Stress-related polyketide metabolism of Dioncophyllaceae and

Stress-related polyketide metabolism of Dioncophyllaceae

and Ancistrocladaceae

Gerhard Bringmann1 and Doris Feineis

Institut fur Organische Chemie der Universitat Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Received 20 February 2001; Accepted 21 May 2001

Abstract

The discovery of a novel biosynthetic pathway toisoquinoline alkaloids is described. The naphthyl-isoquinoline alkaloid dioncophylline A, one of themost prominent representatives of a new class ofstructurally and pharmacologically intriguing sec-ondary metabolites, is shown to originate from acet-ate units, both molecular halves, the isoquinolinepart and the naphthalene portion, being formedfrom identical polyketide precursors. All other tetra-hydroisoquinoline alkaloids previously investigated,ultimately originate from aromatic amino acids.The novel pathway to isoquinoline alkaloids (henceacetogenic) was proved by feeding experiments with13C-labelled precursors administered to callus cul-tures of Triphyophyllum peltatum (Dioncophyllaceae),followed by NMR investigations using the potentcryoprobe methodology. The new pathway is largelystress-sensitive: upon exposure to chemical, bioticor physical stress, T. peltatum stops producing theisoquinoline part, so that the naphthalene moietyaccumulates in the chemical form of naphtho-quinones like plumbagin and droserone and thechiral tetralone isoshinanolone.

Key words: Isoquinoline alkaloids, biosynthesis, plant stress,

Dioncophyllaceae, naphthylisoquinoline alkaloids, polyketide

alkaloids, dioncophylline A.

Introduction

The very small palaeotropical families Dioncophyl-laceae and Ancistrocladaceae including, for example,Triphyophyllum peltatum Airy Shaw from West Africaor Ancistrocladus heyneanus Wall. from India comprise

most peculiar lianas, fascinating to botanists and naturalproducts chemists. They produce a plethora of structur-ally unique naphthylisoquinoline alkaloids, among themdioncophylline A (1) (Fig. 1A) (Bringmann and Pokorny,1995; Bringmann et al., 1998a). These alkaloids are dif-ferent from all the other c. 2500 isoquinolines isolatedfrom plants (Kutchan et al., 1991; Bentley, 1998), not only

1 To whom correspondence should be addressed. Fax: q49 931 888 4755. E-mail: [email protected]

Abbreviations: LC, liquid chromatography; NMR, nuclear magnetic resonance; MS, mass spectrometry; CD, circular dichroism; 2D INADEQUATE,

two-dimensional incredible natural-abundance double-quantum experiment.

Fig. 1. (A) The naphthylisoquinoline alkaloid dioncophylline A (1),derived from acetate. (B) Illustration for morphine (3): the conventionalPictet–Spengler route to plant tetrahydroisoquinoline alkaloids fromdopamine (2) and aldehydes.

Journal of Experimental Botany, Vol. 52, No. 363,Plants under Stress Special Issue, pp. 2015–2022, October 2001

� Society for Experimental Biology 2001

due to their unusual structures (including a mostly rota-tionally hindered biaryl axis between the two molecularparts), but also because of their unprecedented biosyntheticorigin: The structure of 1 does not fit into the generallyaccepted biosynthetic scheme for tetrahydroisoquinolines,which, as exemplified for morphine (3) in Fig. 1B, areknown to originate from aldehydes (or a-keto acids), bya so-called Pictet–Spengler condensation with dopamine(2), thus ultimately arising from aromatic amino acids(Dalton, 1979; Bentley, 1998). The naphthylisoquinolinealkaloid dioncophylline A (1), by contrast, appears to bebuilt up from acetate units, exclusively (Fig. 1A). In agree-ment with earlier hints from biomimetic polyketidecyclization reactions (Bringmann and Pokorny, 1995),it has been postulated that both molecular moieties,the naphthalene and isoquinoline parts, are synthes-ized from joint polyketide precursors (Bringmann andPokorny, 1995).

