Phytochemstry,1973,Vol 12,~~ 1973to 1975 Pcrgamoa PressPr~ntcd~n En&md

ALKALOID

BIOSYNTHESIS
KENNETH L STUART

IN CROTON LINEARIS*

and LASCELVEGRAHAM
Accepted 19 March 1973) alkaloid, crotonosme, hnearwne, blothe

Chemistry Department,

Umverslty of the West Indies, Kmgston 7, Jamaica

(Rewed Recerved 1 March 1973 Key Word Index-Croron synthesis Imearrs, Euphorblaceae,

proaporphme

Abstract-The ablhty of Croton hneans to form crotonosme from hneansme extent of this conversion appears to vary accordmg to the sex of the plant EARLIER

has been demonstrated,

biosynthettc studies on Croton heard demonstrated that crotonosine (I) was formed from (+)-coclaurme (II)or (f)-norcoclaurme (III), and that 8,14-dehydronorsalutandme (IV) could be brosynthestzed from (f)-coclaurme, (f)-norcoclaurme and (f)-tsococlaurme (V) m thts plant There has been some speculation as to the relattonshlp

O /
Me0

NH H

(I)

(II) (III, (PI

R =Me,R,=H,Rz=wH R=R,=H. R2 = -H R=H, R,=Me.R2=-H D

(Ip)
D

0+.
. CH III =

Me

O Me0 D HO P

/ / D 1x3 NH H

mm

(YI) R=R,=R2=3H (IX) R = R, = RP= H

Ic

aur.)

(Xl

between reduced proaporphines and proaporphmes and more recently we demonstrated that crotonosme could be elaborated from 3H3-llneansme (VI).3 On repeating this, we discovered that mcorporation results were consistently different for female plants than for * Part XIV m the series Alkaloids from Crvton specuzs For Part XIII see STUART, K L and GRAHAM.L (1973) Phytochemrstry 12,1967 1 BARTON, D H R , BHAKIJNI. D S , CHAPMAN. G M , KIRBY, G W , HAYNES, L J. and STUAF!T, K L. (1967)1 Chem Sot C, 1295. a HAY-, L J , HUSBANDS, G E M and SNART, K L (1968)1 Chem Sot C, 951 3 STUART, K L and GRAHAM, L (1971) Chem Commun 392 1973

1974

L STUARTand L GR~HA~~

male plants For female plants we observed lncorporntlon talues of 0 01-O 02 :I while In Although these male plants It ranged from no IncorporatIon to 0 002, IncorporatIon experiments may indicate some differences of the blosynthcslzlng enzyme5 between the sexes, other factors such as difference5 m the age of the plantt may habe had some Influence 4 The labelllng pattern of H,-llnearlslne wds determined b> doing pLlr,~llcl c\perlmcnt$ on lmearlsme but using D20 rather than T,O Confirmation of the locatIon of the deutcrlum was by NMR and MS exammatlon Comparison of the NM R spectrum of non-deuterdtcd lmearlsme and deuterated hneLirlslne showed that the deuterated \ampie. obtained by heatmg the alkaloid under nitrogen m dlmethylformamlde with D,O hld the C-3 proton (6 6 43) and two ahphatlc protons (6 2 4-3 5) mlsslng 5 The MS compdrlqon IS shown III Table I and the mterpretatlon IS ba$ed on earher studley on this and reLIted alkaloids A fragment m the spectrum of a deuterdted sample was establlqhed by high resolution MS to h,ive a m/e of 93 0687 and could be due to either of the deuterated fragment? (Vll) or (Vlll), for which the calculated values are 93 0708 nnd 93 0647 reqpectlvely All the foregoing evidence IS consistent with structure (IX) and this labelling pattern, cspecl,lllq wrth regards to the specific replacement of the a-methylene protons In ring D of hnearlclne I$ slmllar to that observed for testosterone. m which case enohzatlon occurred almost evcluslvely towlrd c-2
TABLE 1
MS COMPARISON OF WY-DEUTTRATFD LINEARIIYr AW DWTFRATED

Type Ion M+ (P + 1)
(P -- 1)

Non-deuterated y/, Base peak mlc

299 90 46 5 89 18 5

W/C

Deuteratcd L Base peak

59 23

300
29X 284

302 301

301
2R7 271 259

(P - Me) (P - OMe) (P - NC,H,)

