Biosynthesis of iridoid glucosides inThunbergia alata

Pergamon

0031-9422(94)00536-2

BIOSYNTHESIS

OF IRIDOID GLUCOSIDES

Phytochemistry, Vol. 37, No. 6, pp 159S1603, 1994 Copyright 0 1994 Elsevier Science Ltd Pnntcd III Great Britain. All rights reserved 0031-94x/94 $7.00 + 0.w

IN THUNBERGIA ALATA

SQRENDAMTOFT, LOTTE BOE FREDERIKSEN*and SP~RENROSENDALJENSEN Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark (Received in reoisedform

Key Word Index-77runbergia ‘H NMR.

alata; Acanthaceae;

6 June 1994)

iridoid glucosides; stilbericoside;

biosynthesis;

Ahstraet-Deoxy-[6,7,8,10-2H] loganic acid and 8-epideoxy-[6,7,8,10-2H] loganic acid were fed to Thunbergia alata. The former was incorporated into stilbericoside with retention of the label at H-6, H-7 and H-8. Administration of deuterium-labelled loganic acid, adoxosidic acid, capensioside and 8(S)-bisdeoxy-7,8_dihydroaucubin showed that the first steps in the biosynthesis of stilbericoside from deoxyloganic acid are most probably lo-hydroxylation followed by decarboxylation.

INTUODUCTION Thunbergioideae is a subfamily of the Acanthaceae which, together with Scrophulariaceae, Lamiaceae and a number of other families, form a homogeneous order, designated as the Lamiales by Dahlgren [l]. The biosynthesis of most iridoids found in plants in this order is believed to proceed via 8-epideoxyloganic acid (1). However, some exceptions are known, e.g. Euphrasia cuspidata (Schrophulariaceae) contains iridoids with 8-/3-stereochemistry [l] and cornin (2), found in Verbena species (Verbenaceae) [1,2], are known to be formed from deoxyloganic acid (3) [1,2]. Previously, no biosynthetic work has been performed on compounds lacking C-10. The characteristic iridoids lacking both C-10 and C-11 are only found in a few families: Stilbaceae (Stilbe spp.) [3], Retziaceae (Retzia [4,5]), Acanthaceae (Thwnbergia spp.) [6,7] and Ericaceae (Arbutus unedo) [S]. These families all belong to Lamiales except the last one, which is a member of the Ericales. Owing to the lack of C-11, these compounds would be expected to be formed in a similar way to other compounds with this feature (e.g. aucubin, 4 and antirrhinoside, 5) and thus presumably derived from 1 [l]. However, we have recently isolated thunaloside (6) from Thunbergia alata with 8-&stereochemistry [9] and the related capensioside (7) has been isolated from Retzia capensis [4], casting some uncertainty on the likelihood of 1 being the precursor of these compounds. The purpose of this work was to investigate the biosynthesis of the iridoids from T. alata.

REGULTSANDDISCUSSION In the first experiments deuterium-labelled deoxyloganic acid (d-3) and 8epideoxyloganic acid (d-l) were

administered to Thunbergia alata. The syntheses of these precursors labelled in the 6-, 7-, 8- and lo-positions have been reported elsewhere [2, lo]. The results of the feeding experiments performed in late August 1992 are listed in Table 1 (entries 1 and 2) and showed that deoxyloganic acid (3) was incorporated into stilbericoside (8) and thunaloside (6), while 1 was not. This indicated that the biosynthetic pathway to the iridoid glucosides in T. alata proceeds via 3 and thus is very different from that leading to aucubin (4) and antirrhinoside (5) where 1 is the intermediate. Additionally, the 2HNMR spectrum of stilbericoside (8) from the feeding experiment showed that ‘H apparently was retained in both the 6-, 7-, and 8positions of 8. However, ‘H-7 and ‘H-8 were not separable in the ‘H NMR spectrum. In order to obtain a better separation of the signals from the 7- and 8-protons, stilbericoside (8) was transformed to the hexaacetate 8a. The 2H NMR spectrum of 8a confirmed that both ‘H-7 and 2H-8 were retained during the transformation of deoxyloganic acid (3) to stilbericoside (8). Retention of H8 indicated that geniposidic acid (9) or other compounds without H-8 cannot be intermediates. As incorporation of 3 into 6 also took place (entry l), the latter compound was possibly an intermediate on the route from 3 to 8. If this was indeed the case, three discrete reaction steps were needed when going from 3 to 6 namely decarboxylation of C-l 1, together with 7- and lOhydroxylation. In order to establish the sequence of these three reaction steps, a second series of feeding experiments was performed. Here, we again used 3 (as a reference), adoxosidic acid (10) and loganic acid (11) together with 7 and 12, the decarboxylated analogues of 10 and 3. Deuterium-labelled loganic acid (d-11) was prepared from loganin (13). Thus, 13 was oxidized to ketologanin (14) with Jones’ reagent [ 1l] and the product acetylated

