Biotransformation of sesquiterpenes by cultured cells ofCurcuma zedoaria

Phytochemistry, Vol. 31, No. 1, pp. 143 147, 1992 Printedin Great Britain.

BIOTRANSFORMATION

0031.9422:92 $5.00+0.00 G 1991 Pergamon Press plc

OF SESQUITERPENES CURCUMA

NORIHIRO

SAKUI, MASANORI

KUROYANAGI,*

BY CULTURED

CELLS OF

ZEDOARIA YOKO ISHITOBI, MAKOTO

SATO

and

AKIRA UENO

School of Pharmaceutical Sciences, University of Shizuoka, 395 Yada, Shizuoka-shi, 422 Japan (Received in reoisedform 19 March 1991)

Key Word Index-Curcuma zedoaria; Zingiberaceae; cell suspension culture; biotransformation; germacrone; guaiane; eudesmane.

sesquiterpene;

Abstract-The transformation of a lo-membered ring sesquiterpene, germacrone, into guaiane-type sesquiterpenes by suspension cultured cells of Curcuma zedoaria was investigated. Germacrone was converted into several sesquiterpenes, some of which were isolated from the Curcuma spp. plants through, the key intermediate, (4R,SR)-germacrone 4, S-epoxide, and their structures were determined by spectral and chemical evidence. The configurations of some of the derivatives were opposite to those of the sesquiterpenes isolated from the Curcuma sp plants, C. aromatica, C. longa and C. zedoaria. An eudesmane-type product was also isolated and its structure was determined. From the structure, the transformation of germacrone through the intermediate, (lS, lOS)-germacrone 1, lo-epoxide, was supposed and further confirmed by the transformation of germacrone 1, lo-epoxide into the same product by acid treatment. The structures of two new products were also determined and their transformation mechanism through curcumenone and dihydrocurcumenone was deduced.

INTRODUCTION

Suspension cultured cells of plants are used for biotransformations of organic compounds such as monoterpenes [l], diterpenes [2], steroids [3], triterpenes [4] alkaloids [S] and some synthetic chemicals [6]. The ability of cultured plant cells to transform some organic compounds is useful for mass-production of substances and for biosynthetic experiments on the secondary metabolites of plants. Germacrane type sesquiterpenes are considered to be one of the most important common intermediates for guaiane, eudesmane, pseudoguaiane, elemane and some other types of sesquiterpenes. Of the germacrane type sesquiterpenes, germacrone (1) is the most important and basic sesquiterpene for biosynthesis. In particular the transannular cyclization reaction [7] of the germacrane-type sesquiterpenes is very interesting. We have induced a callus from the shoot tips of Curcuma zedoaria. The C. zedoariu callus showed efficient growth and was subcultured for two years. Also, the isolation of many kinds of sesquiterpenes from the rhizomes of C. aromatica was carried out [8,9]. Some of these constituents have also been isolated from other Curcuma, such as C. zedoariu [lo, 111 and C. bngu [12,13]. From the structures of the constituents, the biogenesis of these sesquiterpenes was assumed in which germacrone was transformed into guaiane type sesquiterpenes through the key intermediate, germacrone 4,5-epoxide as shown in Scheme 1. We carried out the transannular cyclization of the key intermediate, (4S, 5S)-germacrone 4,5-epoxide, under several acidic and thermal conditions [14] to give several guaiane-type sesquiterpenes. The transannular

*Author to whom correspondence should be addressed

cyclization [7J of germacrone into several kinds of guaiane type sesquiterpenes by the callus suspension culture of C. zedouriu is interesting with regard to the confirmation of the biosynthetic pathway. Consequently we have investigated the biotransformations of 1,2 and 5 by the C. zedouria suspension cells. The structures of the resultant products were determined from their ‘H and 13CNMR spectra, and by the identification of some of them with the authentic natural products.

