BIOSYNTHESIS OF TROPIC ACID
Dec. 5, 19G3
any single process. The simple model, however, does provide a useful qualitative picture of the kin-
[CONTRIBUTION FROM THE
SCHOOL O F
4507
etics of helix formation as a nucleation and growth phenomenon.
CHEMISTRY, UNIVERSITY OF
MINNESOTA, b h W E A P O L I S 14, b1INN.I
Further Studies on the Biosynthesis of Tropic Acid1 B Y h l A R Y L. LOUDEN AND EDWARD LEETE RECEIVED JUNE 25, 1962 The administration of ~-phenylaianine-l-C~* and ~ ~ - p h e n y l a l a n i n e - 2 - Cto 1 ~Datura stramonium plants in separate experiments led to the formation of radioactive hyoscyamine and hyoscine which were labeled solely in their tropic acid moieties. Systematic degradation of the side chain of the radioactive tropic acids indicated t h a t the carboxyl group was derived from the carboxyl group of phenylalanine, while the hydroxymethyl group arose from the a-carbon of the amino acid. Administration of sodium bicarbonate-Cl* to Datura plants resulted in general labeling of the alkaloids. Plausible biogenetic schemes for tropic acid are discussed.
In our previous work on the biosynthesis2 of tropic acid3 (I), ~ ~ - p h e n y l a l a n i n e - 3 -was C ~ ~ administered to two-month old Datura stramonium plants resulting in the formation of radioactive hyoscyamine and hyoscine which were labeled specifically a t C-2 of their tropic acid moieties. This result has been recently confirmed by the work of Underhill and Y o ~ n g k e n . ~ We had originally considered that the hydroxymethyl group (C-2) of tropic acid was derived from a onecarbon source. However, the administration of sodium formate-C14, forma1dehyde-Cl4, methionine-methyl-CI4 or serine-3-CI4 to D. stramonium plants did not lead to tropic acid labeled a t C-2. We thus shelved the problem of the origin of the hydroxymethyl group and fed phenylalanine-2CI4 to three-month old Datura plants, fully expecting to obtain tropic acid which would be labeled on its carboxyl group. Radioactive tropic acid was indeed obtained and the degradative scheme which was used to determine the activities 2
3
PhCHCHgOH
I
+PhC=CH2 +PhC=K’OH + H C H O I
I
ICOOH
COOH I1
I
1
COOH
JT
PhKHz C- PhCOOH t P h C N
I’hNIlCOPh
COY--+
Fig. 1.-Dcgradatire
BaC03
J.
Dimedone derivative
[C-3]
COz
J.
BaC03 [C-21 [C-11 sclierne for the radioactive tropic acid.
( 1 ) P a r t of this work has been published in preliminary communications: E. Leete and hl. L. Louden, Clzemisfry E* Industry, 1405 (1961); hl. L. Louden a n d E. Leete, J . A m . Chem. S O L , 84, 1510 (19%‘). This investigation was supported by a research grant, MY2662 from t h e National Institute of Mental Health, U.S.Public Health Service. (2) We accept with enthusiasm t h e suggestion of Rapoport (F. R. Stermitz a n d H. Rapoport, J . A m . Chem. SOL., 83, 4045 (19fjl)) t h a t t h e word biosynthesis should describe in vivo experimental studies on t h e mode of formation of natural products, whilst t h e word biogenesis should be applied t o the hypothetical schemes which organic chemists are so fond of creating t o rationalize t h e formation and structures of natural products. (3) E. Leete, ibid., 81, G12 (1900). (4) E. JV. Underhill and H. W.Youngken, J . Pharm. Sci., 61, 121 (1Dl\2). ( 5 ) L. Marion and A. F. Thomas, Can. J . Chem., 32, 1 1 1 G (195i).