1599

S. DAMTOFTet al.

1600

\ NJ \ 4

COOH

COOMe

0

0

OGlc

4

COOR

COOR

I

HO

OGlc

HO

0

OGIC

5

R=H(I 1) R=Me( 13)

R=H(9) R=Me(lS)

\ Q

COOMe

COOMe

I

\

\

0

0 *

@

OGlc

OGIC

0

HO’

OGlc

14

12

16 COOMe

HO

\ 0 OGlc

OGlc

&

OGlc 21

20

YjOjoc,,:&. 4

OGlc 22

A OGlc

HO

23

to give 14a. Reduction of 14a with NaBD, gave 7epiloganin tetraacetate, selectively labelled at C-7. A Mitsunobu inversion [12] afforded [7-2H] loganin pentaacetate which, after deacetylation and saponification provided [7-‘H] loganic acid (d-11).Labelled adoxosidic acid (d-10) was prepared from geniposide (15) by deuteration with D, over Pd/C in the presence of triethylamine. This afforded a mixture of adoxoside (d-16) and 8epiadoxoside, which after saponification was fractionated by preparative TLC to give pure d-10. Compounds d-7 and d-12 were prepared from the corresponding acids by acetylation followed by heating

with alkaline copper carbonate in quinoline at 200” for 2 hr L-131. Deacetylation afforded the decarboxylated glucosides. The precursors prepared in this way contained the same labelling as the corresponding acids. The labelled compounds were fed to excised leaves of T. alum and the results of the feeding experiments are listed in Table 1 (entries 3-7). The efficient incorporation of adoxosidic acid (10) and the decarboxylated analogue 7 is consistent with the fact that neither loganic acid (11) nor 12 was incorporated. This indicates a biosynthetic pathway in which deoxyloganic acid (3) after lo-hydroxylation is decarboxylated to

Biosynthesis of iridoid glucosides in Thunbergia alata

1601

Table 1. Results of feeding experiments Isolated metabolites (%-incorporation)*

Entry 1 2 3 4 5 6 7

Precursor (mg)

Plant/crude extract (g) (mg)

d-3119 d-1121

11/197 11/104 131207 12/160 11/150 lO/ 120 14/151

d-3133

d-12119 d-10121 d-7110 d-11/18

8 14(12) 15(O) 18(4) 22 (0) 18(11) 15(3) 4(O)

17

6

22


l(3) < l(0) 10(2) 8 (0) < l(0) < l(0) 4 (0)

< l(0) < l(0) l(0) -2(O) < l(0) < l(0) 4(O)

The feeding experiments were performed in early summer except entries 1 and 2, which were performed in late summer. The incorporation was measured on all the fractions shown. tThe metabolite was not separated from the polar compounds. The amount is judged by the peakheight (MPLC) compared to that of stilbericoside.

