Biosynthesis and Metabolism of the Tropane Alkaloids

Home , Cuscohygrine, Littorine, Tropine

Journal of Medicinal Plant Research Editor - in - Chief Editorial Board Hippokrates Verlag E. Reinhard, Univ. Tubingen H. Ammon, Tubingen Stuttgart Pharmazeutisches lnstitut W. Barz. Munster Auf der Morgenstelle E. Reinhard, Tubingen 0. 7400 Tubingen Sticher, Ziirich Vol. 36 H. Wagner, Munchen M. Zenk, Bochurn June 1979 NO.2

Edward Leete .

Natural Products Laboratory, School of Chemistry, University of Minnesota.

Key Word Index: Tropane Alkaloids; Biosynthesis; Metabolism;'Tropic'Acid; Tiglic Acid; Hydroxylation of Tropine.

Abstract Introduction This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

The origin of both the basic (tropane The tropane alkaloids are characteri- derivatives) and acidic (tropic acid, tig- zed by the presence in their molecules of lic acid, benzoic acid) moieties of these the 8-azabicyclo [3.2.1] octane ring sy- ester alkaloids is reviewed. Subsequent stem (I). Its N-methyl derivative is tri- metabolism (mostly by oxidation reac- vially known as tropane: Many of the tions) of the tropine half of these alka- alkaloids are derivatives of tropine (11) loids is discussed. which is tropan-3a-01. Tropine, having 98 Leete

I Tropine I1

"j_ , . qH .:

o\ C-CH-Ph O\C-CHCH~-P~ II I II I 0 CH20H 0 OH

Hyoscyamint Ill Scopolamine IV Littorint V

H!, H!, ,Me H//C o\c/C II 'Me 0 0

Meteloidine V1 Cocaine VII Cochlearine Vlll

Bellendine R:H IX 2P -Btnzyl-3P-acttoxy- Ferrugine R=Me X 2a-Benzoyltropane XI tropane XI1

' Fig. I. Tropa.ne Alkaloids.

a plane of symmetry, is optically inac- are illustrated in Fig. 1. Hyoscyamine This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. tive, however it should be noted that (111) and scopolamine (also known as C-1 (as depicted in 11) has the R-confi- hyoscine) (IV) are found in a large num- guration, and C-5 has the S-configura- ber of Datura species. Atropine is the tion; . and these two positions can be racemic form of hyoscyamine, and race- different with respect to an enzyme. mization of hyoscyamine usually occurs Some of the alkaloids whose biosyn- during its isolation from plants. On the thesis will be discussed in this.review other hand scopolamine seems to be Biosynthesis and Metabolism of Tropane Alkaloids 99

much more resistant to racemization. mine and scoplamine with little change Recently discovered alkaloids contain- in the 14C: 15N ratio, while [2-14C, ing the tropane ring system include bel- a-'5NIornithine yielded tropine con- lendine (IX) [57], and ferrugine (X) taining much less 15N than 14C. These [lo] containing a fused y-pyrone ring. results suggest that it is the &amino Alkaloids (XI) and (XII) are novel in group of ornithine which is a precursor having a benzoyl or benzyl group at of the tropane nitrogen. However the C-2 [52, 531. Extensive work involving calculated '4C specific incorporations labelled precursors has been carried out reported in this publication are too high on both the tropane half of these alka- by a factor of 10. If these results are - loids, and the various acids (tropic, real they indicate that the d-amino tiglic, benzoic) which are present in group of ornithine is ten times more these alkaloids esterified to tropane al- efficient as a source of the tropane nit- cohols. rogen atom, 'than is the carbon skeleton of ornithine a source of the carbons of the pyrrolidine ring. I Origin of the Tropane Ring System believe that extensive loss of 15N from the amino groups'of ornithine occurs by Ornithine, and related amino acids transamination reactions during the fee- (glutamic acid, proline) are precursors ding experiments, all of which involved of the pyrrolidine ring.of tropine [26, root cultures. The incorporation of gln- 27, 39, 48, 491 Ornithine is incorpora- tamic acid and proline is considered to ted-intothe tropane ring system unsym- occur via ornithine, and the metabolic metrically. It was found that tropine relationship of these amino acids is illu- (present in hyoscyamine) derived from strated in Fig. 2. SPENSER[55] has [2-14C]ornithine was labelled only at shown that ornithine is converted to the C-1 bridgehead carbon. On the proline via a-keto-8-amino valeric acid other hand [5-'4C]proline labels only in Datura species, and presumably th,is the C-5 position of tropine [49]. Work reaction is reversible. on the origin of the nitrogen in tropine Figure 3 illustrates how ornithine is is equivocal. We [32] found that both converted to tropine. We presently con- amino groups OP ornithine were appar- sider that ornithine is incorporated via 'ently incorporated intohyoscyamineand 8-N-methylornithine (XIV) [I, 2, 31, scopolamineproduced in a root culture of although it must be admitted that this

