Tropane derivatives from Calystegia sepium

Phytochemistry, Vol.29,No. 7, Pp. 2125-2127, 1990. Printedin Great Britain.

TROPANE

DERIVATIVES

0031-9422/90 $3.00+0.00 0 1990PergamonPressplc

FROM CALYSTEGIA

SEPIUM

ARLETTE GOLDMANN, MARIE-LOUISE MILAT , * PAUL-HENRI DUCROT , t JEAN-YVESLALLEMAND,t MONIQUE MAILLE, ANDREELEPINGLE, ISABELLECHARPIN* and DAVID TEPFER Laboratoire de Biologie de la Rhizosphbe, 78026 Versailles Cbdex, France; +Laboratoire des Mtdiateurs Chimiques, Domaine de Brouessy, Magny-les-Hameaux, 78470,St. R&my-les-Chevreuse,France; TLaboratoire de Synth&e Organique, Ecole Polytechnique, 9 1128 Palaiseau, France (Received in revised form4 January 1990)

Key Word Index-Calystegia sepium; Convolvulaceae; structure; tropane derivatives; calystegins; nutritional mediators; rhizosphere.

Abstract-A family of novel polyhydroxy-nor-tropanes is described. Three of their structures were determined by mass spectrometry and ‘H and ’ 3C NMK spectroscopy. These calystegins occur in two species of the Convolvulaceae and in Atropa belladonna. Their potential ecological significance is discussed.

INTRODUCTION Certain small secondary metabolites of plants are implicated in the establishment and maintenance of specific plant-bacterium relationships. Some of these induce the transcription of genes implicated in the plant-bacterium interaction, for example, virulence genes in the phytopathogen Agrobacterium tumefaciens [l] and nodulation genes in the nitrogen-fixing symbiont Rhizobium [2]. In the former case the inducing molecules are monocyclic phenols such as acetosyringone [3]. In the latter they are complex phenolic flavones or flavonoids [4]. These same substances also serve as chemoattractants [S-7]. Secondary metabolites are implicated in specific nutritional relationships between these bacteria and their hosts. The opines are amino acid derivatives whose synthesis is induced by Agrobacterium in the host through genetic transformation [a]. Homoserine is the major constituent of pea root exudates [9], and rhizopine is an amino-cyclitol synthesized by Rhizobium in nitrogen-fixing root nodules [lo]. We have recently discovered novel secondary metabolites in the root exudates of a member of the Convolvulaceae, Calystegia sepium [ll], which we have named calystegins. In the laboratory calystegins specifically stimulate the growth of R. meliloti strain 41, by serving as a source of carbon and nitrogen. We have suggested [ 1I] that in nature, calystegins could serve as nutritional mediators, that is, secondary metabolites that selectively promote the growth of particular bacteria in the rhizosphere (see [ 121 for an antecedent). In this paper we now show that calystegins are novel tropane derivatives. RESULTSAND

kilograms of transformed C. sepium roots in the space of a few weeks [ 131. Initial purification resulted in a mixture of compounds A and B, contaminated by traces of amino acids. Calystegins A and B were then separated by gel filtration in a volatile buffer; the final purification of each substance was achieved by HPLC. Using HPLC, calystegin A was separated into four peaks suggesting that the product A is a mixture of four different molecules: A, (18%), A, (lo%), A, (60%), A, (12%). In the same manner, compound B was resolved into two peaks, presumably representing two different molecules: B, (40%) and B, (60%). Only the products A,, B, and B, have been studied. High resolution mass spectrometry (EI) gave a value of 159.0895, consistent with a molecular formula of C,H,,O,N for calystegin A, and a value of 175.0844, consistent with a formula of C,H,,O,N for calystegins B, and B,. These results provided good evidence that B, and B, are isomers, and that A, could have the same structure, minus an hydroxyl group. A,, with a mass of 159, is an isomer of A,.