capensioside (7). Since. 3 is also incorporated into thunaloside (6) it is probable that the next step from capensioside is 7-hydroxylation. In Scheme 1 a likely biosynthetic pathway to stilbericoside (8) is given. The formation of 19 from 18 would thus account for the observed retention of *H-B, ‘H-7 and *H-8 in the deoxyloganic acid initially fed. That 19 is a probable intermediate is substantiated by the fact that the 6-hydroxylated analogue, retzioside (u)), has been isolated from Retzia [4,5]. To see whether or not differences in isolated amounts of metabolites were due to the precursors fed or to differences in individual plant specimens, 10 plants of T. alum were worked-up and the contents analysed by HPLC. This showed that all the specimens contained the same compounds in approximately the same relative amounts. Therefore, we conclude that differences in isolated amounts of metabolites must be caused by the precursor fed. A plant-age dependent difference is seen for the incorporation of deoxyloganic acid (3). In late summer (entry l), 3 is almost exclusively incorporated into stilbericoside (8X while in early summer (entry 3) 3 is incorporated to the same degree into stilbericoside (8) and 6-epistilbericoside (17). That neither d-10 nor d-7 is incorporated into 6 may reflect an increased rate of conversion of 6 caused by these two precursors resulting in a very small pool of 6 as seen in experiments 5 and 6 (Table 1). EXPERIMENTAL

Growing specimens of T. alata were. obtained from the experimental station of The Botanical Garden of Copenhagen in TWrup. A voucher is deposited in the Herbarium of The Botanical Museum, Copenhagen (cf. [6]). Prep. chromatography was performed on Merck Lobar reverse phase columns (size B and C) eluting with H,O-MeOH mixts. Peaks were detected by UV at 200 nm. ‘H NMR (250 or 500 MHz) spectra were re-

corded in D20 (HOD-signal at 4.75 ppm was used as int. standard); acetates were recorded in CDCl, using the CDCl, signal at 67.27 as int. standard. In “CNMR (125 MHz), C-6’ was set to 61.5 ppm as the standard [14]. The *H NMR spectra were recorded at 76.8 MHz in H,O or CHCl, with 0.016 and 0.017% *H of natural abundance, respectively. Deoxyloganic acid (d-3). The prepn of d-3 has been described previously [2, lo]. The compound contained 0.5 *H in the 6cl-position, 0.4 *H in the 6/&position, 0.8 *H in the 7c+position, 0.8 *H at C-8 and 2.1 *H at C-10 as judged by ‘HNMR. 8-Epideoxybganic acid (d-l). The prepn of d-l has been described previously [lo]. The deuterium content was as above, except at C-7 where 0.8 *H was found in the ‘IS-position. Ketologanin (14). Loganin (13,581 mg) was suspended in dry Me2CO- (875 ml), an aq. soln of 00, and cont. H2S0, (1.4M; 1.4g CrO, and 1.2ml cont. H,SO,; 1000 pl) was added and the mixt. was stirred at room temp. for 3 min [ll]. Then Na,S,O, was added to remove excess oxidant. The soln was made alkaline with NaHCO, and filtered through act. C over Celite. After washing with Me&O (20 ml) the combined filtrates were evaped to a foam. Fractionation by prep. MPLC (Ccolumn) eluting with 3: 1 afforded first ketologanin (14, 176mg, 30%) then recovered 13 (19 mg, 3%). [7-*HI-7-Epiloganin tetraacetate. Ketologanin (14, 596 mg) was, after acetylation, dissolved in EtOD (30 ml) and stirred with NaBD, (59 mg in 5 ml EtOD) for 30 min. The soln was acidified with HOAc, taken to dryness and redissolved in CHCl, (50ml) and washed with H,O (2 x 25 ml). The organic phase was filtered through act. C over Celite and evapd to a foam (550mg, 79%), which was identified by NMR as [7-*HI-7-epiloganin tetraacetate. [7-*H] Loganic acid (d-11). 7-Epi-[7-*HI-loganin tetraacetate (550 mg) was dissolved in THF (20 ml) and

1602

S. DAMTOFTet al. FOOH

FOOH

$9-Ho5q: yo,q----:>q OGlc

(3)

(7)

(IO)

(6)

t

_q;

(8)

-qc -(1%

LOQ 0 (18)

