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Enantio- and diastereoselectivity in the biotransformation of carveols by Euglena gracilis Z
Phytochemistry,Vol.
Printedin Great
31, No. 6, pp. 2009-2011, 1992 Britain.
0031-9422/92 $S.CO+O.OO Pergamon Press Ltd
ENANTIO- AND DIASTEREOSELECTIVITY IN THE BIOTRANSFORMATION OF CARVEOLS BY EUGLENA GRACILIS Z YOSHIAKI
NOMA
and YOSHINORIASAKAWA*
Faculty of Domestic Sciences and *Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 110, Japan (Received 30 September 1991) Key Word Index-Euglena gracilis Z; biotransformations; monoterpenoids; terpene alcohols; (- )-, (+)- and (+)trans-carveols; (-)-, (+ )- and ( f )-cis-carveols; enantioselectivity; diastereoselectivity.
Abstract-The enantio- and diastereoselective biotransformation of (+)-, (- )- and (_+)-trans-carve01 and cis-carve01 by Euglena gracilis Z cultured photoheterotrophically has been investigated. Though (-)-cis- and (+)-trans-carveols were not transformed at all, both (-)-tram- and (+ )-cis-carveols were dehydiogenated to the corresponding carvones, which were further transformed uia dihydrocarvones to dihydrocarveols. Furthermore, enantio- and diastereoselectivity in the biotransformation of (+)-tram- and (-+)-cis-carveols and the mixture of diastereomers were observed.
INTRODUCTION In previous papers [l, 21, we reported on the biotransformation of terpene and related aldehydes to the corresponding primary alcohols by Euglena gracilis Z cultured photoheterotrophically. In our continuing biotechnological studies of the metabolism of terpenoids by Euglena, we chose carveols (microbial and plant cell metabolites of d-limonene [3--s] and carvone [6]) as substrates with a view to developing a procedure for the useful utilization of Citrus oil as biomass. .Eugl& converted carveols with high enantio- and diastereoselective dehydrogenation to give carvone. We now report on the enantio- and diastereoselectivity in the biotransformation of (-)-tramand (-)-I%carveol(1 and 2), (+)-trans- and (+)-cis-carveol(6 and 7), (+)-tram-carve01 (1 and 6) and (+)-cis-carveol (2 and 7) by E. gracilis Z.
L
5
2
4
6
89
Tune (days)
z f
so
RESULTSANDDISCUSSION The time courses for the biotransformations of compounds 1,2,6,7,1 and 6 and 2 and 7 by E. gracilis Z are shown in Fig. 1. Compound 1 was transformed stereospecifically via (-)-carvone (3) and (+)-dihydrocarvone (4) to (+)-neodihydrocarveol (5) as the major product (Fig. 2). Furthermore, when either 3 or 4 was used as the substrate, 5 was specifically formed. However, 2 was not transformed. On the other hand, in the cases of 6 and 7,7 was transformed via (+)-carvone (8) and (-)isodihydrocarvone (9) to (-)-isodihydrocarveol (10) as major product and (-)-neoisodihydrocarveol (11) as minor product (Figs 1 and 2), whereas 6 was not transformed. Compound 8 was also transformed via 9 to 10 and 11. When the mixture of acetates (12 and 13,49:51, peak area in GC) was added into the culture broth, hydrolysis of both acetates occurred to give 6 and 7 (49:51, peak area in GC), of which only 7 was diastereoselectively transformed in the same manner as
0
2
4
6
8
10
Tune (days)
Time (days)
3
zs
50
E 0
2
4
6
8
10
The WY4
12
14
16 0
2
4 lime
6
8
10 11
(days)
1. Time courses for the biotransformations of ( - )mm- and (1and 2), ( +)-tram- and ciwxveol(6 and 7) and ( + )trans- and cis-carveol (1 and 6, and 2 and 7) by E gracilis Z. 3, (-)-carvone; 5, (+ )-neodihydrocarveol; 10, (-)-isodihydrocarveol; 11,(-)-neoisodihydrocarveol. Fii
cis-carveol
2009
Y. NOMAand Y. ASAKAWA
2010