This paper deals with the naphthylisoquinolinealkaloids, their structures, bioactivities, and ‘producers’and with biosynthetic feeding experiments revealing,exemplarily for dioncophylline A (1), for the first timetheir acetogenic origin; furthermore, the stress-inducedinhibition of this biosynthetic pathway and the produc-tion of acetogenic naphthoquinones and tetralones inAncistrocladaceae and Dioncophyllaceae plants and incell cultures after exposure to chemical, biotic, or physicalstress is described.

Cultivation of the plants

For the biosynthetic experiments to be conducted,living, alkaloid-producing material was essential. Priorto the work described here, nothing was known about thecultivation of Dioncophyllaceae and Ancistrocladaceaeplants in a greenhouse, so reliable growth parameters forthese sensitive tropical lianas had first to be established(Bringmann et al., 1999c, f ). More than ten naphthyl-isoquinoline-producing plants species are growing in theBotanical Garden of the University of Wurzburg,including T. peltatum (Fig. 2), Ancistrocladus abbreviatus(Fig. 3A, B), and A. heyneanus (Fig. 3C, D) which wereused to generate the cell cultures (Bringmann et al.,1998b, 2000a). With these alkaloid-producing systemsavailable, it was possible to probe the biosyntheticpostulate of an acetogenic origin of the alkaloids.

Naphthylisoquinolines, structurallyunique alkaloids

The Dioncophyllaceae and Ancistrocladaceae are widelyused in folk medicine, hinting at the presence of bioactivesecondary metabolites. Indeed, phytochemical investiga-tions have revealed that these plants produce a variety

of structurally unique naphthylisoquinoline alkaloids.At the biaryl axis that connects the naphthalene portionwith the isoquinoline moiety, most of these metabolitesshow the phenomenon of restricted rotation, leading

Fig. 2. The West African liana T. peltatum (Dioncophyllaceae) in theBotanical Garden of the University of Wurzburg. (A) A juvenile plantwith a rosette of c. 15 lanceolate leaves wPhoto: B Wiesenx; (B) a glandu-lar leaf, the insect-trapping organ of T. peltatum wPhoto: H Bringmannx;(C) a ‘clawed’ adult leaf (‘dioncophyllum’) specifically formed onelongated shoots, helping the plant to climb into the canopy of high treeswPhotos: H Bringmannx. For the first time in a greenhouse: T. peltatumflowering (D) and fruiting (E) wPhotos: H Rischerx.

2016 Bringmann and Feineis

to stable atropisomers (¼ rotational isomers). Sometypical examples of the different coupling types by whichthe molecular halves can be joined together are shownin Fig. 4.

For the structural identification of such alkaloids,a broad repertoire of chromatographic, spectroscopic,degradative, synthetic, and computational methods hasbeen used, in particular, a most efficient novel ‘triad’,HPLC-MSuMS-NMR-CD (Bringmann et al., 1999a).Currently, c. 90 such naphthylisoquinoline alkaloidsand their stereostructures are known (Bringmann andPokorny, 1995; Bringmann et al., 1998a).

Bioactivities of naphthylisoquinoline alkaloids

Naphthylisoquinoline alkaloids show promising bioactiv-ities, exerting stress on a variety of human–pathogenicor crop-relevant micro-organisms, for example, againstplant-pathogenic fungi (Bringmann et al., 1992) and anti-feeding and growth-retarding effects against herbivorousinsects (Bringmann et al., 1997). Furthermore, some ofthe alkaloids are efficient agents of potential relevancefor the treatment of severe tropical diseases such asAfrican sleeping sickness, leishmaniasis and, in particular,malaria—in fact, using compounds like dioncophylline C(4, Fig. 4), malaria-infected mice can be fully healed(Francois et al., 1997; Bringmann and Feineis, 2000).Similarly dioncopeltine A (6, Fig. 4) and korupensamineA (5, Fig. 4) are also highly active.

