5472
Biosynthesis and Metabolism of the Hemlock Alkaloids’” Edward Leete* and John 0. O1sonlb Contribution No. 116from the Natural Products Laboratory, School of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455. Received December 14, 1971 Abstract: The administration of 5-oxooctanal-6-14Cand 5-0xooctanoic-6-~~C acid to hemlock (Conium maculatum) resulted in the formation of c0niine-l‘-~4Cand y-coniceine-l’-14C. The high incorporation of activity obtained with these compounds suggests that these are the immediate precursors of the alkaloids. The biochemical relationship of these precursors to other compounds (acetic-I- and -2-l 4Cacid, hexanoic-I-’ 4Cacid, octanoic-I-, -7-, and -8-I4Cacid) which serve as precursors of coniine, and the pattern of labeling obtained with these compounds
are rationalized. (+)-Coniine-2’-14C and (-)-~oniine-2‘-1~Cwere fed to hemlock, and it was found that the natural (+)isomer was dehydrogenated to y-coniceine much more efficiently than its enantiomorph. By administering IjN-labeled (+)-coniine and y-coniceine to hemlock it was established that the nitrogen in these alkaloids is metabolically stable. A new alkaloid, (*)-conhydrinone (1’-oxoconiine), was isolated from hemlock, and its structure confirmed by synthesis. y-Coniceine was shown to be a precursor of conhydrinone.
W
e discovered in 19632 that coniine (IV) is formed from acetic acid, and suggested that the alkaloids of hemlock (Conium maculutum), which are all related to 2-propylpiperidine, are derived from an eight-carbon polyketo acid I produced by the linear condensation of four acetate units. Thus, the administration of acetate-1- 14C to hemlock yielded radioactive coniine labeled equally on the four even numbered carbons. Although there are many natural products which are acetate derived,* few attempts have been made to isolate or characterize the intermediates between acetic acid and the final natural product. We considered that 5-oxooctanoic acid (11) and 5-oxooctanal (111) would be plausible precursors of y-coniceine (V) which is apparently the first alkaloid to be formed in hemlock.5-7 5-0~ooctanoic-6-1~C acid and 5-oxooctanol-6- 14C were prepared by the following method.* Reaction between propyl-1- 14C magnesium bromide and cyclopentanone yielded 1-propylcyclopentanol, which on oxidation with potassium dichromate in sulfuric acid afforded 5-oxooctanoic-6- 14C acid. Dehydration of the 1-propylcyclopentanol yielded 1-propylcy~lopentene~ which was treated with osmium tetroxide to give l-propylcyclopentane-1,2-diol.Cleavage of this diol with sodium metaperiodate yielded 5-0xooctsna1-6-’~C.10 These compounds were administered to hemlock plants” by means of cotton
wicks inserted into the stems of the plant near ground level. The coniine and y-coniceine isolated from these plants were radioactive, excellent incorporations of activity being obtained (see Table I). Degradations Scheme I
HOOCb
18, 528 (1925). ( 1 0) E. Leete, H.V. Isaacson, and H. D. Durst, J . Label. Compounds, 7,313 (1971). (11) Tracer experiments were carried out with both the “Minnesota” variety of Conium maculatum, which contains coniine as the major
alkaloid, and the “Chelsea” variety which has a larger proportion of yconiceine. We thank Professor J. W. Fairbairn, School of Pharmacy, University of London, for seeds of the latter variety.