Scheme 1. Possible biosynthesis of stilbericoside (8). stirred with Ph,P (1.95 g) and HOAc (380 pl). Diethylazodicarboxylate (1060 ~1)was slowly added and the soln was left stoppered under stirring for two days. Evapn of the reaction mixt. afforded a yellow syrup which was deacetylated with 0.1 M NaOMe-MeOH (8 ml, room temp., 4 hr). After neutralization with HOAc and evapn, the foam was partitioned between H,O and EtOAc (75 ml each). The aq. soln was taken to dryness (617 mg) and chromatographed (C-column) affording ‘I-epiloganin (48 mg, 12%), loganin (132 mg, 34%) and 7-acetylloganin (48 mg, 11%) when eluting with 3 : 1,2: 1 and 1: 1, respectively. Saponification of loganin with 1 M NaOH (3 ml, 4.5 hr) gave, after fractionation (B-column; dissolved in 10% HOAc), loganic acid (11,101mg, 18% from ketologanin). The compound contained 1.0 ‘H at C-7. Adoxosidic acid (d-10). A three-necked flask was equipped with a dropping funnel, a stopper and a 3way tap which was connected to a polyethylene bag. Freshly cut Na (1.5-2.0 g) was placed in the flask and the system was evapd. While cooling in an ice-water bath, D,O (6 ml) was added from the funnel. D, was collected in the bag. Geniposide (15, 371 mg) was dissolved in EtOD (5 ml) and stirred with Pd/C (5%, 270 mg) and NEt, (0.1 ml). The flask was equipped with the 3-way tap connected to the plastic bag containing D,. The flask was filled with D, after flushing. Reaction took place overnight. After filtration of the reaction mixt. through Celite, the filtrate was evapd and chromatographed (column size C). Elution with 3: 1 afforded first epiadoxoside (62 mg), then adoxoside (16,100 mg) with an intermediate fr. containing both (52 mg). Adoxoside (16) was saponified with 1 M NaOH (3 ml, 3 hr) and purified by prep. TLC (CHCl,-MeOH-HOAc, 32: 38: 1.2 developments) to give pure d-10 (53 mg: 15% from 15) containing 0.4 2H at C-6or, 0.8 2H at C-7c( and 0.8 2H at C-8. 8(S)-Bisdeoxy-7,8-[6,7, 8,10-2H] dikydroaucubin (d12). [6,7,8, 10-2H] Deoxyloganin (d-21, 197 mg, containing 20% 8-epimer) was, after saponification and acetylation, decarboxylated as described [ 131. Deacetylation and chromatography (B-column) gave d-12 (65 mg,

39%, containing 20% of the 8-epimer), the labelling of which was identical to that of d-3 above. ‘HNMR (500 MHz, D,O): 65.32 (d, J = 3.0 Hz, H-l), 6.18 (dd, J =6.5 and 2.0 Hz, H-3), 4.91 (dd, 5=6.5 and 2.5 Hz, H-4), 2.70 (m, H-5), 1.99 (m, H-6/?), 1.42 (m, H-6a), 1.91 (m, H-73 and H-8), 1.24 (m, H-7fi), 1.78 (dt, J5.9=J,,g=8.0 Hz and J,,,=2.9 Hz, H-9), 1.07 (d, J=6.3 Hz, 3H, H-lo), 4.81 (d, J=8.3 Hz,H-l’),3.31(dd,J=9.3and8.3Hz,H-2’),3.53(t, J=9.3 Hz, H-3’), 3.42 (t. J=9.3 Hz, H-4’). 3.50 (ddd, J =9.3, 5.8 and 2.3 Hz, H-5’), 3.95 (dd, J= 12.5 and 2.3 Hz, H-6’), 3.75 (dd, J = 12.5 and 5.8 Hz, H-6’). 13CNMR (62.5 MHz, D,O): 696.3 (C-l), 136.1 (C-3), 110.3 (C-4), 32.6 (C-5), 32.9* (C-6). 31.4* (C-7), 35.2 (C-8), 49.5 (C-9), 19.9 (C-lo), 99.2 (C-l’), 73.6 (C-2’), 76.4 (C-3’), 70.4 (C-4’), 70.4 (C-4’), 77.0 (C-5’), 61.5 (C-6’). *Signals are interchangeable. [6,7, 8-2H] Capensioside (d-7). This precursor was prepared by decarboxylation as above. Thus from d-16 (88 mg) pure d-7 (18 mg, 24%), containing the same labelling as d-10, was obtained after purification on a Bcolumn. The 13CNMR spectrum was similar to that reported previously [14]. ‘HNMR: 65.13 (d, 3=4.5 Hz, H-l), 6.17 (dd,J=6.9 and 3.0Hz, H-3), 4.82 (dd, J=6.9 and 3.2 Hz, H-4), 2.62 (m. H-5), 1.79 (m, H-6), 1.28 (m, H-6), 1.79 (m, H-7 and H-8), 1.39 (m, H-7), 2.03 (m, H-9), 3.5-3.3 (m, 2H under Glc, H-lo), 4.71 (d, J=8.3 Hz, H-l’), 3.25 (t, 5=8.3 Hz, H-2’), 3.48 (t, J=8.3 Hz, H-3’), 3.38 (t, J =8.3 Hz, H-4’), 3.41 (m, H-5’), 3.88 (dd, J- 12.5 and 2.8 Hz, H-6’), 3.68 (dd, J= 12.5 and 7.6 Hz, H-6’). Feeding expts. The precursor was dissolved in H,O (l-2 ml) and the leaves (freshly cut under water) were allowed to absorb the soln. More water was added in 2 ml portions and after absorbtion of a total amount of 6-8 ml the leaves were transfered to a larger beaker with water. If the leaves started to dry out, they were kept in a chamber with moist air. After 6 days the leaves were worked-up (Table 1). The isolated frs were analysed by ‘H, 2H and 13C NMR spectrometry. Stilbericoside hexaacetate @a). Labelled stilbericoside (8, 14 mg) from expt 1 (Table 1) was acetylated with

