiterpene lactones in trichomes of Parthenium hysterophorus L. (Compositae), Experientia, 32 (1976) 236. 14 T.R. Narasimhan, M. Ananth, M. Naryana Swamy, M. Rajendra Babu, A. Mangala and P.V. Subba Rao, Toxicity of Parthenium hysterophorus L. to cattle and buffaloes, Experientia, 33 (1977) 1358. 15 K.-H. Lee, T. Ibuka, R.-Y. Wu and T.A. Geissman, Structure-antirnicrobial activity relationship among the sesquiterpene lactones and related compounds, Phytochemistry 16 (1977) 1177. 16 P.V. Subba Rao, A. Mangala, G.H.N. Towers and E. Rodriguez, Immunological activity of parthenin and its diastereoisomer in persons sensitized by P. hysterophorus L., Contact Dermatitis, 4 (1978) 199. 17 E. Rodriguez, The chemistry and distribution of lactones and fiavonoids in Parthenium (Compositae), Ph.D. Thesis, Univ. of Texas, Austin.
83
FORMATION OF ADDUCTS OF PARTHENIN AND RELATED SESQUITERPENE LACTONES WITH CYSTEINE AND GLUTATHIONE
ANNA K. PICMANa, ELOY RODRIGUEZ b and G.H.N. TOWERSa aDepartment of Botany, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada) and bDepartment of Ecology and Evolutionary Biology, University of California, Irvine, CA (U.S.A.) (Received May 18th, 1978) (Revision received June 4th, 1979) (Accepted June 19th , 1979)
SUMMARY
Parthenin, the major sesquiterpene lactone of Parthenium hysterophorus, a weed responsible for dermatitis in man is primarily restricted to leaf and stem trichomes. Parthenin forms a m o n o a d d u c t with L-cysteine through the a-methylene group of the 7-1actone and a biadduct with the endocyclic double bond on the cyclopentenone ring. Studies with other sesquiterpene lactones support the view that the types of adducts formed are correlated with the biological activity of the sesquiterpene lactones. INTRODUCTION Parthenium hysterophorus L., a widespread composite of the Southern U.S.A., the Caribbean, Australia and parts of South America, has become a serious agricultural pest as well as a medical hazard in India where it is the cause of an epidemic of allergic contact dermatitis [1]. It is generally recognized that partbenin, the major sesquiterpene lactone of this species, is responsible for the dermatological reaction associated with this plant [1]. Sesquiterpene lactones, characteristic of the Compositae, are an important group of terpenoids which exhibit cytotoxic, antineoplastic, allergenic and other biological activities [2]. These compounds, by Michael-type addition, form adducts with amino acids containing SH groups such as cysteine [3,4] or with lysyl or histidyl residues [5]. It has been suggested that the conjugated a-methylene~/-lactone moiety of sesquiterpene lactones is responsible for cytotoxicity [6,7] as well as for allergenicity [8,9]. A structural requirem e n t for significant cytotoxic activity is the O=C--C--CH2 moiety which could be part of an ester, ketone or lactone [10]. Results of tests for aUergenicity with different sesquiterpene lactones indicated differences in response between patients [11]. The fact that
84
A
B
b H
a I HS--C½--#--COO+NH3
C
.~.~
D HOOC
°'-c°/ I s - ~
NH2
COOH
~
'-- N H2
coon Fig. 1. Parthenin, cysteine and their adducts (A-parthenin, B-cysteine, C,monoadduct, D-biadduct).