Atropa belladonna. However the speci- amino acid has not yet been detected in This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. fic incorporation of 14C(located at C-2 Datura or related species. Earlier work 'of ornithine) was about three times [58] which .apparently implicated a-N- higher than the nitrogen (located at the methylornithine rather than the 8-iso- a- or 8-positions). SPENSER[68] found mer as a precursor of tropine, was dis- similar results. On the other hand LIE- counted when it was shown [2O] that BI~CHand SCHUTTE[49] reported that these authors were using non-authentic [2-14C,d-15Nlornithine was incorpora- a- and S-N-methylornithines in their ted into the tropane moiety of hyoscya- tracer experiments. It was shown that 100 . Leete

C L HOOC

Glutarnic acid Glutarnic -y-semialdehyde Ornithinc

0- HOOCm= N HOOC N H

Proline A'-Pyrrolinc-2-carboxylic a-~eto-6-arninovalcric acid acid Fig. 2. Metabolic Relationship of Glutamic Acid, Ornichine and Proline.

COOH COOH cN42 - cNH2 NH2 NHMe NHMe NH2

Ornilhine Xlll

4-Methylaminbbulaml XIX

Tropine (+) -(2R)-Hygrinc Dehydrohygrinc Tropinone This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. XX XXI XXll 11 Fig. 3. ~etabolisrnof Ornithine to Tropine.

[methyl-14C]-6-N-methyl-[2-14C]~~rni-positions indicated that the 6-N-methyl- thine was incorporated into hyoscya- ornithine was incorporated without any mine labelling C-1 and the N-methyl cleavage of the N-methyl group. The group. The ratio of activity at these two next step is a decarboxylation to yield Biosynthesis and Metabolism of Tropane Alkaloids 101

N-methylputrescine (XV) an establi- rine (XX) an established precursor of shed precursor of tropine [45, 471. Pu- tropine [46, 591. It has been recently trescine (XVI) has .also been shown to shown [54] that it is the (+)-(2R)-hyg- be a precursor of the tropane alkaloids rine (XX) enantiomer which serves as a [23, 36, 48, 501. However putrescine precursor of the tropane alkaloids of cannot be a free intermediate between Datura innoxia. The incorporation of ornithine and the tropane skeleton, (-)-(2s)-hygrine, although lower than ' since such a symmetrical intermediate its 2R isomer (3.7 to 10.7'times less effi- would result in the formation of tro- cient), was never-the-less significant. pine labelled equally at C-1 and C-5 The incorporation of the 2s-isomer after the administration of [2-l4C]orni- could be rationalized by suggesting that thine. We suggest that the utilmization of some racemization occurred during -the putrescine represents an "aberrant" bio- feeding period (7 days). Racemization synthesis. By this we mean that putres- occurs readily in neutral or basic solu- cine is not part of the biosynthetic path- tion by the mechanism illustrated in way, however the plant contains enzy- Fig. 4. It is of interest to note that (2R)- mes which are capable.of converting the hygrine is formed by an attack of the administered putrescine to N-methyl- acetoacetate on the pyrrolinium salt on putrescine. Oxidation of the primary the opposite side from the one involved amino. group of (XV) affords 4-methyl- in the formation of natural (-)-2's)- aminobutanal (XIX), which was detec- nicotine (XXIII). This result is illustrated 'in Datura plants which had been ted in Fig. 5. Final steps to yield tropi- fed [2-'4C]6rnithine [56]. The cyclized none (XXII) are considered to involve form of this amino aldehyde is the N- the hypothet.ica1 intermediate dehydro- methyl-A'-pyrrolinium salt (XVIII). hygrine (XXI). Tropinone has been iso- Carbons 2, 3, and 4 of tropine are deri- lated from Cyphomandra species [16]. ved from acetic acid [24, 461 and it is Stereospecific reduction of tropinone assumed that the acetate is incorporated yields tropine. via acetoacetic acid or some suitably It is instructive to follow the biosyn- activated derivative such as its coenzy- thetic pathways whereby [l-14C]acetate me A ester. The result of such a conden- is incorporated into tropine (Figure 6). sation between (XVIII) and acetoace- It has been shown that glutamic acid tate is hygrine-a-carboxylic acid (XVII) isolated from wheat plants was labelled which on decarboxylation affords hyg- mostly at C-1 and C-5 [Ill. This result This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Fig. 4. Mechanism for:the Racernization of Hygrine. 102 Leete

Nicotinic acid

\ COOH

\ Me Me

Fig. J. Nucleophilic Attack of N-Methyl-dl-pyrrolinium Salt from the a- and p-face.