Nitrogen-free metabolites in otherDioncophyllaceae: first hints atan acetogenic origin of the alkaloids?

By far the most active antimalarial naphthylisoquinolinealkaloids have been isolated from T. peltatum whichconstitutes a rich source of c. 20 such secondarymetabolites, all of them structurally unprecedented(Bringmann et al., 1998a). It therefore seemed rewardingto investigate other phylogenetically related plants fromthe same family. However, besides T. peltatum, only twoother Dioncophyllaceae species are known: Habropetalumdawei (Hutch. & Dalz.) Airy Shaw from Sierra Leoneand Dioncophyllum thollonii Baillon from Gabon (AiryShaw, 1951). Phytochemical investigations on thesevery rare plants yielded, unexpectedly, no sign of naphthyl-isoquinoline alkaloids, but abundant amounts ofnaphthalene-related, quite wide-spread, known naturalproducts (Fig. 5) like isoshinanolone (10), plumbagin(11), and droserone (12) (Bringmann et al., 1999a, b, d ).

However, by applying the efficient analytical ‘triad’LC-MCuMS-NMR-CD (Bringmann et al., 1999a),naphthylisoquinoline alkaloids were detected in lowquantities in these species, including known representa-tives like dioncophylline A (1), but also (mainly) newones.

The bicyclic 10–12 proved to be valuable indicatorsof an acetogenic origin of the naphthylisoquinolinealkaloids, since one of them, plumbagin (11), is already

Fig. 3. Morphological peculiarities of Ancistrocladaceae plants, cul-tivated in the Botanical Garden in Wurzburg. (A) A characteristichooked branch (here the West African species A. abbreviatus Airy Shaw)wPhoto: H Bringmannx; (B) A. abbreviatus flowering: cincinnus inflores-cence with five petals, generally ten stamens wPhoto: H Rischerx; (C) theIndian liana A. heyneanus Wall. developing its typical fruits: nuts withfive ‘wings’ (enlarged sepals) wPhoto: B Wiesenx; (D) a plantation ofc. 400 hydrocultured specimens of A. heyneanus wPhoto: C Schneiderx.

Stress-related polyketide metabolism 2017

Fig. 4. A selection of naphthylisoquinolines representing the biaryl coupling types detected in Dioncophyllaceae (red arrows) and Ancistrocladaceae(green arrows). In most cases (except for example 8, whose axis can freely rotate), the biaryl axis is configurationally stable and represents anadditional element of chirality.

Fig. 5. Proposed biosynthesis of naphthylisoquinoline alkaloids like dioncophylline A (1), and the stress-induced (or constitutional) formation of thenaphthalene-related compounds isoshinanolone (10), plumbagin (11) and droserone (12).


known to be formed from 14C-labelled acetate (Durandand Zenk, 1971).

The concept of acetogenic isoquinoline alkaloids

The hypothesis of an acetogenic origin of naphthyl-isoquinoline alkaloids as the first solely acetate-derivedtetrahydroisoquinolines, is outlined in Fig. 5. Accord-ingly, both molecular halves of the alkaloids, the naph-thalene part 15 and the isoquinoline portion 16, originatefrom six acetate units each, even via joint polyketideintermediates 13. The naphthoquinones and tetralones10–12 abundantly occurring in H. dawei and D. tholloniiare apparently derived from the free naphthalene part 15

of the alkaloids (Fig. 5), oxidized to give plumbagin (11),further oxygenated to droserone (12), or reduced to giveisoshinanolone (10). Enhanced formation of 10–12

must relate to a block (or non-existence) of the reductiveincorporation of nitrogen into the postulated jointdiketo precursor 14 to give the dihydroisoquinoline 16.The capability to undertake this presumed key bio-synthetic step, is restricted to the Dioncophyllaceaeand Ancistrocladaceae, exclusively. Other taxonomicallyrelated plant families (Fig. 5, drawn in blue), includingthe closest phylogenetic neighbours to these plants, such asthe Drosophyllaceae, the Droseraceae, the Nepenthaceae(all likewise carnivorous), and the Plumbaginaceae,cannot perform this remarkable biosynthetic step. Theyall produce nitrogen-free compounds like 10–12, but nonaphthylisoquinoline alkaloids.