parthenin (Fig. 1A) possesses two potentially active sites (an exocyclic methylene C13 and an C2 C3 endocyclic double bond allylic to C4 carbonyl group) at which Michael-type additions could occur, might explain toxic effects on cattle and the potent allergenicity of this compound [1,12]. We have examined the reaction of parthenin and of several closely related sesquiterpene lactones w i t h cysteine and:g!utathione in order to determine whether more than one type of adduct is formed. We also have reexamined the distribution of parthenin in aerial parts of P. hysterophorus. MATERIALS AND METHODS
Plant material. P. hysterophorus plants were grown from achenes obtained from Austin, TX, U.S.A. Achenes germinated in wet sand In 1--3 weeks and when leaves had formed, seedlings were transfered to pots and maintained at 30°C during the day and 20°C at night. Light period was 16 h. Detection o f parthenin in plant material. Parthenin was isolated from Parthenium hysterophorus by the method of Rodriguez [12]. Plant material (achenes or seedlings) were extracted with chloroform for 24 hrs and the presence of parthenin checked by thin-layer chromatography (TLC) with a known sample (Eastman Si gel plates with benzene--acetone, 1 : 4, fumed with iodine vapors, Rf = 0.57). Crushed or intact plant material gave identical results. Detection o f parthenin in trichomes and stems o f P. hysterophorus. From
85 ten fresh stems 10 mg of trichomes were removed with a spatula and transfered to distilled methanol (10 ml). The stems, with all trichomes removed (1 g), were cut into small pieces and extracted with distilled methanol (25 ml) overnight. Both extracts were concentrated to minimum volumes and checked by TLC for presence of parthenin. TLC of sesquiterpene lactone-thiol adducts. Eastman cellulose plates, without fluorescent indicator, and the upper phase of n-butanol/acetic acid/ water (4 : 1 : 5) were used. Plates were sprayed with 0.25% ninhydrin (acetone), heated for several minutes at 80°C and then exposed to iodine vapors to detect sesquiterpene lactones w~hich give brown spots. The monoand biadducts of parthenin give violet colored ninhydrin spots with Rf = 0.55 and Rf = 0.19, respectively. (Rf cysteine = 0.36, Rf cystine = 0.13). Reactions o f sesquiterpene lactones with L-cysteine and reduced glutathione. To 0.01 mmol of each of the sesquiterpene lactones shown in Fig. 2 in aqueous ethanol 0.04 mmol of an aqueous solution of L~ysteine (Sigma Chemical Company) was added and the mixture allowed to stand at room temperature. The formation of adducts was checked by TLC. Parthenin and helenalin (0.01 mmol and 0.04 mmol of each of them in water) were allowed to react with 0.04 mmol of an aqueous solution of glutathione (Sigma Chemical Company) in the same way. Isolation and identification of: (a) Monoadduct -- equimolar aqueous solutions of parthenin (5.84 mg/0.58 ml) and L-cysteine (2.42 mg/0.24 ml) were mixed and allowed to stand at room temperature. After 6 h, when TLC showed only one positive spot with ninhydrin, the solution was shaken with Chloroform to remove remaining parthenin. The aqueous phase when freezedried yielded 8.2 mg of a white powder soluble in water, methanol, ethanol, n-butanol, acetone but insoluble in chloroform. It turned brown at 210--212°C and decomposed at 247°C. NMR {15.4 MHz, D20 with DSS): 8 7.67 (H-2, d), 6.17 (H-3, d), 5.06 (H-6, d), 4.03 (Hb, s), 3.10 (Ha, s), 1.27 (C5-Me, s), 1.10 (C10-Me, s). IR (KBr disc): kmax 3300--2900 (aminoacid, s), 1755 (vs), 1715 (cyclopentenone, vs), 1650--1580 (COOH, s), cm -1. (b) Biadduct-aqueous solution of parthenin (17.5 mg representing 0.06 mmol, in 1.75 ml) and L-cysteine (14.5 mg, representing 0.12 mmol, in 1.45 ml) were mixed and allowed to react at room temperature for 6 h and then freeze~lried. The residue, dissolved in water, was chromatographed on a column (25 × 2 cm) of Whatman cellulose powder which was packed and eluted with the upper phase of the solvent system n-butanol/water (4 : 5). After fast moving compounds were removed from the column, methanol/water (1 : 1) was used to elute the cysteine-parthenin biadduct. The fractions containing this product were pooled and freeze
86
CO
O..CO/---
par t h e n i n
H
hymenin
i
/
o"
o1-t
//
helenalin
damsin
H
6~ °--co cumambrin- B
tetraneurin-D
I
H
'
0
0
tenulin
H
I
.?0
,
CO
isotenulin
Fig. 2. Sesquiterpene lactones selected for the reaction with L-cysteine.
(H-3, d) 6.28 (H-13b, d), 5.63 (H-13a, d), 5.06 (H-6, d), 1.27 (C5-Me, s), 1.10 (C10-Me, s), IR (KBr disc): kmax 3420 (OH, us), 1742 (7-1actone, vs), 1718 (cyclopentenone, vs), 1662 (7-1actone, w), 1595 (cyclopentenone, w) am
-1
.
L-cysteine NMR: (15.4 MHz, D20 with DSS): 6 4.03 (Hb, s), 3.10 (Ha, s). IR (KBr disc): kmax 3300--2900 (aminoacid, s), 2560 (SH, m), 1650--1580 (COOH, s) cm -~ .