DISCUSSION

Calystegins were initially detected as two spots (A and B) using paper electrophoresis at pH 1.9 and silver nitrate staining [ll]. In order to produce these substances in large quantities, we took advantage of the capacity of roots genetically transformed by A. rhizogenes to grow in vitro. Fermenter cultures were used to produce several 2125

OH

2126

A. GOLDMANN

The mass spectrometry data were used to derive the molecular formula C,H,,O,N, where five hydrogens can be exchanged for the calystegin B,. ‘H and 13C NMR studies at high field with extensive ‘H-‘H decoupling inexperiments and 13C multiplicity determination dicated a polyhydroxylated tropane structure for this molecule (formula 1). A first spin system of five coupled protons is identified and assigned to H-2 (d, 63.12, J = 9 Hz), H-3 (m, 63.95, J = 7.5,7 Hz), H-4 and H-4’ (m, 6 1.33 and 1.88) and H-S (m, 63.12). A second spin system of three protons is also visible in the spectra: H-6 (4,63.95, J = 7 and 8 Hz), H-7 (m, 6 1.30) and H-7’ (m, 62.35). A small long range coupling constant can be detected between H-7 and H-2 (J = 1.5 Hz). The structure of calystegin B, is in agreement with the observation that two vicinal protons H-5 and H-6 do not display any coupling, due to a diedrial angle close to 90” (bridgehead and endo protons). The 13C spectrum with carbon multiplicity editing, displays seven resonances corresponding to a quaternary carbon C-l (93 ppm), four C-H carbons (82, 73, 70, 60 ppm) tentatively assigned to C-2, C-3, C-5 and C-6 and two CH, groups (25, 32 ppm, C-4 and C-7). Structures of calystegins B, and A, were elucidated in a similar fashion (2 and 3). The coupling constant measurements indicated that the three hydrogens on C-2, C-3 and C-4, bearing the OH groups, are axial (J = 10 Hz). Comparison with synthetic compounds where the stereochemistry is well known and study of the different coupling constants indicate a chair conformation for calystegin B,. It is not possible at this time to specify the absolute stereostructure of this molecule. Radiotracer experiments revealed that exogenous 14Cputrescine serves as the precursor for the biosynthesis of calystegins. Uptake of radioactive putrescine by C. sepium roots grown in vitro resulted in the appearance of labelled compounds that react with the silver nitrate reagent and have the same R, as calystegins after electrophoresis. These labelled compounds were further identified by biological means. Catabolic tests were performed with the R. meliloti strain 41, bearing the plasmidencoded genes encoding calystegin degradation [ 11). Controls consisted of parallel tests with the same bacterium after removal of the catabolic plasmid [14]. The bacterium containing the catabolic genes degraded the labelled substances that co-migrate with calystegins, indicating that the substances labelled by feeding with labelled putrescine are indeed calystegins, and that the biosynthesis of the tropane ring of calystegins would be similar to that already described for the tropane alkaloids [15]. Other potential precursors (such as sugars or amino acids) did not give efficient labelling of calystegins. We suggest that the hydroxyl groups at the 1, 2 and 4 positions in the calystegins could be introduced after formation of the tropane ring, as described for the alkaloid meteloidine [16]. The three species known to produce calystegins (C. sepium, C. arvensis and Atropa belladonna) synthesize the tropane alkaloids cuscohygrine, hyoscyamine, atropine and scopolamine [ 131. Calystegins were not detected in other species that synthesize tropane alkaloids, although we did not examine another family, the Erythroxylaceae, which synthesizes cocaine and related compounds. Besides the opines and homoserine, the calystegins constitute a third class of plant secondary metabolites thought to be involved in nutritional relationships with

et al.

Rhizobiaceae in the rhizosphere. They are novel in their chemical structure and are of particular interest because they are products of the normal metabolism of plants, rather than being induced by transformation and symbiosis. EXPERIMENTAL