RESULTS AND

DISCUSSION

The callus of Curcuma zedoaria was induced only from shoot tips, and subcultured for more than two years in Murashige-Skoog’s agar medium [lS] supplemented with 1 mgl- ’ of 2,4-dichlorophenoxyacecetic acid and 0.2 mg l- 1 of kinetin. (4S,SS)-Germacrone 4,5-epoxide (2) was administered to the suspension of cultured cells and incubated for 10 days to produce 3-6,10 and 11. The ‘H NMR and mass spectra of these products indicated that 3-5,lO and 11 are zedoarondiol, isozedoarondiol, curcumenone, procurcumenol and 5-hydroxyprocurcumenol, respectively, and they were identical with the authentic samples. The ‘HNMR spectrum of 6, C,,H,,O1, revealed a similar signal pattern to that 5 except for the presence of a carbinyl proton (63.89m) and a doublet methyl group Me-14 (61.18, 3H, d, J=6.4Hz) instead of a carbonyl group and acetyl group at C-4. This was also revealed in the 13CNMR spectrum. These results indicated that the compound is a reduced product of the ketone group on the side chain of 5. Curcumenone (5) was reduced with sodium borohydride to give a monoalcohol, which was identical with the transformed product 6.

143

N.

144

SAKUIet al.

\/ \ FT0

1

Scheme 1. Hypothetical biogenesis of the sesquiterpenes of Curcuma sp.

The possibility of the transformation of 2 without the cultured cells to guaiane derivatives was observed. Therefore, a time course experiment was conducted comparing the results of change of 2 in both suspension mediums with and without the cells. Both media produced similar products, but the amounts of 5 and 10 decreased after seven days incubation in the medium with the cells, but did not decrease in the medium without cells. This suggested that 5 and 10 were further metabolized into other compounds. These facts indicated that spontaneous transformation of 2 partly contributes to the formation of guaiane type sesquiterpenes by the transannular cyclization reaction. The ability of the cells to reduce substances was anticipated. Consequently, the transformation of 5 by suspension of cultured cells was investigated to produce 6,12 and 13. The mass spectra of compounds 12 and 13 showed the fragments m/z 232 and 234 which were supposed to be the dehydration fragments of the molecular ions. This was suggested from the 13C NMR spectrum of 12 and 13 (Table 1). Thus, the molecular formulae of 12 and 13 were concluded to be C15HZ203 and C15H2_,03, respectively. The ‘H NMR spectrum of 12 and 13 showed the presence of newly arisen olefine protons (67.25, lH, d, J = 2.0 Hz and 7.24, lH, d, J = 2.0 Hz) and singlet methyl (61.34, 1.35 and 1.35, 1.36) corresponding to the two methyl groups. Other signals were similar to those of 5 and 6. The 13C NMR spectrum of 12 and 13 showed the presence of carbinyl carbons (671.7 and 71.8, respectively), which were assignable to the C-l 1 carbon. Other r3CNMR chemical shifts of 12 and 13 (Table 1) supported the assigned structures. The products 12 and 13 were assumed to be transformation products from 5 and 6, respectively, as shown in Scheme 2. Compounds 5 and 6 yielded 12 and 13 during the incubation with the

Table 1. “%NMR

spectral data of the sesquiterpenea (CD’&)

C

12

13

8

5

6*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

27.0 30.1 43.1 201.9 41.7 147.0 138.3 199.2 45.6 21.4 71.7 29.0 28.7 23.3 18.7

27.1 25.5 38.9 67.7 42.8 147.5 138.1 199.4 45.8 21.5 71.8 29.0 28.8 23.8 18.7

78.0 28.3 40.7 71.5 50.4 26.0 130.5 204.0 56.7 41.2 146.0 22.3 22.8 23.6 12.7

29.8 24.2 43.8 208.2 24.3 28.0 128.0 201.2 48.9 20.0 146.9 23.4 23.4 23.2 18.9

24.1 25.0 39.3 67.4 24.7 28.0 128.1 201.1 48.9 19.8 147.0 23.4 23.4 24.0 19.0

25.2 39.4 67.6

*Compound 6 is a mixture of epimers at C-4, so gave separate signals at C-2, C-3 and C-4 based on the epimers suspension cells, it is suggested that the cells carried out a redox reaction at the C-4 position of 5 and 6.