described above. When either (f )-trans-carve01 (1 and 6) or ( -I)-cis-carve01 (2 and 7) was added to the culture broth, enantioselectivity of the biotransformation was observed (Fig. 1). The same enantioselectivity for 1 and 6 and 2 and 7 and diastereoselectivity for 1 and 2 as well as 6 and 7 were observed when different concentrations (50-500 ppm, 50 ppm intervals) of each substrate were added to the cultures. In contrast to Pseudomonas oualis strain 6-l [A, Streptomyces bottropensis SY-2-1 [S-lo] and Aspergillus niger (Noma and Asakawa, unpublished results) which are unable to bring about either the diastereo- or the enantioselective transformation of carveols, Euglena has the ability to bring about the efficient enantio- and diastereoselective transformation of trans- and cis-carveols. The same result had been obtained for the biotransformation of carveols with cultured cells of Nicotiuna tabacum [ll] (Table I). On the basis of the above result, it is considered that Euglena is a good bioreactor for the enantioselective and diastereoselective transformation of monoterpenoids. EXPERIMENTAL Cultrvation and biotransformation. Euglena gracilis Z was photoheterotrophically (light illumination at ca 2OOG3000 lux) and statically at 25-30” for 7 days m 50 ml of a
cultivated
medmm containmg KH,PO, (0.4 g). (NH&HPO, (0.2 g), MgSO,. 7 H,O (0.5 g), CaCO, (0.2 gh !x-malate (2 g), Naglutamate (5 g), EDTA .2 Na (SOmg), &SO,. 7 H,O (22 mg), MnSO, H,O (5.8 mg), FeSO,(NH,), SO, 6 H,O (5.7 mg). Na,MoO,.H,O (1.5 mg), C&O,.5 H,O (1.6 mg), CoSO,.7 H,O (1.5mg), H,BO, (11.4mg), Vitamm B, (2.5mg) and Vitamin B,, (0.02 mg) in 1 I distilled water (pH 3.3, adjusted with HCl) [12]. After full growth, substrate (5C-500ppm) and aliquots (5 ml) of the cultured broth of Euglena were added in the test tube cell and biotransformation was carned out for l-12 days. After reaction, Euglena was removed by centrifugation at 1000 rpm for 5 min. and the broth extracted with 2 ml Et,O. Each extract was analysed by CC and GC-MS. The products were identified by comparison of R,s and mass spectra with those of authentic speamens. Acknowledgements-The authors wish to express their gratitude to Nippon Terpene Chemical Co. Ltd. for supplying the terpenes and Emeritus Prof. Shouzaburo Kitaoka, University of Osaka Prefecture, for supplying the culture of Euglena
REFERENCES 1. Noma, Y., Takahashi, H. and Asakawa, Y. (1991) Phytochemistry 30, 1147. 2. Noma, Y., Okajlma, Y., Takahashi. H. and Asakawa, Y. (1991) Phytochemistry 30, 2969. 3. Noma, Y.. Yamasaki, S. and Asakawa, Y. (1992) Phytochemistry (in press). 4. Dhavalikar, R. S. and Bhattacharyya, P. K. (1966) Zndian J. Biochem. 3, 144. 5. Bowen, E. R. (1975) Florida State Horticultural Sot. 88,304. 6. Fujita, S. and Nedzu, K. (1986) 30th Symposium on the Chemistry of Terpenes, Essential Oils and Aromatics of Japan, Symposium Paper, p. 201, Hiroshima. 7. Noma, Y. (1977) Nippon Nogeikagaku Knrshi 51,463. 8. Noma, Y., Nishimura, H., Hiramoto, S., Iwami, M. and Tatsumi. C. (1982) Agric. Biol. Chem. 46, 2871.
Biotransformation
of carveols
2011
11
13
6
Fig. 2. Metabolic pathways for the biotransformation of (- )-tram- and (+ )-cis-carveol(1 and 7) by E. gracilis Z. 2, (-)-cis-Carveok 3, (-)-carvone; 4, (+)-dihydrocarvone; 5, (+ )-neodihydrocarveol; 6, (+)-trans-carveol; 8, (+ )carvone; 9, (-)-isodihydrocarvone; 10, (-)-isodihydrocarveol; 11, (-)_neoisodihydrocarveok 12, (+ )_Cis-CarVeYf acetate; 13, (+ )-trans-carveyl acetate.
9. Nishimura, H., Hiramoto, S., Mizutani, J., Noma, Y., Furusaki, A. and Matsumoto,T. (1983) Agric. Biol. Chem. 47, 2697. 10. Noma, Y. and Nishiiura, H. (1987) Agric. Biol. Chem. 51, 1845.
11. Suga, T. and Hirata, T. (1983) J. Chem. Sot. Jpn, Chem. Ind. C/tern. 1345. 12. Hutner, S. H. (1959) J. Protozool. 6, 23.
