Aug. 20, 195s
BIOGENESIS OF
[CONTRIBUTION FROM THE
DEPARTMENT OF
THE
4393
NICOTIANAALKALOIDS
CHEMISTRY, UNIVERSITY OF CALIFORNIA, L O 5 h N G E L E S ]
The Biogenesis of the Nicotiana Alkaloids. VI. The Piperidine Ring of Anabasine1 BY EDWARD LEETE RECEIVED MARCH10, 1958 Cadaverine-l,5-Cl4 dihydrochloride was fed to intact Nicotiana glauca plants resulting in the formation of radioactive aiiabasirie. Systematic degradation of the active anabasine indicated t h a t the cadaverine was incorporated into the piperidine ring with maintenawe of the integrity of the five carbon chain, There was negligible activity in the pyridine ring. The radioactive cadaverine was also incorporated into anabasine by excised shoots of N . glauca, the percentage incorporation being much higher than that of l y ~ i n e - 2 - C ' ~ .The significance of these results is discussed.
Introduction IVe have shown previously2 t h a t lysine is a precursor of the piperidine ring of anabasine, an alkaloid of AT. glauca. The metabolism of lysine in this plant was not analogous to t h a t of ornithine in LV. tabacum in which the main alkaloid is nicotine. I n the latter species the administration of ornithine2-C14 led to the production of nicotine labeled equally on the two a-positions of the pyrrolidine ring, whereas lysine-2-CI4 yielded anabasine labeled only on c - 2 of the piperidine ring.2 We have shown recently4 that putrescine, like ornithine, is a n efficient precursor of the pyrrolidine ring of nicotine. It was thus of interest to examine cadaverine (1,5-diaminopentane) as a possible precursor of the piperidine ring of anabasine. Radioactive cadaverine previously has been prepared biosynthetically from l y ~ i n e ,but ~ . ~in the present work it was obtained conveniently by the catalytic hydrogenation of 1,3-dicyanopropane which was derived from the reaction of 1,3-dibromopropane with potassium cyanide-C'*. T h e overall yield was 70y0. ilronoff7 has reported t h a t there was a negligibly small amount of radioactivity in the anabasine obtained from excised leaves of N.glauca which had been fed lysine-2-C14. Since this result conflicts with our work using intact plants2 we have also fed l y ~ i n e - 2 - Cto ' ~ excised shoots of N . glauca. We also fed cadaverine-1,5-C14to the excised shoots for varying lcngths of time wheii we discovered t h a t it was a good precursor of anabasine in tlic intact 1)la11t . Experimental Cadaverine-1,5-CL4 Dihydroch1oride.----1,:j-I )iIm1iiiopropaue ( 0 . 5 5 nil.) was added to a solution of potassium cptiiide (0.65 g., with an activity of 4.0 X lo8 c.p.m.8) and potassium iodide (0.05 9.) in 75'4 ethanol (4 ml.) and the mixture refluxed for 45 minutes. The solution, which had deposited considerable solid, was diluted with 30 ml. of water and extracted with ether t o remove urircacted di(1) T h e title of this series has been changed t o t h e present one to iuclude S i c o t i a n a alkaloids o t h e r t h a n nicotine. T h i s work has been supported b y a g r a n t f r o m t h e Research Corporation, New York, a n d was presented in p a r t a t t h e 133rd meeting of t h e American Chemical Society, San Francisco, April 13-18, 1958. ( 2 ) E. Leete, THISJ O U R N A L , 78, 3520 (105G). (3) E. Leete a n d K. J. Siegfried, ibid., 7 9 , 4529 (195i). (1) E. Leete, i b i d . , 80, 2162 (1958). ( 5 ) H. R. V. Arnstein, G. D. H u n t e r , H. M. M u i r a n d A. Keuberger, J . Chenz. SOL.,1320 (1952). ( 6 ) R . W Schayer, R. L. Smiley a n d J. Kennedy, J . B i d . C h r n z . , 206, 461 (1954). ( 7 ) S. Aronoff, Ploiil Phvsiol., 31, 355 (1956). ( 8 ) All counts were carried o u t in a windowless flow (>.XI. counter (Kuclear-Chicago Co. Model D-46 A ) using " Q gas" as the quencher. Determinations n e r e carried o u t on samples (If finite thickness, making coriwtiuns f u r eiiiiiciency a l ~ dself absorption.