The incubation of germacrone (1) with the suspension cell culture of C. zedouria gave products 2’-6’,7- 9. These products were purified by silica gel column chromatography and high performance liquid chromatography (HPLC). The ‘HNMR and mass spectra of 2-6 and 7 suggested that these products are germacrone 4,5epoxide, zedoarondiol, isozedoarondiol, curcumenone, dihydrocurcumenone and 13-hydroxygermacrone, respectively. These structures were further confirmed by a

145

Sesquiterpenes biotransformations

0 H.OH

Scheme 2. Mechanism for the formation of compounds 12 and 13 from 5 and 6.

direct comparison

with the authentic samples by TLC, spectrum. Some of the compounds have asymmetric centres and, therefore, were measured by circular dichroism (CD). The CD spectrum of 3’ and 4’ gave a negative Cotton effect at 320 and 312 nm, respectively, based on rz-+z* transition of the a,~-unsaturated ketone [ 161. The CD spectrum of compound 2’ gave a positive Cotton effect at 309 nm, based on n-+8* transition of the &y-unsaturated ketone [17]. The CD spectra were opposite to the CD spectra [8] of 2-4 isolated from Curcumu. These results indicate that the absolute configurations of r-5’ are opposite to those of the 2-5 isolated from the plants. The transformation of I by the cultured cells of C. ze~~ri~ produced the enttype sesquiterpenes via (4R,5R~germacrone 4,5-epoxide, which was the opposite configuration of the 4,5-epoxide isolated from the parent plants. The ‘HNMR spectrum of 8, C,,H,,O,, showed the presence of four singlet methyl groups at 60.90,1.22,1.86 and 2.13. Of these, the methyl group at 60.90 is assignable to an angular methyl group of the eudesmane-TV of derivative and the methyl group at 6 1.22 to the carbinyl methyl group. The other two methyl groups were suggested to be vinyl methyl groups from their ‘HNMR chemical shifts. The methyl groups at 60.90 had a long range coupling with one of the methylene protons at C-9. This suggested that the methyl group is most probably axial. The signal pattern of the methylene protons at C-6 indicated that the H-S proton should be axial. Thus, 8 was shown to be a truns-eudesmane derivative. The CD spectrum of 8 indicated the positive Cotton curve at HPLC

and the ‘HNMR

322 nm based on ~-+IE* transition of an a,~-un~turated ketone. This positive CD curve [16] suggested the righthanded helicity between the carbonyl and olefin groups. The absolute configuration of 8 was revealed to be as shown in structure 8. This structure was further confirmed by synthesis from germacrone &IO-epoxide (9). The epoxide 9 was treated with acid in methanol to produce the methyl ether {14) of 8, but in dioxane-water the product was 8, which was identical with the original compound 8 in all respects. The epoxide has not been isolated as a pure compound from the incubated suspension medium, but the presence of 9 in the reaction solution with the suspension culture was determined by HPLC and TLC. Generally, acid treatment of germacrone+-epoxide produces several guaiane-type derivatives, but germacrone-l,lO-epoxide produces some eudesmane-type derivatives [18]. From the biotransformation of germacrone with the C. zedoaria suspension of cultured cells, the same results were obtained. From the transformation mechanism for the conversion of compound 9 to 8, the absolute co~guration of the intermediate epoxide (9), was deduced to be lS,lOS as shown in Scheme 3. The suspension of cultured cells was able to transform 1 to some guaiane-type derivatives through germacrone 4,5-epoxide and to a eudesmanetype sesquiterpene through germacrone 1, lo-epoxide. In the transformation of germacrones having no chirality to guaiane type se~uite~n~ that have chirality, the epoxidation by some kind of monooxygenase is a very important step for the establishment of the stereochemistry of the sesquiterpenes resulting from transannular cycliz-

R’

R’

OH bk,WH

Me OH.loH

Scheme 3. Transannular

cyclization mechanism for guaiane and eudesmane sesquitcrpenes from germacrone through the epoxide intermediates.

N. SAKUI et al.

146

6’

(4R,5R)- @‘nmc~ne 4,5_epoxide(2’) I

_______,

3’

I

1

[@Ji,~J-q/ II-

germacrone (1)

--_--_--_-____I

r___‘_-_____‘-

____

-,

I IL,__--__,_,______-__,l (5) c”rc”me”O”e

I =OH

(1S,1o.s)-germacrone 1,IO-cpoxide (9)

7

Scheme 4. Biotransformation

of germacrone and curcumenone by suspension cultured of C. zedoaria.

ation. Thus, the configurations of the guaiane type sesquiterpenes depend upon the stereochemistry of the epoxidation.