Printedin Great
31, No. 6, pp. 2009-2011, 1992 Britain.
0031-9422/92 $S.CO+O.OO Pergamon Press Ltd
ENANTIO- AND DIASTEREOSELECTIVITY IN THE BIOTRANSFORMATION OF CARVEOLS BY EUGLENA GRACILIS Z YOSHIAKI
NOMA
and YOSHINORIASAKAWA*
Faculty of Domestic Sciences and *Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 110, Japan (Received 30 September 1991) Key Word Index-Euglena gracilis Z; biotransformations; monoterpenoids; terpene alcohols; (- )-, (+)- and (+)trans-carveols; (-)-, (+ )- and ( f )-cis-carveols; enantioselectivity; diastereoselectivity.
Abstract-The enantio- and diastereoselective biotransformation of (+)-, (- )- and (_+)-trans-carve01 and cis-carve01 by Euglena gracilis Z cultured photoheterotrophically has been investigated. Though (-)-cis- and (+)-trans-carveols were not transformed at all, both (-)-tram- and (+ )-cis-carveols were dehydiogenated to the corresponding carvones, which were further transformed uia dihydrocarvones to dihydrocarveols. Furthermore, enantio- and diastereoselectivity in the biotransformation of (+)-tram- and (-+)-cis-carveols and the mixture of diastereomers were observed.
INTRODUCTION In previous papers [l, 21, we reported on the biotransformation of terpene and related aldehydes to the corresponding primary alcohols by Euglena gracilis Z cultured photoheterotrophically. In our continuing biotechnological studies of the metabolism of terpenoids by Euglena, we chose carveols (microbial and plant cell metabolites of d-limonene [3--s] and carvone [6]) as substrates with a view to developing a procedure for the useful utilization of Citrus oil as biomass. .Eugl& converted carveols with high enantio- and diastereoselective dehydrogenation to give carvone. We now report on the enantio- and diastereoselectivity in the biotransformation of (-)-tramand (-)-I%carveol(1 and 2), (+)-trans- and (+)-cis-carveol(6 and 7), (+)-tram-carve01 (1 and 6) and (+)-cis-carveol (2 and 7) by E. gracilis Z.
L
5
2
4
6
89
Tune (days)
z f
so
RESULTSANDDISCUSSION The time courses for the biotransformations of compounds 1,2,6,7,1 and 6 and 2 and 7 by E. gracilis Z are shown in Fig. 1. Compound 1 was transformed stereospecifically via (-)-carvone (3) and (+)-dihydrocarvone (4) to (+)-neodihydrocarveol (5) as the major product (Fig. 2). Furthermore, when either 3 or 4 was used as the substrate, 5 was specifically formed. However, 2 was not transformed. On the other hand, in the cases of 6 and 7,7 was transformed via (+)-carvone (8) and (-)isodihydrocarvone (9) to (-)-isodihydrocarveol (10) as major product and (-)-neoisodihydrocarveol (11) as minor product (Figs 1 and 2), whereas 6 was not transformed. Compound 8 was also transformed via 9 to 10 and 11. When the mixture of acetates (12 and 13,49:51, peak area in GC) was added into the culture broth, hydrolysis of both acetates occurred to give 6 and 7 (49:51, peak area in GC), of which only 7 was diastereoselectively transformed in the same manner as
0
2
4
6
8
10
Tune (days)
Time (days)
3
zs
50
E 0
2
4
6
8
10
The WY4
12
14
16 0
2
4 lime
6
8
10 11
(days)
1. Time courses for the biotransformations of ( - )mm- and (1and 2), ( +)-tram- and ciwxveol(6 and 7) and ( + )trans- and cis-carveol (1 and 6, and 2 and 7) by E gracilis Z. 3, (-)-carvone; 5, (+ )-neodihydrocarveol; 10, (-)-isodihydrocarveol; 11,(-)-neoisodihydrocarveol. Fii
cis-carveol
2009
Y. NOMAand Y. ASAKAWA
2010