bromopropane. The aqueous solution was acidified Kith 20 ml. of 2 A: sulfuric acid and extracted with chloroform in a continuous extractor for 24 hr. The chloroform extract was taken to dryness and the residue dissolved in a mixture of methanol (50 ml.) and concentrated hydrochloric acid ( 5 ml.) and hydrogenated a t a pressure of 45 Ib./sq. in. in the presence of Adams catalyst (0.5 g . ) for 2 hr. The coagulated catalyst was filtered off and the filtrate taken to dryness in vacuo to leave a residue which was crystallized from a mixture of ethanol and ether yielding colorless hygroscopic needles of cadaverine-1,5-C" dihydrochloridc, m . p . 235-237" cor. having a n activity of 4 . 3 X lo5 c.p.ni./ mg. The yield was 0.610 g. (70%). Anul.9 Calcd. for CsH14N2.2HC1: C, 34.20; 11, 9.21. Found: C, 34.26; H, 8.84. Administration of Cadaverine-l,S-C14 to the Intact h:. g1auca.-The N . glauca plants were about 5 months old and were grown in a n inorganic nutrient solution as previously described.2 Cadaverine-l,5-C'* dihydrochloride (350 mg. (2.0 m.W) with an activity of 1.50 X lo8 c.p.m.) was divided equally between the nutrient solutions of three of the plants. The cadaverine was absorbed rapidly by the roots and after 5 days the nutrient solutions had only 2.5% of their original activity. Two weeks after feeding the cadaverine, the plants were harvested and the anabasine isolated and degraded by established methods.* The yield of anabasine from the three plants (wet wt. 800 8.) was 555 tng. The aqueous ammoniacal sap had an activity of 4.4 X 106 c.p.m. The activities of anabasine and its degradation products are recorded in Table I.
TABLE I
ANABASINEA S D
ITS DEGRADATION PRODVCTS SpeciBc activity
x 10-5,
c . p m./m.M
Atiabasiiie tliperclil~iratc Anabasine dipicrate Anabasine dipicroloiiatc Sicotinic acid Sicotinic acid hytlrociil~ride Pyridine picrate10
I :19 1.45 I .39
75 0,Rt
0
0 (I1
Administration of Cadaverine-I ,5-C14 and Lysine-2-C14 to Excised Shoots of h'.g1auca.-The shoots were obtained
from the tops of 6 months old N. glaucu plants. The shoots were cut a t a n angle of 45" with a razor blade and irnmediately placed in 25 ml. of distilled water containing only the radioactive compound. The average length of the shoots was 30 cni. and they had a fresh wt. of about 25 g. The shoots vvere exposed t o continuous illumination. .Ss the aqueous solution was absorbed by the shoots it was replenished with distilled water. The shoots were harvested a t the times indicated in Table 11. The fresh shoots were macerated in a IVaring Blendor with a mixture of chloroform (100 ml.) and 15 N ammonia solution (20 ml.). Inactive anabasine (100 mg.) was added to the mixture as a carrier. The Blendor was washed out with a further 300 ml. of chloroform and the plant allowed to stand in contact with the chloroform and ammonia for 24 hr. The marc was then filtered off and the filtrate separated into aqueous and organic layers. The aqueous layer was assayed for activity. The anabasine was recovered from the chloroform in the usual way.2 It was found that the anabasine (9, Carried o u t by h l i s H e a t h r r King of Ihpse laboratories. (10) Obtained lrom the dec.rrboi)latiun vf the nicotinic ncid.'
-i3M
Vol.
