GLUCOSINOLATES I N THE GENUS ZILLA (BKASSICACEAE) B. EIrh!kSSH.%nI,31.K a ~ &YnA and G. WASSEL Pharmaceutical Scaences Laboratorzes, h-atzonal Research Centre, Dokka-Cairo, Egypt
J. R E I ~ C H Instztutfur Pharmazeutische Chemze, 1l.estfalzchen TTilhelms- Cntr ersztat, 44 M u n s t e r , IT-. Germany
ANDERSK J A E R ~ Department of Organic Chemistry, T h e Technical L-nioersity of D e n m a r k , 2800 L y n g b y , Denmark
Traditionally, the genus ZiZZa Forsk. (Brassicaceae) is conceived as composed of the species Z . macroptera Cosson (A), Z . biparmata 0. E. Schulz (B), Z . spiltosa (L.) Prantl (C), and a variety of the latter: Z. spinosa v. microcarpa Dur. & Sch. (D) (1, 2). Some authors, bolt-ever, regard the genus as monotypic, comprising two subspecies: Z . spinosa (L.) Prantl, subsp. spiitosa ( = Z . myagroides Forsk. =subsp. costala lIaire et Reiller), and subsp. macroptera (Cosson) Maire et Weiller ( = Z. macroptera Cosson = Z . biparmata 0. E. Schultz) (cf. e.g. Ref. 3 ) . -411 are spiny shrubs indigenous to the desert regions stretching from llorocco to Arabia and reputed, among the local populations, ac useful remedies in the treatment of ailments e.g. kidney stones. We have qtudied the taxa (B), (C), and (D) for their content of glucosinolates (l), a characteristic class of ions ubiquitously present within the family Bra--' saaceae (4, 5 ) . Paper chromatography disclo3ed the presence of only one, and apparently the same, glucosinolate in extracts of seed-containing siliques of Z . biparmata. Z . spinosa, and Z . spiiiosa v. microcarpa. For each species, the glucosinolate fraction n as isolated, an aliquot converted into a crJ-stalline acetate (IC-salt), and the remainder
subjected to enzymic hydrolysis, followed by isolation and chromatographic purification of the sulfur-containing hydrolysis product. In each case, chemical and spectroscopic evidence disclosed the identity of the glucosinolate as 2-hydroxy-3butenylglucosinolate (2, and/or 3), characterized as the crystalline penta0-acetyl derivative (K-salt), as n-ell as by its ability to undergo myrosinasecatalyzed hydrolysis to the corresponding isothiocyanate, spontaneously cyclizing to give a dextrorotatory 5-vinyloxazolidine-2-thione (5) The optical rotation data for the isolated products are presented in table 1. I n a previous paper, Z . spiizosa \yas described as a species producing the levorotatory enantiomer (4) (goitrin) on enzymic hydrolysis (8).* (R)-P-Hydroxy-3-but enylglucosinolate (2), affording the antithyroid (S) ( - )-5-vinyl-oxazolidine-2-t hione (4) (goitrin) subsequent to enzymatic h>-drolj-~is,~ is an established constituent 'The (S)-configuration (4)was establjshed for the levorotatory enantiomer (goitrin) several years ago ( 6 , 7 ) . *Though considerable variation in enantiomeric composition with strain, season, environment efc. is throughout conceivable, an experimental error in the sign of rotation of a preparation, brought t o enantiomeric homogeneity b y fractional crystallization, cannot be excluded. 3 0 n the reasonable assumption that the configuration is retained during the cyclizat ion
'To n-hom inqiiiries should be addressed. 534
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19801
EL-3iESSH-IIi-1 ET .iL.