described above. When either (f )-trans-carve01 (1 and 6) or ( -I)-cis-carve01 (2 and 7) was added to the culture broth, enantioselectivity of the biotransformation was observed (Fig. 1). The same enantioselectivity for 1 and 6 and 2 and 7 and diastereoselectivity for 1 and 2 as well as 6 and 7 were observed when different concentrations (50-500 ppm, 50 ppm intervals) of each substrate were added to the cultures. In contrast to Pseudomonas oualis strain 6-l [A, Streptomyces bottropensis SY-2-1 [S-lo] and Aspergillus niger (Noma and Asakawa, unpublished results) which are unable to bring about either the diastereo- or the enantioselective transformation of carveols, Euglena has the ability to bring about the efficient enantio- and diastereoselective transformation of trans- and cis-carveols. The same result had been obtained for the biotransformation of carveols with cultured cells of Nicotiuna tabacum [ll] (Table I). On the basis of the above result, it is considered that Euglena is a good bioreactor for the enantioselective and diastereoselective transformation of monoterpenoids. EXPERIMENTAL Cultrvation and biotransformation. Euglena gracilis Z was photoheterotrophically (light illumination at ca 2OOG3000 lux) and statically at 25-30” for 7 days m 50 ml of a
cultivated
medmm containmg KH,PO, (0.4 g). (NH&HPO, (0.2 g), MgSO,. 7 H,O (0.5 g), CaCO, (0.2 gh !x-malate (2 g), Naglutamate (5 g), EDTA .2 Na (SOmg), &SO,. 7 H,O (22 mg), MnSO, H,O (5.8 mg), FeSO,(NH,), SO, 6 H,O (5.7 mg). Na,MoO,.H,O (1.5 mg), C&O,.5 H,O (1.6 mg), CoSO,.7 H,O (1.5mg), H,BO, (11.4mg), Vitamm B, (2.5mg) and Vitamin B,, (0.02 mg) in 1 I distilled water (pH 3.3, adjusted with HCl) [12]. After full growth, substrate (5C-500ppm) and aliquots (5 ml) of the cultured broth of Euglena were added in the test tube cell and biotransformation was carned out for l-12 days. After reaction, Euglena was removed by centrifugation at 1000 rpm for 5 min. and the broth extracted with 2 ml Et,O. Each extract was analysed by CC and GC-MS. The products were identified by comparison of R,s and mass spectra with those of authentic speamens. Acknowledgements-The authors wish to express their gratitude to Nippon Terpene Chemical Co. Ltd. for supplying the terpenes and Emeritus Prof. Shouzaburo Kitaoka, University of Osaka Prefecture, for supplying the culture of Euglena
REFERENCES 1. Noma, Y., Takahashi, H. and Asakawa, Y. (1991) Phytochemistry 30, 1147. 2. Noma, Y., Okajlma, Y., Takahashi. H. and Asakawa, Y. (1991) Phytochemistry 30, 2969. 3. Noma, Y.. Yamasaki, S. and Asakawa, Y. (1992) Phytochemistry (in press). 4. Dhavalikar, R. S. and Bhattacharyya, P. K. (1966) Zndian J. Biochem. 3, 144. 5. Bowen, E. R. (1975) Florida State Horticultural Sot. 88,304. 6. Fujita, S. and Nedzu, K. (1986) 30th Symposium on the Chemistry of Terpenes, Essential Oils and Aromatics of Japan, Symposium Paper, p. 201, Hiroshima. 7. Noma, Y. (1977) Nippon Nogeikagaku Knrshi 51,463. 8. Noma, Y., Nishimura, H., Hiramoto, S., Iwami, M. and Tatsumi. C. (1982) Agric. Biol. Chem. 46, 2871.
Biotransformation
of carveols
2011
11
13
6
Fig. 2. Metabolic pathways for the biotransformation of (- )-tram- and (+ )-cis-carveol(1 and 7) by E. gracilis Z. 2, (-)-cis-Carveok 3, (-)-carvone; 4, (+)-dihydrocarvone; 5, (+ )-neodihydrocarveol; 6, (+)-trans-carveol; 8, (+ )carvone; 9, (-)-isodihydrocarvone; 10, (-)-isodihydrocarveol; 11, (-)_neoisodihydrocarveok 12, (+ )_Cis-CarVeYf acetate; 13, (+ )-trans-carveyl acetate.
9. Nishimura, H., Hiramoto, S., Mizutani, J., Noma, Y., Furusaki, A. and Matsumoto,T. (1983) Agric. Biol. Chem. 47, 2697. 10. Noma, Y. and Nishiiura, H. (1987) Agric. Biol. Chem. 51, 1845.
11. Suga, T. and Hirata, T. (1983) J. Chem. Sot. Jpn, Chem. Ind. C/tern. 1345. 12. Hutner, S. H. (1959) J. Protozool. 6, 23.