on the side chain carbons of tropic acid is illustrated in Fig. 1. Dehydration of tropic acid by refluxing with concentrated aqueous potassium hy&oxide6 yielded atropic acid (11) which was oxidized in alkaline solution with sodium metaperiodate and a catalytic amount of osmium tetroxide yielding formaldehyde collected as its dimedone derivative, and phenylglyoxylic acid isolated as its oxime (111). This oximino acid was refluxed in water yielding benzoiiitrile and carbon dioxide,7 collected as barium carbonate. The benzonitrile was hydrolyzed with potassium hydroxide yielding benzoic acid which was subjected to the Schmidt reaction affording aniline, assayed as its benzoyl derivative, and carbon dioxide, collected as barium carbonate. We were agreeably surprised to discover that the tropic acid derived from phenylalanine-2-C1 was labeled solely on the hydroxymethyl group (cf. Table I). While OUT work was in progress, Goodeve and Ramstad6 reported that the administration of tryptophan-3-CI4 to D. stramonium yielded radioactive hyoscyamine which was labeled specifically on the carboxyl group of its tropic acid moiety. This was an astonishing result and difficult to reconcile with our observations. It seemed unlikely that tropic acid was being formed in the same plant by two quite different biosynthetic mechanisms, and we interpreted their work as follows. The radioactive tryptophan could undergo metabolic breakdown in the plant yielding some radioactive carbon dioxide. This could then participate in a carboxylation reaction with a metabolite derived from phenylalanine leading to carboxyl labeled tropic acid. We attempted to test this hypothesis by feeding sodium bicarbonateC14 to Datura plants in the hope that we would obtain preferential incorporation of carbon- 14 into the carboxyl group of tropic acid. Radioactive hyoscyamine and hyoscine were obtained. However, assay of the degradation products of the alkaloids ( c j . Table I) indicated that the distribution of activity between the tropic acid and the tropane bases was almost proportional to the carbon content of the two halves of the alkaloids. The distribution of activity in the side chain of (C) J. E’.Baker and A. Eccles, J . Chem. Soc., 212.5 (1927). (7) -4.AhmaB and I. D. Syenser, C U J IJ. . C k n . , 39, 1310 (1961). ( 8 ) A. M. Goodeve and E. Ramstad, Experiemfia, 17, 12-1 (l9til).
~ I A RL. I ' LOUDEN A N D EDWARD LEETE
430s
the tropic acid was not completely uniform. However, there was not a significantly larger amount of activity located in the carboxyl group. For some reason, the distribution of activity in the side chain of the tropic acids derived from hyoscyamine and hyoscine was not the same, a more uniform distribution being present in the tropic acid from the latter alkaloid. \Ye then considered that the branched side chain of tropic acid might arise by a rearrangement of the phenylalanine side chain. This possibility was tested by feeding L-phenylalanine-l-C14 to Datura plants. The incorporationg of tracer into the alkaloids was relatively high (0.23%) and degradation showed that the tropic acid was labeled solely on the carboxyl group. This significant result may indicate that an intramolecular rearrangement of the side chain of phenylalanine is occurring (reaction A, Fig. 2 . ) . Alternatively, oxidative cleavage of the side chain could take place (reaction B) yielding carbon dioxide (perhaps attached to an enzyme or coenzyme such as biotin) and a CS-C, unit such as phenylacetaldehyde. Carboxylation of the phenylacetaldehyde, or related metabolite, with the carbon dioxide complex (reaction C) followed by reduction of the aldehyde function would yield tropic acid. If reaction B does occur, one could claim that the radioactive carbon dioxide derived from phenylalanine-l-C1' never got very far away from the C8-C2 unit, and as a result never reached the photosynthetic cycle with consequent genera labeling.
---. ' PhCH2-CH-t:OOH
COOH
I
W I
PhCHCH20H
I
1
"2
A
COOH I
I'h('€IpC"O -1- C o r -complex A PhCHCHO Fig. 2.--Plausible biogenetic pathways from phenylalanine t o tropic acid.
The recent results of Underhill and Youngken4 favor the participation of a C6-Cs compound in the biosynthesis of tropic acid. They found that phenylacetic acid- l-C14 was a precursor of tropic acid. Furthermore they found that the tropic acid was not labeled on the carboxyl group. Unfortunately, they did not carry out a more extensive degradation of the radioactive tropic acid. I t should be mentioned that the plants used in the present investigation were between two and three months old, and in all cases both the hyoscyamine and the hyoscine became labeled, indicating that both alkaloids were being synthesized in plants of this age. This result is in agreement with earlier studies.IO
Experimental Administration of Tracers to D. stientonium plants and Isolation of the Alkaloids .-The tracers were administered
Vol. 54
to the plants which Irere gruivitig iii soil in d by means of a cotton wick inserted through the stems with a n English sewing needle. The alkaloids r+ere extracted from the plants without dilution b y methods previously described,3J0 two weeks after administration of the tracers. Details of the amount of tracers fed, and activities of the alkaloids are recorded in Table I . T h e radioactive alkaloids Rere diluted prior to degradation.