CH~EOOH CH~~OOH ~H~~OOH PCOOH I I . O=C-COOH - HO-C-COOH +++ CH2 CH~~OOH O=C-COOH I I I,- ---- I - I. CH2COOH CH2COOH 04C t:OOH CH2COOH CHZCOOH

Oxaloacctalc Citratc a-K~togIuIarate Succinale

. . ,COOH CHzCOOH CH2COOH CH~~OOH I I I. . HO-C-COOH /c;-cH\ .NH2 CH2 . - CH2 - CH2-CH2 1. 1 I 'NH~ ,CH-COOH ,C-COOH CH~EOOH H2N 0' This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Tropine Fig. 6. The Incorporation of [1-14C]Acetate into Tropine. Biosynthesis and Metabolism of Tropane Alkaloids 103 is in accord with the expected incor- [46] yielded radioactive cocaine. Ho- poration of acetate into the Krebs cycle. wever most of the activity (- 60b/o) The ornithine derived from this gluta- was apparently located on the ester me- mate would be labelled at C-1 and C-5. thyl 'group. Another 30 O/o was present Incorporation of this ornithine as pre- on the carbonyl group of the benzoyl viously described yields tropine label- residue, and only small amounts were led at the C-5 bridgehead position with present in the tropane ring system. We no label at C-1 [12]. Labelling at C-3 [42] and others [69] have also failed to results from the incorporation of the observe the incorporation of likely [I ,3-i4C]acetoacetate, and the higher precursors (putrescine, ornithine, N- level of activity found at this position methyl-dl-pyrrolinium salt) into the (81 010) is in accord with the administer- pyrrolidine ring of cocaine. ed acetate being closer biochemically to Reports [25] that labelled succinic this part of the molecule. acid was incorporated into hyoscyami- It seems reasonable to propose that ne, led BARELLEand GROS[3] to repeat the tropane moiety of cocaine (VII), this experiment, with the finding that ie. ecgonine (XXIV) is produced by a non-specific labelling occurred. slight modification of the biosynthetic The tropane alkaloids having a C,- route illustrated in F.ig. 3, whereby the C, group attached to C-2 of the tropane carboxyl group of hygrine-a-carboxylic molecule are plausibly derived by reac- acid (XVII) is retained, as illustrated in tion of the N-methyl-dl-pyrrolinium Fig. 7. However no definitive results salt with a poly-ketide having benzoic have been obtained on.the biosynthais acid as its starter unit, as illustrated of the tropane moiety of this important schematically in Fig. 8. This Figure also medicinal agent. The administration of depicts the formation of bellendine (IX) [l-14C]acetate to Erythroxylon coca from the pyrrolinium salt and a poly-

COOH COOH This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

Cocaine Ecgonine VII XXIV Fig. 7. Hypothetical Biosynthesis of Cocaine. 104 Leete

Ph Ph

COOH

NHMe

4:-Ph0 qcn2;0-C-Me H

Bellendine IX

Fig. 8. Hypothetical Biosynthesis of Some Novel Tropane Alkaloids. acetyl chain which has undergone C- 3a-tigloyloxytropan-7B-01 (XXVII). methylation (from methionine) at a rea- One problem in studying the origin of sonable position (between two carbonyl these tigloyloxytropanes is the facile groups). hydrolysis which these compounds un- dergo in the plant in the course of feeding experiments. The dihydroxylation Oxidation of the Tropane skeleton which occurs at C-6 and C-7 in the formation of meteloidine proceeds with It was shown that the pyrrolidine retention of configuration [38]. This re- ring of meteloidine (VI) is derived from sult is typical of direct hydroxylations ornithine [41] and the hydroxyl groups at saturated carbon atoms [7], and was 6 at the and 7-positions are apparently established by. labelling tropine stereo- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. introduced at a late stage in the biosyn- specifically wtih tritium at the 68 and thetic sequence, after formation of the 7B-positions. tropine ring system [28]. Extensive A related oxidation of the tropine work by WOOLLEYand coworkers [6, ring system occurs in the formation of 8, 91 and ourselves [37] indicate that scopolamine. Long ago it was esta,bli- meteloidine is formed by the hydroxy- shed that this alkaloid is a metabolite lation of 3a-tigloyloxytropane (XXV) of hyoscyamine [19, 63, 64, 65, 661, (also known as tigloidine), probably via and the reaction proceeds via 6-hydro- Biosynthesis and Metabolism of Tropane Alkaloids 105

Tmpine I1 3a-Tigloyloxytropane XXV

H 0 Tig 0 Tig

XXVlll Meteloidine V1

Fig. 9. Biosynthesis of Some Tigloyloxytropanes.