Plant materials. Transformed root cultures of C. sepium were established as reported in ref. [ 131.They were grown in a fermenter in the liquid medium of Monnier with the vitamins of Morel [13]. Bacterial strains. The A. tumefaciens strain B6 806, was provided by J. Tourneur; strains R. meliloti 41 and GM113 were provided by .I. Wnarit. GM113 is a derivative of the AK631 strain cured ofpRme4la, which no longer catabolizes calystegins [ll]. Stock cultures of bacteria were maintained on tryptoneyeast extract medium (TY). Radiochemicals. [1,4-‘4C]Putrescine (100 mCi/mM) was obtained from Amersham International. Extraction and purification. Calystegins were isolated from an aq. extract of 2.5 kg of transformed roots of C. sepium. They were extracted with the amino acid fr. by treatment with Dowex 50 X8 H+ form followed by a biological enrichment using A. tumefaciens B6 806 strain [ll]. Calystegins A and B were then sepd by filtration on a 60 x 2 cm GF 05 column (I.B.F. France) using the volatile buffer trimethylammonium carbonate at pH 7. Calystegins in the effluent were sepd by paper electrophoresis and detected with the AgNO, reagent as previously described [ll]. Frs containing each of the calystegins were collected and evapd to dryness. Further purification was carried out by HPLC at 2500 psi using a column of 9.4 mm i.d. x 25 cm (Zorbax NH, 6 p and MeCN-H,O, 4: l), flow rate 2.5 ml min- ’ and RI detection. Calystegin B,. %Azabicyclo(3.2.l)octan-1.2.3.6-tetraol (1). C,H,,O,N: Found 175.0842. Calc. 175.0848. CIMS (NH,) 158 [M+H-H,O]+. ‘+ZNMR (D,O) m/z 176 [M+H]+, (6 in ppm): 25.32 (C-4, C-7); 60 (C-5); 70, 73, 82 (C-2, C-3, C-6); 93 (C-l). ‘HNMR (D,O): 1.3 (H-4, H-7); 1.88 (H-4’); 2.35 (H-7’); 3.12 (H-5); 3.2 (H-2); 3.3 (H-3k 3.95 (H-6). J,,, = 9 Hz; ; *,,, =1.5Hq J,.,=7Hz; J,,,.=7.5Hz; J,,,.=13Hz; ,,,=2.5Hz; J,.,,=3Hz; J,.,=7Hz; J,,,,=8Hz; J,,,. =15Hz. Calystegin B,. 8-Azabicyclo(3.2.l)octan-1.2.3.4-tetraol (2). C,HiJO,N: Found 175.0844. Calc. 175.0848. CIMS (NH,) m/z 176 [M+HJ+, 158 [M+H - H,O]+. ‘%ZNMR: 24, 31 (C-6, C-7); 58 (C-5); 76 (2C), 80 (C-2, C-3, C-4); 93 (C-l). ‘H NMR: 3.41 (H-4); 3.25 (H-3); 3.17 (H-2); 3.12 (H-5); 1.8 (H-6, H-6’); 1.58 (H-7); 1.37 (H-7’). J,., = 8.5 HZ J,,, = 3.5 HZ J,., = 9 Hz; J ,.,.=~.~Hz;J,,,=~Hz;J,,,=~Hz;J,,,.=~Hz;J ,.,, =5Hz;J, ,.=8Hz. Calystegh A,. 8-Azabicyclo(3.2.l)octan-1.2.3-trio1 (3). C,H,,O,N: Found 159.0895. Calc. 159.0900. CIMS (NH,) m/z 160 [M+H]+, 142 [M+H - H,O]+. “CNMR: 29; 31.5; 42.5 (C-4, C-6, C-7); 54 (C-5); 82.5; 72.5 (C-2, C-3); 93 (C-l). ‘H NMR: 1.4; 1.9 (6H) (H-4, H-4’, H-6, H-6’, H-7, H-7’); 3.3 (H-2); 3.4 (H-5); 3.6 (H-3). Incorporation of radioactive putrescine. Labelled putrescine (2 @A)was added to 3 ml of liquid culture medium containing transformed roots of C. sepium maintained in the dark in Petri

dishes. After four days, 200 mg of roots were removed, washed with H,O and homogenized in 0.02 M HCl as described in ref. [ll]. The extract containing calystegins was subjected to HV paper electrophoresis. Labelled compounds were detected by autoradiography and electrophoretograms were stained with the AgNO, reagent [ill.

Tropane derivatives from Calystegia sepium Confirmation of identity of labelled cafysystegins.Labelled root extract was purified by treatment with Dowex 50 resin prior to catabolic tests performed as previously described [ll]. The reaction mixture (200 ~1) contained the purified root ext, inorganic salts of SM medium and R. meliloti strains 41 or GM1 13. Calystegin catabolism was assayed using HV paper electrophoresis followed by autoradiography. Acknowledgement-We are indebted to C. Descoins (Laboratoire des Mtdiateurs Chimiques, 1.N.R.A) for his support, both in organizing the collaborations necessary to this work and in giving scientific advice. REFERENCES 1.

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