Stress-induced naphthoquinone formation

This assumed transamination (plus cyclization) step14 £16 in Ancistrocladaceae and Dioncophyllaceae can,however, be easily blocked (Fig. 5, pointed out in blue),by all sorts of chemical (e.g. by elicitors like methyljasmonate), physical (e.g. in plants wounded mechan-ically), or biotic stress (see below), thus leading to theformation of the isocyclic compounds 10–12, apparentlyneeded as ‘chemical weapons’.

An illustration of this, hinting at a possible chemo-ecological role for all these acetogenic products in theinteraction between the plants and their herbivores, is,for example, the East African species A. robertsoniorum.When wounded by insects, this species produces largequantities of pure crystalline droserone (12) of such ahigh quality that one can perform an X-ray structureanalysis (Fig. 6), directly on these ‘biogenic crystals’(Peters et al., 1995).

Another example is A. heyneanus: when attacked bythe holoparasite Cuscuta reflexa, it forms large amountsof plumbagin (11) around the haustoria (Fig. 7), thusultimately killing the parasite and also some of its owncells, so that a brown scar remains, but the parasite

is repelled and Ancistrocladus can survive (Bringmannet al., 1999e).

Biogenesis of acetogenic naphthoquinonesand tetralones

The stress-induced naphthalene formation is alsopredominant in cell cultures of A. heyneanus and can,

Fig. 6. A. robertsoniorum: production of crystalline droserone (12) intoinsect holes—pure needles of sufficient quality even for an X-raystructure analysis.

Fig. 7. A. heyneanus: accumulation of orange-coloured plumbagin (11)at the site of invasion by Cuscuta reflexa (C) between the phloem (P) andthe xylem (X) of the host.


in turn, be used for biosynthetic feeding experimentswith doubly 13C-labelled acetate. These investigationsconfirm the postulated acetogenic origin of the threebicyclic compounds 10–12 (Fig. 8), with the full foldingmode of the polyketide chain, including the site whereone C-atom gets lost by decarboxylation (Bringmannet al., 1998b).

This result was, however, only partially satisfying. Itdid give concise information on the polyketide origin ofthe nitrogen-free compounds 10–12; an acetogenic originof the alkaloids, however, although more plausible,still remained unproved, just because the cell cultures ofA. heyneanus did not produce enough dioncophylline A (1).

Finally proved: the acetogenic polyketide origin ofdioncophylline A—a new biosynthetic pathway toisoquinoline alkaloids!

The breakthrough finally came from cell cultures ofT. peltatum, which have recently been established forthe first time, starting from fresh seeds (Fig. 2E). Besidesagain forming naphthoquinones (Bringmann et al.,2000a), these tissue cultures produced sufficient quantitiesof dioncophylline A (1), so that feeding experimentswith 13C2-labelled acetate became possible. Subsequentlya small quantity (c. 1 mg) of 1 was isolated and invest-igated by NMR, and this time, a clear incorporation of13C2-units was found over the naphthalene part. This wasto be expected from the structural relationship to thenaphthalene analogues 10–12, but has now been verifiedfor the first time for a naphthylisoquinoline alkaloid.