H H 0 Tropyl 0 Tropyl

Hyoscyamine 111 6-Hydroxyhyoxyamine XXX XXXI This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.

6q G H Enyme-S 0 Tropyl - 0 Tropyl qH0 Tropyl Scopolamine 1V . 6,7-Dehydrohyoscyomine -XXXll XXXlll Fig. 10. Metabolism of Hyoscyamine to Scopolamine. . Leete xyhyoscyamine (XXX) and 6,7-dehy- scopolamine revealed the presence of drohyoscyamine (XXXII). This overall satellite peaks, due to spin-spin coupling reaction also involved the loss .of both of contiguous 13C atoms which arose by the ,B-hydrogens at C-6 and C-7 [38]. a 1,2-intramolecular rearrangement. This result indicates that the conver- The mechanism of this rearrangement sion of 6-hydroxyhyoscyamine to the is unknown. Compounds which are me- dehydro-compound (XXXII) involves tabolically related to phenylalanine, a cis-dehydration. This is unusual, but such as phenylpyruvic acid (XXXV) not without precedence [13]. We sug- and phenyllactic acid (XXXIV) are gest that the dehydration takes place by also incorporated into tropic acid [17, a two step mechanism illustrated in Fig. 331. Phenyllactic acid is esterified with 10. A phosphorylated 6-hydroxyhyos- tropine in the alkaloid littorine (V) and cyamine (XXXI) could be attacked by its formation from phenylalanine has a nucleophilic reagent (represented here been established [I 83. When phenylala- by the SH-group of an enzyme) to af- nine was labelled with tritium in its side ford (XXXIII). Then the double bond chain it was found [67] that the tritium would be generated by a trans-elimina- at C-2 was not incorporated into tropic tion. acid, however one of the tritiums at C-3 was retained in the ultimate tropic acid [43]. DALTON[15] has proposed a Origin of the Acid Moieties of the reasonable hypothesis for this rearran- Alkaloids gement, and it is reproduced in Fig. 11. Tropic Acid and Related Aromatic One experimental fact which this sche- Acids me does not take into account is the fin- Tropic acid having the (S)-configura- ding [31] that 2-formylphenylacetic - tion (XXXRIII) is found in the alka- acid (XXXVII) failed to serve as a pre- loids hyoscyamine and scopolamine. Its cursor of tropic acid in Datura. biosynthesis has been the subject of ex- The same rearrangement of the phe- tensive investigations by several groups nylalanine side chain is involved in the in the past 18 years. This work has biosynthesis of the fungal product ten- been reviewed [29] and I will confine ellin (XL). The intramolecular nature my remarks to recent developments. of this reaction was also established by Tropic acid is formed by the rearrange- means of I3C NMR [35]. 2-Formylphe- ment of the side chain of phenylalanine nylacetic acid, or its coenzyme A ester (XXXVI) and it has been shown that (IXL) would be a plausible interme- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. this rearrangement is intramolecular diate to condense with a polyketide [MI. This important result was obtain- chain derived from acetate and the me- ed by labelling phenylalanine at C-1 thyl groups of methionine as illustrated and C-3 with carbon-13, so that the ad- in Fig. 12. Hydroxylation of the phenyl ministered amino acid contained 81°/o ring apparently occurs at a late stage in of the doubly labelled species. Exami- the biosynthesis. nation of the ISC NMR spectra of the Despite claims to the contrary [22, resultant labelled hyoscyamine and 701 we [34] have been unable to sub- Biosynthesis and Metabolism of Tropane Alkaloids 107

COOH ,COOH . COOH / Ph-CH - Ph-CH - ~h-c-H \ \ CH CHO CH20H __--II--..

N 2-Formylphenyl- (S)-Tropic acid This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. I acetic acid XXXVll

Fig. 11. Hypothetical Memanism for the Formation of Tropic Acid from Phenylalanine. 108 Leete