EDWARD LEETE
so
Tram tlic cadaverine fcedings was contarnitiatcd with a trace cadaverine. Therefore the distilled anabasine was dissolved in benzene and chromatographed on alutnina.ll 7'hc mahasine was eluted with a solution of 170 methanol i i i hcnzenc. Cadaverine remained on the column until pure nieth:riiol was used as the eluting solvent. T h c fractioiis containing anabasine (detected by paper chromatography) lverc combined and distilled. The recovery of analiasiiie from each experiment was about 80 mg. The incorporation of C14 (shown as a percentage in Table 11) vias calculated by dividing tlie total activity found in the anabasine by the total activity absorbed by the excised shoot. Tlic small amount of anabasine (ca. 10 mg.) in the 25 g. of shoots was neglected in calculating the $70 incorporation n.liich w:is based on a loo'% recovery of the inactive anabasine added as a carrier. Details of these experiments are recorded in Table 11. The radioactive anabasine diperchlorate obtained from the ly~ine-2-C'~ fed had a specific activity of 2.5 X l o s c.p.m./mM. This was oxidized with nitric acid to yield nicotinic acid with a n activity of 2.6 X l o 3 c.p.m./mA.l. If it is assumed that there is no activity in the pyridine ring of the nicotinic acid, this result is consistcnt w i t l i otir previous findings.2
slioots for 4 days was extrenicly low niid just within the limits of accurate counting with a G.M. flow counter. It is thus not surprising that .Ironoff reported' negligible activity in the anabasiiie after feeding lysine-2-Cl 4 to an excised leaf. T h e activities of the degradation 1)roducts (Table I) of the radioactive anabasine are consistent with the hypothesis t h a t t h e cadavcrine-1,JC'*is incorporated into the piperidine ring without any breakdown of the 5-carbon chain. Half the activity of the aiiabasine was located at C-2 of the piperidine ring and we assume t h a t the rest of the activity is a t C-G. \York is proceeding to confirrii this. \Ye consider that the iiiimcdiate precursor of the piperidine ring of anabasine is A'-piperideine which is derived from cadaverine b y oxidation to 5 aminopentanal followed by cyclization. This oxidation has been accomplished by M a n ~ i aiid Smithies13 using, a s catalyst, an :miirie oxidase TABLEI1 isolated from pea seedlings. More recently Hasse and Berg1*have obtained anabasine by the oxidation of cadaverine in the presence of a pea extract. _..,of Activity Schapf, ct nl.,'5 have shown t h a t A1-piperidcine will A c t i v i t y feed7% of a m o u n t fed in: undergo dinicrization in tlitro to yield tetrahytlroWt., X 10-7, i n g , N u t r i I'n-curvm mg. c.p.m. d a y s ent Sap Anabasinc anabasine. However, since the aiiabasine obtained in our experirncnts had negligible activity 2 23 19 0.078 C';idaverinc2.5 1.07 in the pyridine ririg, analogous reactions iiiust not 4 15 22 .26 I,j-C'*-di25 1.07 occur in N . g!auca. Nicotinic acid has been shown IICl 25 1.oi 6 0 20 .49 to be a precursor of the pyridine ring of i i i c o t i t ~ c ' ~ ~ ' ~ I,y\iiie-2-Cl4 atid it seems rc:rsoiiable to assume t h a t i t is also a I IC1 26 0.49 4 12 28 .03G precursor of the pyridine ring of aiiabnsinc. The incorporation of l y ~ i n e - 2 - Cinto ' ~ the pipcriDiscussion cline ring cannot proceed *Lli(i,frcc cadaverine since ' I l c iiicorporatioii of catlavcriiic-1,5-C:'" into this would result i t i r:intl(~riiizntioiiof activity beaiiabasiiic 14 days after feeding to the intact A'. twccii C-2 and C-0 of allab:isitlc, wltcrcns 311 the giuiicn plant was 0.337, which compares favorably activity was found a t (2,-2. Hen-ever i t is coiiccivwith t h a t of l y ~ i r i e - 2 - Cwhich ~ ~ was only 0.00-30 able t h a t the lysine-2-C'* could lic converted to ; i i i d 0 . 0 4 6 ~ , 5 and 16 days after feeding.* T h e a'-pil,eritleinc-"C' 1 ~ i ( catla\-criiie i if it is assuiuetl cxcisetl shoots were also capable of synthesizing that the cadaverine is bound to the ciizyiiic surface :mnbasiiie froiii cadaverine or lysinc, confirming so t h a t randomization cannot occur, tlie cy-carboii t h e coiiclusions of Dawson12t h a t alkaloid synthesis of lysine becoming the nldchyde group in ,"mininooccurs in the aerial parts of N . glauca as well as in pentanal. Cadaverirle m t y be a l x t t e r precursor the roots. The percentage incorporation of cada- of the piperidine ring than Ipsiiie because of its Ycrine in the shoots illcreased slowly with time, be- metabolic proxitnity to :i-nn~inopcntan:~l. ing highest after G days. .ittempts to extend the Acknowledgment. -\Yetliaiik tlic 1 ) c ~ ) ; t r t i i i e i i t lime of feeding beyond this were unsuccessful since oI I h t a n y of this Uiiiversity for cultivatioli or tlic tlic shoots became flaccid. Previous results3~* &ucts. h a v e indicated t h a t nicotine is synthesized a t a s. slow rate in .V.tabncum. It may be a general 1,os . \ N (.; I : I ,.l ~ s 2.1, C.\l pheiioinenon t h a t alkaloid synthesis occurs a t a 1 1 ) I