87 RESULTS
P. hysterophorus achenes and seedlings, with the first true leaf bearing trichomes, were found to contain parthenin. Younger seedlings with cotyledons only and lacking trichomes did n o t contain detectable amounts of parthenin. A comparison of methanolic extracts of trichomes which had been carefully excised from the stems with extracts of stems that had been depilated confirmed that parthenin is restricted to trichomes [13]. TLC of a mixture of aqueous solutions of parthenin and L~cysteine yielded t w o additional ninhydrin positive spots. One of them, identified as a monoadduct (Fig. l b ) , was obtained directly from an equimolar mixture o f both reactants. The second one, a biadduct (Fig. lc), was isolated b y column chromatography from a reaction mixture containing an excess of L-cysteine. The NMR spectrum of the m o n o a d d u c t did not show the characteristic signals for = CH2 of parthenin. Instead new signals due to a cysteine residue (Ha and Hb) were apparent. Proton signals corresponding to H2 and H3 in the cyclopentenone ring remained. The IR spectrum exhibited the cyclopentenone chromophore of parthenin with the additional absorption of the cysteinyl carboxyl group. This adduct therefore contains one molecule of cysteine attached through a sulfhydryl link to parthenin on the = CH2 carbon of the 7-1actone ring. The NMR spectrum of the second adduct showed the disappearance of the signals for = CH2 as well as for the protons on carbons of the cyclopentenone ring and in addition two new signals for L~cysteine (Ha and Hb). This c o m p o u n d was identified as a biadduct containing one molecule of cysteine at each active site. Rapid decomposition of the biadduct to m o n o a d d u c t and to cysteine, cystine and parthenin was detected by TLC in the first hour after isolation. There was also a slower transforma-
TABLE
I
POTENTIAL ACTIVE SITES THEIR ADDUCT FORMATION Sesquiterpene
OF SELECTED SESQUITERPENE WITH L-CYSTEINE
a-CH2-7-1actone
Cyclopentenone
LACTONES
AND
Adducts of sesquiterpene lactones with L-cysteine
Parthenin
Hymenin Helenalin Damsin Cumambrin-B Tetraneurin-D Tenulin Isotenulin
+ + + + + + ---
+ + + ---+ +
monoadduct
biadduct
+ + + + + + + +
+ + ÷ ------
88
tion of the isolated monoadduct to biadduct and also to parthenin, cysteine and cystine. These transformations were detectable by TLC 24 h after isolation and indicated a higher stability of the monoadduct. TLC of reaction mixtures of selected pseudoguaianolides (Fig. 2) with L-cysteine indicated the formation of monoadducts with iactones containing only one potential active site, i.e., either the exomethylene function on the lactone ring or the cyclopentenone ring. Biadducts were obtained with parthenin, hymenin and helenalin, compounds which possess both potentially active sites. This is shown in Table I. Equimolar quantities of parthenin or helenalin with reduced glutathione gave only monoadducts. When 4-fold quantities of glutathione were used, helenalin formed a biadduct. Parthenin, however, formed only a monoadduct even after standing for six days. DISCUSSION Parthenin was found only i n seedlings older than 10 days and its presence was associated with the development and differentiation of trichomes. The restriction of parthenin to the trichomes of P. hysterophorus was confirmed [13]. The higher concentration of this lactone in leaves [17] may be explained, in part, by the fact that leaves elaborate more trichomes than do other parts of the plant. In the present study we have demonstrated that parthenin forms two unstable adducts with L-cysteine. The monoadduct contains a cysteinyl moiety attached to parthenin through the C13 and the biadduct possesses an additional molecule of cysteine on the C2 position. The formation of the biadduct was obtained with higher concentrations of cysteine in the reaction mixture. The amounts of thiols, including proteins with sulphydryl groups, in living cells are probably high enough so that both active sites of parthenin or of certain other sesquiterpene lactones become involved. The results suggest that the presence of two active sites in parthenin is related to the fact that it is a strong sensitizer in man. A similar case may be made for helenalin which has antibiotic properties [15] and which is also reported to be a good sensitizer in delayed hypersentitivity [9]. However, hymenin, a diastereoisomer of parthenin, which also forms two adducts, is only a weak sensitizer in some cases [16] suggesting that additional factor(s) influence allergenicity. ACKNOWLEDGEMENTS We thank Dr. C.-K. Wat for the helpful discussions and Pat Jamieson and the Department of Chemistry, U.B.C. for measurements and help with inter. pretation of the NMR spectra. This study was supported by the National Research Council of Canada.
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