5 MeCOOH

0000 ------+ I ---___ Me CHO

Mes~NH2COOH

XXX\Ill Tenellin XXXX Fig. 12. B'iosynthesis of Tenellin. stantiate the incorporation of phenyl- The benzoyl moiety of cocaine is acetic acid into tropic acid. Several derived from phenylalanine [21], by groups [17, 431 have reported that cin- cleavage of the side chain between C-2 namic acid does not serve as a precursor and C-3. The formation of benzoate by of tropic acid. We also found that cin- the ,&oxidation of cinnamic acid (for- namoyltropine is not converted directly med from phenylalanine by the action to the tropane alkaloids of atb bra stra- of the enzyme phenylalanine-ammoni- monium. The results obtained [33] indi- alyase) has been established by ZENK cated that this ester was hydrolysed in [73]. A C,-C, fragment derived from the plant to tropine and cinnamic acid, phenylalanine is also involved in the only the former compound being incor- biosynthesis of ephedrine [72]. porated into hyoscyamine and scopol- The m-hydroxybenzoic acid residue amine. However it has been recently re- found in the alkaloid cochlearine (VIII), Fig. 1 is also formed from phenylalani- ported [61.] that [2-i4C]cinnamic acid ne, apparently via m-tyrosine [44]. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. yielded hyoscyamine which was labelled specifically on the hydroxymethyl group Tiglic Acid of tropic acid. However I have grave This compound which is the acid doubts about the validity of this obser- moiety of meteloidine and several other vation, especially after examination of alkaloids is formed from L-isoleucine the dissertation [60] from which the (XLI) [40, 711 in Datura species, and published results were ~resurnabl~ob- current evidence suggests that the path- tained. way whereby it is formed is similar to Biosynthesis and Metabolism of Tropane Alkaloids 109

Me'coo,

Angelic acid (+)-(2S)-2-Methyl- Tiglic acid butanoic acid XXXXIlI XXXXIV . xxxxv -