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(1) (a) Presented in part at the 162nd National Meeting of the American Chemical Society, Washington D.C., Sept 1971. (b) Based in part on the Ph.D. Thesis of J. 0. O., University of Minnesota, 1971. (2) E. Leete,J. Amer. Chem. Soc., 85,3523 (1963); 86,2509 (1964). (3) One possible exception is 2-methyl-Af-piperideine, which B. T. Cromwell (Biochem. J., 64, 259 (1956)) has claimed, on the basis of
chromatographic evidence, to be a minor alkaloid of hemlock. (4) Cf.J. H. Richards and J. B. Hendrickson, “The Biosynthesis of Steroids, Terpenes and Acetogenins,” W. A. Banjamin, New York, N. Y.,1964. (5) S. M. C. Dietrich and R. 0. Martin, Biochemisfry,8,4163 (1969). (6) J. W. Fairbairn and P. N. Suwal, Phytochemistry, 1, 38 (1961). (7) E. Leete and N. Adityachaudhury, ibid., 6,219 (1967). ( 8 ) A preliminary account of this work has appeared as a communication: E. Leete and J. 0. Olson, Chem. Commun., 1651 (1970). (9) F. Eisenlohr and G. Gorr, Fortschr. Chem. Phys. Phys. Chem.,
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Ix were carried out on these alkaloids as previously de~ c r i b e d , ~and , ~ it was established that essentially all the activity was located at C-l’, indicative of the direct incorporation of these compounds into the alkaloids. The incorporation of 5-oxooctanal-6- 14C into coniine was significantly higher than that of 5-oxooctanoic-614C acid, a result which is consistent with the former
Journal of the American Chemical Society / 94.15 / July 26, 1972
54
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Leete, Olson / Biosynthesis and Metabolism of Hemlock Alkaloids
5474
compound being the immediate precursor of the alkaloids. We suggested l that the 5-oxooctanal undergoes transamination at the aldehyde group yielding 5-oxooctylamine (VI) which then cyclizes nonenzymatically to y-coniceine. Recently, Roberts13 has been able to isolate from hemlock leaves (Chelsea variety) an enzyme which catalyzes a transamination between 5-oxooctanal and L-alanine yielding y-coniceine and pyruvic acid. Prior to our work with 5-oxooctanal we serendipitously discovered that the administration of octanoicl-14C acid to hemlock yielded coniine which had almost all its activity located at C-6.14 The coniine derived from octanoic-7-14C was labeled mostly at C-2’. On the other hand the coniine isolated from hemlock plants one day after feeding octanoic-814Chad only 48 % of its activity at C-3’ with considerable randomization of activity on the other carbons. The randomization of activity was even greater in the coniine isolated 7 days after feeding octanoic-8-14C acid (only 28% at C-3’). We rationalized these apparently paradoxical results in the following way. 1 2 It is considered that the labeled octanoic acids are incorporated intact into coniine via 5-oxooctanoic acid. Evidence in favor of this was obtained by adding nonradioactive 5-oxooctanoic acid to an extract of hemlock plants which had been fed octanoic-l-14C acid. The reisolated 5-oxooctanoic acid was radioactive, and degradation established that essentially all the activity was located on the carboxyl group, indicative of a direct formation from octanoic acid. The labeled octanoic acids are also considered to undergo 0 oxidation affording labeled acetic acid. Thus, octanoic-land -7-14Cacid would yield acetate-l-14C. Entrance of this acetate-l-14C into the Krebs cycle should result in labeling of the compounds in this cycle. This proved to be the case. The fumaric acid isolated from hemlock plants which had been fed octanoic-l-1*C for 1 day was highly radioactive (4.4 % incorporation), with all the activity located on the carboxyl groups (see Table 11). The 0 oxidation of octanoic-8-14C leads Table 11. Incorporation of Activity in Fumaric Acid and Distribution of the Label
Expt no. 1 2 6 7 9 4
AbsoDistribution of lute inc activity in fumaric of acid, % Compd fed activity, CarC-2 (duration of feeding. days) Z boxvls C-3
+
Acetic-l-14C acid (1) Acetic-2-14C acid (1) Octanoic-l-14C acid (1) O ~ t a n o i c - l - ~acid ~ C (7) Octanoic-8-14C acid (1) Hexanoic-I-14C acid (7)
0.91 0.72 4.4 0.94 0.51 0.82
99 35 100 98.5 30 97.9