EXPERIMENTAL The ‘H NMR spectra were recorded at 400 and 90 MHz; the r3C NMR spectra were recorded at 67.9 and 22.5 MHz. HPLC was carried out on reversed phase column and using MeCN-H,O solvent system. Prep. TLC was carried out on silica gel PF,,, (200 x 200 x 0.8 mm) and hexane-EtOAc solvent system. Plant tissue cultures. The callus of Curcuma zedoaria was induced from the shoot tips of the plant in Murashige and Skoog’s agar medium supplemented with some plant growth regulator combinations. Curcuma zedoaria callus showed efficient growth. The callus, after being s&cultured for more than 2 years, was transferred into Murashige-Skooge’s suspension culture supplemented with 2,4-dichlorophenoxyacetic acid-kinetine (l-O.2 mgl-I). Biotransformation of (4S,SS)-germacrone 4,5-epioxide (2), germacrone (1) and 5. Substrate 2 (40 mg) in 0.8 ml of EtOH was poured into a 200ml suspension culture of C. zedoaria cells

through a Milipore filter. A total of 200 mg of 2 was incubated for 8 days. Substrate 1 was very hydrophobic and hardly dissolved in H,O. Subsequently 20 mg of 1 was dissolved in 1 ml EtOH and poured into each 200 ml of suspension culture of C. zedoaria cells in 500 ml conical flasks through a Milipore filter; a total of 200 mg of 1 was incubated for 10 day at 25 ’ under 12 hr tight per day. Substrate 5 was incubated in the same way.

Isolation and purification of the products. The incubated suspension cultures with 2 were separated into medium and cells and both were separately extracted with EtOAc. The EtOAc extracts showed almost the same TLC patterns. Subsequently the two extracts were combined and evapd to give a pale yellow oil (250 mg). The extract was sepd on prep. TLC to give 8 frs. Some of these frs were further purified on HPLC (YMC, ODS-7, 30-50% MeCN, detection at 220 nm) to give products 3-6’,10 and 11. The incubated suspension culture of 1 was also separated by the same procedure as used in the case of 2 to give products z-6’, 7-9. The incubated suspension culture of 5 was simrlarly separated by the same procedures as above to give products 6,12 and 13. Structkes of 3,4,5,6,10 and 11. From the ‘H NMR spectra of the products the structures were deduced to be &6,10 and 11, respectively. These compounds were identical with the authentic samples on TLC, HPLC and by the ‘H NMR spectra. Structures of 2’, 3’, 4, S’, 6’ and 7. The structures were assigned from the ‘H NMR spectra of the products and identify with their authentic samples on TLC, HPLC and the ‘HNMR spectra. The CD spectrum of 2’. 3’ and 4’ were shown as [f?] 569, mm f901 and [8],,2 + 1040 (MeOH) respecttvely?‘Product 6. MS m/z: 236 [M]’ for ‘c1s H 24 0 2 ‘HNMR (CDCl,); 1.12 (3H, s, Me-15), 1.18 (3H, d, J=6.4Hz, Me-14), 1.793, 2.09 (3H each, s, Me-12, Me-13), 3.78 (lH, m, H-4). Product 8. MS m/z: 252.1714 [M]’ (252.1725, Calcd. for CISH,,O,). ‘H NMR (CD&); 60.90 (3H, s, Me-15), 1.22 (3H, s, Me-14), 1.86, 2.03 (3H each, s, Me-12 13), 2.13 (lH, d, J =15.2Hz, H-10), 2.24(1H, brt, J=15.OHz, H-6), 2.56(1H,d, J = 15.2 Hz, H-lo), 2.98 (lH, dd, J=4.4,15.0 Hz, H-6). CD; [B],,, +3000 (MeOH). The 13CNMR spectrum is shown in Table 1.