Nbte

0 CH20C-C-C-OH Me I' H-C-OHI \ Me 0 I Me I I Heliosupine 0

XXXXVl Fig. 13. Biosynthesis of Tiglic and Angelic Acids. the one previously established in animal A novel aliphatic acid, 1,2-dithiolane- systems (pig heart and rat liver) [62]. 3-carboxylic acid is present in the alka- The immediate precursor of tiglic acid loid (XLVII) which was isolated from is 2-methylbutanoic acid [5, 301 and it th,e tropical mangrove Bruguiera sexan- seems probable (although this has not gula [51]. No work has been carried out been proven) that it is the (2s)-enantio- on its bios~nthesis,however its forma- mer (XLIV) which is involved. Another tion from butanoic acid would seem point of interest, is which hydrogen (the reasonable, analogous to the formation pro-R or pro-S) is lost from C-3 in the of lipoic acid from octanoic acid. final dehydrogenation to yield tiglic This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. acid. Angelic acid (XLIII), the geome- tric isomer of tiglic acid, is present as an Acknowledgments ester in the alkaloid heliosupine (XLVI), and CROUT[I41 has shown that it is also formed from isoleucine. BA~EYand Our work described in this review (Contri- bution No. 158) was supported by a research WOOLLEY[5] found that angelic acid grant, GM-13246 from the National Institutes was not isomerized to tiglic acid in Da- of Health, U. S. Public Health Service. I de- tura innoxia. dicate this paper to Leo Marion with whom I Leete started work on the biosynthesis of the tropane and threonine arising from acetate-1-1% alkaloids some 25 years ago at the National and -2-"C. ibid., 35, 365-371 (1957). Research Council of Canada. 12. Bothner-By, A. A., R. S. Schutz, R. F. Dawson and M. L. Solt: The asymmetric incorporation of isotopic label in the bio- References genesis of hyoscyamine. J. Am. Chem. Soc. 84, 52 (1962). 1. Ahmad, A. and E. Leete: Biosynthesis of 13. Butler, J. R., W. L. Alworth and M. J. the tropine moiety of hyoscyamine from Nugent: Mechanism of dehydroquinase ca- 6-N-methylornithine. Phytochemistry 9, talyzed dehydration. I. Formation of a 2345 (1970). Schiff base intermediate. J. Am. Chem. Soc. 2. Baralle, F. E. and E. G. Gros: Biosynthesis 96, 1617 (1974). of cuscohygrine and hyosciamine in Atropa 14. Crout, D. H. G.: Pyrrolizidine alkaloids. belladonna from DL-a-N-methyl-[sH]orni- Biosynthesis of the angelate component of thine and DL-6-N-methyl-[3H]ornithine. heliosupine. J. Chem. Soc. (C) 1233 (1967). Chem. Commun. 721 (1969). 15. Dalton, .D.: "An Alkaloid 'Primern, New 3. Baralle, F. E. and E. G. Gros: Biosynthesis York 1978, Marcel Dekker. of cuscohygrine and hyosciamine in Atropa 16. Evans, W. C., A. Ghani and V. A. Wool- belladonna from DL-a-N-methyl-$H-orni- ley: Alkaloids of Cyphomandra betacea thine, DL-6-N-methylornithine and succi- SENDT.J. C. S. Perkin I. 2017 (1972). nic acid-1,4-"C. Anales Asoc. Quim. Ar- 17. Evans, W. C. and J. G. Woolley: Biosyn- gentina 18, 299 (1970). thesis of C,-C, acids in Datura innoxia. 4. Basey, K. and J. G. Woolley: Biosynthesis Phytochemistry 15, 287 (1976). of the tigloyl esters in Datura: The role of 18. Evans, W. C. and V. A. Woolley: Biosyn- 2-methyl butyric acid. Phytochemistry 12, thesis of the (+)-2-hydroxy-3-phenylpro- 2197 (1973). pionic acid moiety of littorine in Datura 5. Basey, K. and J. G. Woolley: Biosynthesis sanguinea and Anthoceucis littorea. Phyto- of the tigloyl esters of Datura: Cis-trans chemistry 8, 2183 (1969). isomerism. Phytochemistry 12, 2883 (1973). 19. Fodor, G., A. Romeike, G. Janzso and I. 6. Basey, K. and J. G. Woolley: Biosynthesis Koczor: Epoxidation experiments in vivo of ditigloyl esters of di- and tri-hydroxy with dehydrohyoscyamine and related com- tropanes in Datura. Phytochemistry 14, pounds. Tetrahedron Lett. No. 7, 19 (1959). 2201 (1975). 20. Gilbertson, and Leete: Biosynthesis 7. Bentley, R., in "Molecular Asymmetry in T. J. E. of the Nicotiana alkaloids. XII. The in- Biology", Vol. 2, p. 259, 274, 1970 Aca- demic Press. corporation of alpha and delta-N-methyl- 8. Beresford, P. J. and J. G.,Woolley: Bio- ornithine into the pyrrolidine ring of nico- synthesis of 3a,68-ditigIoyloxy tropane and tine. J. Am. Chem. Soc. 89, 7085 (1967). 3a,6,8-ditigloyloxytropan-78-01in Datura. '21. Gross, D. and H. R. Schiitte: Phenylalanin Phytochemistry 14, 2205 (1975). als Vorstufe von Tropasaure und Benzoe-. 9. Beresford, P. J. and J. G. Woolley: Compe- saure. Arch. Pharm. 296, 1 (1963). titive feeding experiments with tropine in 22. Hamon, N. W. and J. L. Eyolfson: Biosyn- Datura. Phytochemistry 14, 2209 (1975). thesis of tropic acid in Datura innoxia root This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. 10. Bick, R. C., J. W. Gillard and M. Wood- tissue. J. Pharm. Sci. 61, 2006 (1972). ruff: Ferrugine: a novel tropane alkaloid. 23. Kaczkowski, J. and L. Marion: The incor- Chem. and Ind. (London) 794 (1975). poration of putrescine into hyoscyamine. 11. Bilinski, E. and W. B. McConnell: Studies Can. J. Chem. 41, 2651 (1963). on wheat plants using carbon-14 compounds. 24. Kaczkowski, J., H. R. Schiitte and K. Mo- 111. The utilization of acetate for amino thes: Die Rolle des Acetats in der Biosyn- acid biosynthesis. Can. J. Biochem. Physiol. these der Tropanalkaloide. Biochim. Bio- 35, 357-363 (1957); IV. Distribution of phys. Acta 46, 588 (1961). carbon-14 in glutamic acid, aspartic acid, 25. Kalantyr, M. S. and Y. B. Tikhonov: Suc- Biosynthesis and Metabolism of Tropane Alkaloids 111