1603

Biosynthesis of iridoid giucosides in Thunbergiaalata

Ac,O-pyridine (1: 1) (4 ml, room temp. 10 days) in the usual way to give stilbericoside hexaacetate (8a, 22 mg, 91%), which was diluted with carrier (Sa, 88 me;) and purified by prep. TLC (Et,O). Extraction of the band at R, = 0.54 and evapn afforded pure stilbericoside hexaacetate @a, 72 mg, 65%), mp 148.0-148.5” (lit. 144-146” E3Ik Eali$o- 154.7”(CHCI,; c 0.8). ‘HNMR showed signals at 5.1, 3.8 and 3.5 ppm (ca 2:3:3) showing incorporation in the 6-, 7-, and &positions, respectively. ‘H NMR (500 MHz, CDCl,): 55.62 (d, J = 2.2 Hz, H-l), 6.39(d,J=6.5Hz,H-3),5.52(dd,J=6.5and lSHz,H-4), 5.05 (d, J= 3.0 Hz, H-6), 3.75 (r, f= 3.0 Hz, H-7), 3.49 (d, J = 3.0 Hz, H-8), 2.76 (brs, H-9), 4.85 (d, f=8.0 Hz, H-l’), 5.04(dd,J=9.3and8.0Hz,,H-2’),5.20(c,J=9.3Hz,H-3’), 5.08(~,5=9.3Hz,H-4’),3.73(ddd,J=9.3,4.5and2.3 Hz, H-5’),4.26(dd,J=12.5and4.5Hz,H-6’),4.15(dd,J=12.5 and 2.3 Hz, H-6’). “CNMR (125 MHz, CDCl,): 691.2 (C-l), 143.4 (C-3), 102.5 (C-4), 79.3 (C-5), 74.5 (C-6), 57.3* (C-7), 56.7* (C-8), 46.5 (C-9), 95.4 (C-l’), 70.2 (C-2’), 72.1 (C-3’), 68.0 (C-4’), 72.4 (C-S), 61.5 (C-6’). *Signals are interchangeable. A single plant of Thunbergia alata (13 g) was worked-up as described earlier [9] and the crude extract (O.lOg) analysed by HPLC (gradient program) and then fractionated by MPLC (B~olumn). Eiution with water afforded 17 (9 mg, 0.07%) and continued elution with 25: 1 gave 8 (13 mg, 0.10%); 15: 1 afforded first 22 (1 mg, 0.006%) and then 23 (c 1 mg, <0.005%) and finally elution with 10: 1 gave 6 (2 mg, 0.01%). Nine other individual plants of T. al&u were worked-up and analysed by HPLC showing the same composition of iridoids as above.

PHYTO 37-6-I

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