100 10
5 20

268 256

100

Because the utlhzatlon hnearlslne for the elaboratron of crotonosme either Involved the mtermedlacy of an lmme or enamme, it was Important to cstabhsh that m the deuterdted compound no racemlzatlon had occurred durmg the convcrclon This was done by showmg that the specific rotation of the deuterated compound was the same as that for the nondeuterated lmearlsme ([all, + 117 5) In an attempt to estabhsh the location of the trltlum atoms m the Isolated crotonoslne, the acid rearranged product, apocrotonosme (X), was deuterated This resulted m the removal of 887; of the radIoactIvIty If the actlvlty was equally distributed between two labelled posltlons, then this represents a 94 % exchange at each trltlated site The converslon of lmearlsme (Vl) to crotonosme (1) must Involve N-demethylatlon, Inversion of the asymmetrlc centre at C-6a as well as oxldatlon of carbons 8 and 9 In ring D
D (1968) Compehensrce Btochemrstry (FLORKIN, M and STOTZ,E H , eds ), p 237, Elsevrer, New York 5 GRAHAM,L (1972) Ph D Thesis, Unlverstty of the West Indies, p 113 for spectra BALDWIN, M , LOUDON,A G , MACOLL,A, HAYYES, L J and STUART, K L (1967) J Chem Sot C, 154 MALHOTRA, S K and RINGOLD, H J (1964) J Am Chem Sot 86, 1997
4 SPENSER, I

Alkaloid blosynthesls

m Croton Imeurrs

1975

It has been assumed m the past that reduced proaporphmes are dewed from proaporphmes, but these experiments clearly mdlcate that the reverse process can also occur
EXPERIMENTAL Deuteratlon and frttratron of lmearcsme Lmeansme (90 mg) which was isolated from Croton Imearls Jacq usmg the techmque earlier published* was dissolved m chmethylformamlde (2 ml), DzO (2 ml) added, and the solution sealed under N1 m a thick glass tube The tube was heated at 100 (72 hr) The mixture was then cooled to room temp chilled m ice and opened After evaporation under reduced pressure, the solution was purified on a short alumma column wth CHC13 Evaporation of the CHC13 yielded lmeansme (31 5 mg) Trltlation was achieved in a similar manner when TzO was used instead of DtO, and matenal obtained m several expenments ranged from 20 0 to 56 1 &I Feeding of 3H3-lmearume andexrracrron of crotonosme Male plants and female plants were fed m separate experiments by the wick feeding technique, and m a typlcal feeding, the fed branch (130-250 g) was milled and extracted Hrlth 2 % tartanc acid 8 After bastication, contmuous extraction with CHC13, removal of the solvent and addition of a few drops of acetone yielded crotonosme Radloisotoprc punty was achieved by prepanng the NO-dlacetyl denvative 8 Male plants showed mcorporatlon values varying from no mcorporatlon to 0 002 % while female plants had mcorporatlon values of 0 01-O 02 % Preparurlon of deurerated apocrotonoane This expenment was performed m an attempt to locate all the trltlum atoms m crotonosmk Radioactive crotonbsme (30 mg, -actlvlty = 7 5 x IO-@ based on the dlacetate) was dissolved m 6 N HCl (1 5 ml) and heated at 100 for 1 hr 9 The resultma anocrotonosme (7 4 x 1om3 ~0) was heated at 100 (i2 hr) 41th K t-butoxlde (30 mg) and D20 (1 ml) under-N2 m a sealed tube The NMR of the product was consistent with structure (x), but scmtdlatlon counting carried out as outhned m Part XIII O still showed some residual actlvlty (8 88 x low4 pB or 12 /,) Acknowledgements-We thank Professor J P Kutney, Umverslty of British Columbia, Dr G M Husbands, Wyeth Laboratories, Pennsylvania for obtaining MS data on deuterated hneansme, and one of us (L G ) acknowledges a postgraduate scholarship from the International Order of the Daughters of the Empire 8 HAYNES,L J and STUART,K. L (1963) J Chem Sot 1784 BARTON,D H R , BHAKUNI.D S , CHAPMAN,G M , KIRBY, G. W , HAYNES,L J and STUART,K L (1967)J C/rem Sot C, 1265 lo STUART.K L and GRAHAM,L (1973) Phytochemlslry 12,1967