:
GLUCOSISOL.ITES I S ZILLA
535
I
OH
R 1
2: R = ( R ) CHz=CH-CH(0H)-CHp-
R'= CHZ=CH-; RZ= H 3: R1= H : R2= CH,CH-
4:
3: R = (S) CH?=CH-CH(OH)-CHz-
of many Brassica species (turnip, cabbage, kale, and rape) (9), whereas the epimeric (S)-glucosinolate (3), giving rise to (R) (+)-5-vinyloxazolidine-2thione ( 5 ) , has been encountered previousl!- only in seeds of Crambe abyss i i i i c ~ ( 1 0 ) . Reported rotation values of the penta-0-acetyl derivative of the (R)-glucosinolate (2): ([cx]*~D-9.5") (11): and the (S)-glucosinolate (3),
The identical glucosinolate patterns seem to support the monotypic treatment of the genus ZilZa. Classification of Z . biparmata ( = Z . macroptera) as a subspecies of Z . spinosa was also suggested recently on the basis of chromosomal siniilaritie3 (13). I n rape plants, the biosynthesis of (2) proceeds from 2-amino-6-methylthiohexanoic acid (6). which. in its
T ~ B L1.E Rotation data for penta-0-acetyl derivatives of glucosinolates (K-salts) and 5-vinyl-oxazolidine-2-thiones, from Ziiln species. 1
Zilla species
1
Z . biparniata. . . . . . . . . . . I
Z. spinosa... . . . . . . . . . Z . spinosn v. microcnrpn. .
'
[ a ] * ~for o penta-0acetyl derivatives [ a ]for ~ 5-vinyl-oxain Kiter ( e , 2.4-2.6) 1 zolidine-2-thiones -14.6" -14.5O -14.6"
1 ~
+61.5'* +61 . 3 O b , +61.lCb
aAt 26"; e, 2.5; 1IeOH. '.kt 25'; C , 2.3-2.5: CHClj. CPreviously reported ( 7 ) : - X C , at 31"; in CHC13 ( c j . footnote 2 ) .
turn, derives from methionine ( 7 ) by ([cx]?~D- 14.8" and - 13.1') ( l o ) , totwo-fold homologization (14, 15); the gether with those of the isolated and side-chain hydroxylation seems to ocsynthetic (S) -5-vinyloxazolidine-2-t hicur at a late stage along the synthetic one (4), [ ~ ! ] ~ ~ D - i o . j " (NeOH) (9); route (14, 15). Assuming a siniilar [ C ~ ] ~ ~ D - T(JIeOH) ~ . S " (synthetic) (12); pathway in Zilla: one is left with tn-o [ ~ r ] * ~ D - i 6 . S ' (CHC1,) (lo), and the (R)-enantiomer (Z), [ ~ ~ ] ? ~ D + i 0 . 5 ' possibilities:4 (1) that tn-o stereo(JIeOH) (synthetic) (12) : [ ~ ~ ] ~ j D + i 5 . 1 " specific hydroxylases are simultaneou.slj- operating. or (2) that the hy(CHC1,) (10). justif- the concluqion that the three Z . species all contain the 4Epimerization of the glucosinolate fracepimeric (S)-and (R)-gluco+iolates, tion, or racemization of the 5-vinyl-osazoli(2) and (3). and in approximately the dine-2-thiones, during the isolation and purisame ratio, si;. 9:1, based on the rotafication procedure seem highly unlikely in tion values (table 1). the present case.
JOCRSAL OF KATURAL PRODUCTS
536
droxylase in Zilla species is lacking stereospecificity. KO choice between these possibilities can be made on the available evidence. Me-S-[CH2] .-CH (NHa+)-CO%-
6: n = 4 5: n=2
EXPERIIIENTXL PLI~XT~\r.kTERI.kL.-Seed-COntaining
siliques of all three Zilla species were collected in the Egyptian desert from plants authenticated by (the late) Professor I-.Tackholm, Cairo University. DETECTION A K D E X T R . I C T I O N . - Pchro~~~~ matography of concentrated methanolic silique extracts was performed as previously reported (8). In each extract, one glucosinolate spot only could be seen. Powdered and carbon tetrachloride-defatted, seed-containing siliques (200 g) of the various Z i l l a species n-ere extracted with three 250 ml portions of ’io% methanol. After removal of methanol iii m c i t o , water was added to a total volume of 2 liters, and the solutions were slowly passed through a column of anionotropic alumina (Woelm) (150 gm); the column was eluted with a 3yG KqSOa-solution and the glucosinolate-containing fractions were taken to dryness and freed of most inorganic material by extraction into hot 90% ethanol. CH.iRACTERIZ.kTIOiX A K D EiXZY311C HYDROLYsIs.-One half of the glucosinolate fraction was subjected to acetylation in pyridine ( 5 ml) and acetic anhydride (5 ml) at 25” for 12 hours. The pentaacetates, recrystallized once from 95% ethanol, proved identical (uv, ir, pc) with an authentic specimen of the penta-0-acetyl derivative of the glucosinolate (3) isolated from Crambe seeds (10). Rotation: c j . table 1. The second half of the glucosinolate fraction was dissolved in a citrate buffer (pH 6.8) and subjected to enzymic hydrolysis by the addition of a few drops of a myrosinase solution. After 6 hours, the solutions were extracted with chloroform, and the extracts were purified by chromatography on silica gel with benzene as the eluent and Grote’s reagent a s a diagnostic reagent for 5-vinyl oxazolidine-2-thiones. The still oily fractions containing the latter were again subjected to chromatography, now on silica gel (Rlerck ‘Fertigsaule B’), with ethyl acetate a s an eluent, (6 ml/min) and uv-
[VOL.