TABLE I
Tracer ied Wt., m.g Activity,'S mc. h l o n t h of expt. S o of plants Fresh wt. of plants, g , Act. of aq. sap (70of total fed)
o~-PhenyIaIanine-Z-C'( hydrochloride12
L-Phenylalanine- 1C14 1 3
36.4 0.1 May 20 409
1.21 Dec. 10 109
10
7
Sodium bicarbonate-C" 1 4
24.8 1.0 June 20 418
0.05
:!I
Hyosc)-amiiic atid its degradation produc t s (ilct. ind.p.m. / d l .) Incorporation, r z Hyoscyamine, HCI Tropine picrate Tropic acid Formaldehyde-dimedone Phenylglyoxylic acid oxime Barium carbonate (from oxime) Benzoic acid BaC03 (from benzoic acid) S-Phenylbenzamide
0.046
0.005
1.5 X 106 0.00 1 . 3 X 100 1 . 6 X 100
0.11 3 . 6 X 106 0.00 3 . 6 X 108 0.00
5.1 2.1 3.1 0.5 X
x x
x
10; 105 105 106
0 . 0 3 X 10,
3 . 4 X 100
2.5
x
10;
X 106
0.5 2.0
x
x
105
3.:
0 00
0.1 2.1
x
x
10: 106 105
Hyoscine and its degradation products Incorporation, io Hyoscine.HC1 Oscine picrate Tropic acid Formaldehyde-dimedone Phenylglyoxylic acid oxime BaC03 (from oxime) Benzoic acid BaCOa (from benzoic acid)
0,022 4.7 10' 0.00 4.8 10; 4 . 3 X 105
x x
0.13 3 . 0 x 100 0.00 i3.0 1oc
x
0.00
x x
3.0 3.1 0.00
in0
100
0,003 4.2 1oj 1 7 x 105 2.6 X 1 0 5 0.2 10"
x
x
3.2 0.3 1.6
x x
x
10' 10' 10'
0 2 x 10s S o t isolated, or degradation i i r i t carried out to this stagc because of uegligible activitj- or lack of 1n:Lterial.
Degradation of the Radioactive Alkaloids. ( a ) Hydrolysis.-The following typical procedure gave inore reliablc results than the method previously described.3 Hyiisci~ic hydrochloride (570 nig.) was refluxed with 352 barium hydroxide solution (21 ml.) in a nitrogen atmosphere for 45 min. The cooled solution was acidified with hydrochloric acid and extracted with ether. T h e residue obtaiued 0 1 1 evaporation of the dried ether extract was crystallized from a mixture of benzene and petroleum ether ( b .p. 60--70") yielding tropic acid (219 mg., 815,). m.p. l16.r5-l18'. The solution from which t h e tropic acid had been extractctl was made alkaline with potassium hydroxide and extracted with ether yielding oscine which was purified by subliination in ' I * ( I C U O ; 102 mg., 395;, m.p. 108-111". The picrate crystallized from ethanol without dificulty (6. ref. 4), 111.1). 237-240". Hyoscyamine was hydrolyzed with dilute aqueous methanolic potassium hydroxide yielding tropine and tropic acid. (I 1) We thank Robert C. McLeester of the Botany Department of t h e University of Minnesota for the cultivation of t h e D a t w a plants. (12) Purchased from Tracerlab, Inc., Waltham, Mass. (13) Purchased from S i c h e m , Inc., Bethesda, hId. T h e L-isomer was fed since the m-phenylalanine-l-C1J wac, not commercially available. ( 1 4 ) Purchased irom Iwtupes Specialties C C I, Inc., Burbank, Calif. (I.->) liadicmcti\.ity measurements were carried o u t in a SuclearChicagi, model C-1 13 lc,w hackgruund 0 Eas flr,w coiinter. 1)etcriiiinati