Sesquiterpenes biotransformations Product 12. MS m/z: 232 [M-H,O]+, Ci5Hz402. ‘HNMR (CD&): 60.43 (lH, dt, 5=4.4, 7.6 Hz, H-l), 1.24 (3H, s, H-15), 1.34 and 1.35 (3H each, s, H-12 and H-13), 1.73 (2H, dt, J=7.6, 7.5 Hz, H-2), 2.15 (3H, s, H-14), 2.52 (lH, d, J=18.0Hz, H-9), 2.54 (2H, t, J=7.5 Hz, H-3), 2.78 (lH, dd, J= 1.4, 18.0 Hz, H-9), 7.2 (lH, d, J=6.3 Hz, H-6). The ‘“CNMR is shown in Table 1. Product 13. MS m/z: 234 [M -H,O]+, (CisHzzOz). ‘HNMR (CD&): 60.43 (lH, m, H-l), 1.21 (3H, d, J =6.4 Hz, H-14), 1.24 (3H,s, H-15), 1.35 and 1.36(3H each, s, H-12 and H-13), 2.52(1H, d, J= 18.0 Hz, H-9), 2.72 (IH, d, J= 18.0 Hz, H-9), 3.80 (lH, m, H-4), 7.22 (lH, d, J=6.3 Hz, H-6). The ‘“CNMR is shown in Table 1. NaBH, reduction ofcurcumenone (5). To the soht of 100 mg of 5 in 5 ml of MeOH, NaBH, was added. TLC was employed to check for the formation of monoalcohol derivatives. The addition of NaBH, was stopped at the stage when the presence of 5 was no longer indicated. The reaction soht was poured into icewater and extracted with EtOAc. The EtOAc extract was purified by silica gel CC to give 80 mg powder. The ‘“C NMR spectrum is shown in Table I. Product 6 and synthetic 6 were identical to each other on TLC, HPLC and the ‘HNMR SpCtlllIIl.

Epoxidation of1. To a soln of 200 mg of germacrone in 40 ml of CHCl, was added m-chloroperbenzoic acid (1.1 equivalent), and the mixture stirred for 30 min at room temp. The reaction soln was evapd in uacuo, then purified by prep. TLC to give two epoxides, l,lO-epoxide (33 mg) and 4,S-epoxide (144 mg), respectively. The 4,5-epoxide was identical with the authentic natural germacrone 4.5-epoxide. Gcrmacrone -l,lO-qoxide. MS m/z: 234 [M]’ for C15”r40,. ‘HNMR(CDC1,): 6 1.27 (3H, s, Me-15) 154(3H,s, Me-14), 1.67, 1.77(3Heach,s,Me-12,Me-13~5.00(1H,dd,J=5.4,10.8Hz,H5). i3CNMR (CDCI,): 815.5 (C-15), 17.3 (C-14), 19.9 (C-13), 22.1 (C-12), 23.4 (C-3), 29.8 (C-2), 36.3 (C-6), 55. 3 (C-9), 57.6 (C-lo), 64.3 (C-l), 123.6 (C-5), 129.9 (C-7), 134.0 (C-4), 138.1 (C-11). Acid treatment of germacrone 1,10_epoxide. To the soln of racemic 9 (10 mg) in 2 ml of MeOH was added 0.4 ml of 1M aq. HCl and the mixture was stirred for 1 hr at room temp. To the reaction soht, ice- Hz0 was added followed by extraction with CHCl, to give a pale yellow oil, which was purified by prep. TLC to give 5 mg of 14. ‘H NMR (CD&): SO.95(3H, s, Me-15), 1.18 (3H, s, Me-14), 1.83,2.03 (3H each, s, Me-12, Me-13), 3.18 (3H, s, MeO). 13CNMR(CDCls): 613.0,18.3,22.0,23.3,23.4,25.9,28.3, 34.5, 41.1, 43.7, 48.0, 57.0, 130.6, 144.4, signals of two carbinyl carbons and one carbonyl carbon could not be detected because of the scarcity of the sample. The same reaction was carried out

147

in dioxane-Hz0 soln to give racemic 8. This was identical with product 8 in all respects. Acknowledgements-We thank Dr K. Shimomura (Tsukuba Medicinal Plant Research Station, National Institute of Hygienic Sciences) for the suggestion about cell culture and Dr K. Koyama (Meiji College of Pharmacy) for the measurement of 400 MHz NMR spectrum. REFERENCES

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