43,
NO.
4
detection of the eluted oxazolidinethiones. The crystalline residues, mp ca. 50°, representing the total quantity of the latter, exhibited uv, ir (in solution), and ms data indistinguishable from those of an authentic specimen of (- )-5vinyl-oxazolidine-2-thione (4); for rotations, cf. table 1. ACKSOIT’LEDGRIENTS The authors are indebted t o Dr. R l . E. Daxenbichler, The Northern Regional Laboratory, Peoria, Ill., U.S.A., for an authentic specimen of the penta-0-acetyl derivative of (S)-2-hydroxy-3-butenylglucosinolate (Ksalt), isolated from Crambe seeds, to Dr. B. W.Christensen, in the Danish laboratory, for help with the chromatographic purification, and to Dr. A. Amin, The Department of Botany, El-Mansura University, Egypt, for her kind assistance in plant collecting. One of the authors (B.E.-M.) acknowledges his gratitude t o the University of Munster for a Post-Doctoral Fellomhip (1975-7). R e c e z e d 26 S o ~ e i n b e r1979. 1.
2. 3.
4. 5.
6.
LITERilTURE C I T E D Engler and H . Harms, “Dze -Va/ztrlzcheu Pjaizzetifanzilzet~’, Duncker 8: Humblot, Berlin, 1936, vol., 17b, 371. T-. Tackholm. “Students Flora o f Egypf”, Cairo tniversitj-, 1974, 197. S. $1. H. Jafri, F1. Lib., 23,i 2 (1977). A. Kjaer, Forfschr. Chenz. Org. 1-afursto-f, 18,122 (1980). RI. G. Ettlinger and A. Kjae;, “Recent Adeaiices in Phvfochenzistr?’ 1-01. 1. Appleton-Century-Crofts, 3e&York; 1968, p. 58. 4 . Kjaer, B. W.Christensen and S. E. Hansen, A c t a Chenz. Scand., 13, 144
.
(1959).
7 . -4. A . T.’Bottini, T’. Dev and hf. Stewart, J . Org. Chevz., 28, 156 (1963). 8. 1I.S.Karawvya, G . 11.Wassel and B. S. Elmenshavi, Acta Pharnt. Sueczca, 10, Elmenshavi,-Acta 477 (1973). 9.
E. B. Astwood. R l . -4.Greer and 11.G.
Ettlinger, J . ’Biol. Chenr., 181, 121 11919’1. 10. 31.E: Daxenbichler, C. H . Van Etten and I. -4.Wolff, Biochemzstry, 4, 318 19G5): cf. also ibid. 5 , 692 (1966). 11. 0.-E.‘ Schultz and W. Wagner, A r c h . Phnrni.. 289/61.597 (1961). 12. 11.G. Ettlingei, J . dnz. Cheni. Soc., 72, 4792 (1950). 13. A. Amin, Egypt. J . Bot., 16, 501 (1973). 14. G.S. Serif and L. A . Schmotzer, Phytochemistr?, i,1151 (1968). 15. E. Josefsson, P h y s i o l . Plant.,, 24,, 161 (1971). 16. C.-J. Lee and G. S. Serif, Biochinz. B i o p h y s . i l c t a , 230,462 (1971). I
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