cinic acid as a precursor of tropane alka- 38. Leete, E. and D'. H. Lucast: Loss of tritium loids. Khim. Prir. Soedin 3, 395 (1967). during the biosynthesis of meteloidine and 26. Leete, E.: The stereospecific incorporation scopolamine from [N-methyl-"C,6p,7p- of ornithine into the tropine moiety of SH,]tropine. Tetrahedron Lett. 3401 (1976). hyoscyamine. J. Am. Chem. Soc. 84, 55 39. Jeete, E., L; Marion and ,I. D. Spenser: (1962). The biogenesk of alkaloids. XII. The mode 27. Leete, E.: Biosynthesis of hyoscyamine. of formation of the tropine base of hyos- Proof that ornithine -2-"C yields tropine cyamine. Can. J. Chem. 32, 1116 (1954). labelled at C-1. Tetrahedron Lett. 1619 40. Leete, E. and J. B. Murrill: Biosynthesis of (1964). the tiglic acid moiety of meteloidine in 28. Leete, E.: Biosynthesis of meteloidine. Phy- Datura meteloides. Tetrahedron Lett. 1727 tochemistry 11, 1713 (1972). (1967). 29. Leete, E.: Biosynthesis of tropic acid, in T. 41. Leete, E. and S. J. Nelson: Biosynthesis of A. Geissrnan (ed.): Biosynthesis, Specialist teloidine moiety of meteloidine in Datura Periodical Report, Chemical Society, Vol. meteloides. Phytochemistry 8, 413 (1969). 2, pp. 115 (1973). 42. Leete, E. and M. L. Yu: Unpublished work 30. Leete, E.: Biosynthetic conversion of a-rne- (1978) on the biosynthesis of cocaine invol- thylbutyric acid to tiglic acid in Datura ving feedings of [2-14C]-ornithine and [2- meteloides. Phytochemistry 12, 2203 (1973). 14C]-N-methyI-A~-pyrroliniumsalt to Ery- 31. Leete, E. and E. Bellion: Unpublished work throxylon coca. (1970). 43. Liebisch, H. W.: Formation of tropic acid 32. Leete, E. and E. G. Gros: In discussion of in vivo and in virro. 7th IUPAC meeting paper by J. C. Woolley. The biosynthesis on Natural Products, Riga. Abstracts p. of the tigloyl esters in Datura. Abh. Dtsch. 557 (1970). Akad. Wiss. Berlin, K1. Chem., Geol., Biol., 44. Liebisch, H. W., H. Bernasch and H. R. 3, 538 (1966). Schiitte: Zur Biosynthese der Tropanalka- 33. Leete, E. and E. P. Kirven: Biosynthesis of loide XII; Die Biosynthese des Cochlearins. tropic acid: Feeding experiments with cin- 2. Chem. 13, 372 (1973). namoyltropine and littorine. Phytochemis- 45. Liebisch, H. W., W. Maier and H. R. try 13, 1501 (1974). Schiitte: Zur Biosynthese der Tropanalka- 34. Leete, E., N. Kowanko and R. A. New- loide VI. N-Methylputrescine als eine m6g- mark: Use of carbon-13 nuclear magnetic liche Vorstufe des Pyrrolidinringes in Hyos-- resonance to establish that the biosynthesis cyamin und Scopolamin. Tetrahedron Lett. of tropic acid involves an intramolecular 4079 (1966). rearrangement of phenylalanine. J. Am. 46. Liebisch, H. W., K. Peisker, A. S. Radwan Chem. Soc. 97, 6826 (1975). and H: R. Schiitte: Zur Biosynthese der 35. Leete, E., N. Kowanko, R. A. Newmark, Tropanalkaloide. XI. Die Bildung der C,- L. C. Vining, A. G. McInnes and J. L. C. Briicke des Tropins. 2. Pflanzenphysiol 67, Wright: The use of carbon-13 nuclear rna- 1 (1972). gnetic resonance to establish that the bio- 47. Liebisch, H. W., A. S. Radwan and H. R. synthesis of tenellin involves an intramole- Schiitte: Zur Biosynthese der Tropanalka- cular rearrangement of phenylalanine. Te- loide, X. Uber die Bildung des Cuskhygrins. trahedron Lett. 4103 (1975). Annalen 721, 163 (1969). This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. 36. Leete, E. and M. C. L. Louden: Incorpora- 48. Liebisch, H. W., H. Ramin, I. Schijffinius and H. R. Schiitte: Zur Biosynthese der Tropanalkaloide. IV. Z. Naturforsch. 20b, tion of putrescine into the tropine moiety 1183 (1965). of hyoscyamine. Chem. and Ind. (London) 49. Liebisch, H. W. and H. R. Schiitte: Zur 1725 (1963). Biosynthese der Tr~~analkaloide.VIII. 37. Leete, E. and D. H. Lucast: Biosynthesis of Vorstufen des Pyrrolidinringes. 2. Pflanzen- meteloidine from 3a-tigloyloxytropane in physiol. 57, 434 (1967). Datura innoxia. Phytochemistry 14, 2199 50. Liebisch, H. W., H. R. Schutie and K. Mo- (1975). thes: Zur Biosynthese der Tropanalkaloide, Leete