In the isoquinoline part, one clear single 13C2-unit(C5–C6) was identified, in perfect agreement with thebiosynthetic hypothesis, but it was impossible to assignany further acetate units because of an unfavourablesignal-to-noise ratio. After renewed extensive purificationof the alkaloid and elimination of the last minor para-magnetic impurities and, in particular, by applying the2D-INADEQUATE technique (Buddrus and Bauer,

1987; Podkorytov, 1999), in combination with the novelNMR cryoprobe methodology (Styles et al., 1984, 1989),the full polyketide folding pattern was clearly andunambiguously identified. The spectrum (Fig. 9) unequi-vocally shows the entire carbon skeletons of both halvesof dioncophylline A (1) to be derived from acetate units,with identical incorporation patterns for the two molecu-lar portions (Bringmann et al., 2000b). DioncophyllineA is thus the first unambiguously proven acetogenictetrahydroisoquinoline alkaloid: a novel, though highlystress-sensitive, pathway to plant isoquinoline alkaloidshas been discovered.

Summary and outlook

With the acetogenic nature of naphthylisoquinoline alkal-oids established, further work on these interesting second-ary metabolites will now focus on other remarkable(mostly unique) details of the biosynthesis, such as

(1) the identification of the polyketide synthase, which(unless influenced by stress) always seems to producea 1 : 1 ratio of the naphthalene and isoquinolinebuilding blocks,

(2) the unique incorporation of nitrogen into a ketoneprecursor (not into an a-keto acid or an aldehyde, asusual), and

(3) the remarkable coupling enzymes, which, for most ofthe alkaloids, join the two molecular halves togetherwith high regio- and stereoselectivity.

Of similar interest is the correlation of chemo-taxonomical results with those of molecular phylogenyof African and Asian naphthylisoquinoline-producingplants (Meimberg et al., 2000), a rewarding task for thefuture.

Fig. 8. Biogenesis of isoshinanolone (10), plumbagin (11) and droserone (12) from acetate, from feeding experiments in callus cultures; single13C-labelled carbon atoms are marked as red points, intact 13C2-units are indicated by a red bold bond line.


Acknowledgements

This work has been supported by the Deutsche Forschungs-

gemeinschaft (SFB 251 ‘Okologie, Physiologie und Biochemiepflanzlicher und tierischer Leistung unter Stress’), by theFonds der Chemischen Industrie, by the Max-Buchner-Stiftung,and by the UNDBuWorld BankuWHO Special Programmefor Research and Training in Tropical Diseases (TDR). Astimulating and intensive co-operation with partners of manyyears’ standing, in particular with Professor L Ake Assi (Centrede Floristique, Abidjan, Ivory Coast), Professor V Mudogo(University of Kinshasa, Republic of Congo), Dr K Peters(MPI fur Festkorperforschung, Stuttgart), Professor G Heubl(Institut fur Systematische Botanik, Munchen), Dr G Francois(University of Antwerp, Belgium), Dr R Kaminsky andDr S Brun (Swiss Tropical Institute, Basel, Switzerland), andmany others, is gratefully acknowledged. We thank Dr D Marekand Dr D Moskau (Bruker AG, Fallanden, Switzerland),who made the cryoprobe technique available to us, as well asDr D Scheutzow and Dr M Grune (Universitat Wurzburg)for valuable advice. Cordial thanks are also due to the fellowworkers of our group dealing with this field of research, ofwhich we would like to mention in particular Dr M Rubenacker,Dr T Ortmann, Dr C Gunther, Dr D Lisch, R Zagst, W Saeb,A Hamm, K Messer (phytochemical analysis), Dr M Ruckert(NMR, LC-triad), Dr J Schlauer, H Rischer (plants, cellcultures, biosynthesis), M Wohlfarth (NMR, LC-triad, bio-synthesis), Dr K Wolf (plants, NMR imaging techniques),Dr S Busemann, J Kraus (CD calculations), B Wiesen (plants,cell cultures, biotests), Dr J Holenz (chemical modification, bio-tests), and M Michel, P Henschel, J Mies for reliable technicalassistance.

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Stress-related polyketide metabolism of Dioncophyllaceae and