IV. Putrescin als mijgliche Vorstufe des Pyr- 63. Romeike, A.: Ober die Mitwirkung des rolidin-Rings der Tropan-alkaloide. Anna- Sprosses bei der Ausbildung des Alkaloid- len 668, 139 (1963). spektrums. I. Epoxydbildung beim Hyos- 51. Loder, J. W. and G. B. Russell: Tropine cyamin durch Datura ferox; L. Flora 143, 1,2-dithiolane-3-carboxylate, a new alka- 67 (1956). loid from Bruguiera senangula. Tetrahedron 64. Romeike, A,: Uber die Mitwirkung des Lett. 6327 (1966). Sprosses bei der Ausbildung des Alkaloid- 52. Lounasmaa, M.: On the minor alkaloids of spektrurns. 11. Bildungsstatte und Biogenese Knightia deplanchei. Planta Medica 27, 83 des Scopolamins in Datura ferox. L. Flora (1975). 148, 306 (1959). 53. Lounasmaa, M., P. M. Wookulich and E. 65. Romeike, A.: Uber das Vorkommen von 6- Wenkert: Structures of some Knightia de- Hydroxyhyoscyamin in Datura. Naturwis- planchei alkaloids. J. Org. Chem. 40, 3694 senschaften 49, 281 (1962). (1975). 66. Romeike, A. and G. Fodor: The biogenesis 54. McGaw, B. A. and J. G. Woolley: Stereo- of hyoscine in Datura stramonium L. Te- chemist'ry of tropane alkaloid formation in trahedron Lett. No. 22, 1 (1960). Datura Phytochemistry 17, 257 (1978). 67. Schiitte, H. R. and H. W. Liebisch: Zur 55. Mestichelli, L. J. J., R. N. Gupta and I. D. Biosynthese der Tropanalkaloide. Zum Me- Spenser: The biosynthetic route from orni- chanismus der Bildung der Tropasaure aus thine to proline. J. Biol. Chem. 254, 640 Phenylalanin-(1 -"C,2-SH). Z. Pflanzenphy- (1979). siol. J7, 440 (1967). 56. Mizusaki, S., T. Kisaki and E. Tarnaki: 68. Spcnser, I. D.: The biosynthesis of alkaloids Phytochemical studies on the tobacco alka- and of other nitrogenous secondary meta- loids. XII. Identification of y-methylamino- bolites, in M. Florkin and E. H. Stotz (eds): butyraldehyde and its precursor role in ni- Comprehensive Biochemistry, Vol. 20, Chap. cotine biosynthesis. Plant Physiol. 43, 93 VI, pp. 231, ref. (116), 1968. (1 967). 69. Tamprateep, P., E. H. Tayler and E. Ram- 57. Motherwell, W. D. S., N. W. Isaacs, 0. stad: Biosynthesis of the alkaloids of Ery- Kennard, I. R. C. Bick, J. B. Bremner and throxylon novogronatense. Lloydia 26, 203 J. Gillard: Bellendine, the first proteaceous (1963). alkaloid, a y-pyronotropane: X-ray struc- 70. Underhill, E. W. and H. W. Youngken: ture determination by direct methods. Chem. Biosynthesis of hyoscyamine and scopola- Commun. 133 (1971). mine in Datura stramonium. J. Pharm. Sci. 58. Neumann, D. and H. B. Schroter: N-Me- 51, 121 (1962). thylornithin als Vorstufe des Pyrrolidin 71. Woolley, J. G.: The biosynthesis of tigloyl rings in Tropanalkaloiden. Tetrahedron Lett. 1273 (1966). esters in Datura. Abh. Dtsch. Akad. Wiss. 59. O'Donovan, D. G. and M. F. Keogh: The . Berlin K1. Chem., Geol., Biol., Nr. 3, pp. role of hygrine in the biosynthesis of cusco- 531 (1966). hygrine .and hyoscyamine. J. Chem. Soc. 72. Yamasaki, K., T. Tamaki, S. Uzawa, U. (C) 223 (1969). Sankawa and S. Shibata: Participation of 60. Prabhu, B. V.: The biosynthesis of tropic C,-C, unit in the biosynthesis of ephedrine

acid. Ph. D. Thesis, University of Georgia in Ephedra. Phytochemistry 12,2877 (1973). This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. (1974). 73. Zenk, H. M.: Metabolism of prearomatic 61. Prabhu, B. V., C. A. Gibson and L. C. and aromatic compounds in plants, in H. Schramm: Biosynthesis of tropic acid. I. Wagner and L. Horhammer (eds): Pharma- Incorporation of cinnamic acid-2-'4C in cognosy and Phytochemistry, pp. 330-339, ~atura'innoxia. Lloydia 39, 79 (1976). Berlin 1971, Springer Verlag. 62. Robinson, W. G., B. K. Bachhawat and M. J. Coon: Tiglyl coenzyme A and a-methyl- Address: Professor Edward Leete, acetoacetyl coenzyme A, intermediates in School of Chemistry,

' the enzymic degradation of isoleucine. J. University of Minnesota, Biol. Chem. 218, 391 (1956). Minneapolis, 35455, USA