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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.51 no.2 Concepción June 2006

http://dx.doi.org/10.4067/S0717-97072006000200004 

 

J. Chil. Chem. Soc., 51, Nº 2 (2006) , pags: 855-857

 

REGIO AND STEREOSELECTIVE HYDROXYLATION OF 5a-HYDROXY-14-EUDESM-11-EN-3-ONE EUDESMANE BY BIOTRANSFORMATION OF RHIZOPUS NIGRICANS.

 

SERGIO ÁGUILA1, JULIO ALARCÓN2, CAROLINA CONEJEROS2 AND JOEL B. ALDERETE1,*

1Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad de Concepción, Concepción, Chile.
2Departamento de Ciencias Básicas, Facultad de Ciencias, Universidad del Bío-Bío, Chillán, Chile.


ABSTRACT

This paper describes a combined synthesis of b-hydroxy-sesquiterpen eudesmane derivative using a microbiological hydroxylation sesquiterpen eudesmane derivative as the key reaction. The stereochemistry of the biohydroxylation was identified using mono and bi-dimensional 1H-NMR experiments.

Keywords: Biotransformation, Sesquiterpenes, Stereoselective Hydroxylation, Agarofurans


INTRODUCTION

Sesquiterpen eudesmane derivative is a pathway in the chemical synthesis of natural agarofurans isolated from plants of the family Celastraceae. Several of these compounds exhibit insecticide, insect antifeedant, cytotoxic, antitumor and inmunosuppresive activities1-10. It has been reported that when increasing the amount to hydroxyl groups in the molecule, a increment of biological activity can be observed11. Of the several routes for the production of synthetic agarofurans12-14, we have employed annelation of Robinson combined with microbiological hydroxylation in the synthesis of hydroxylated agarofuran12 .The present work reports the dehydration of the C5 position and the incorporation of hydroxyl groups in the 5a-hydroxy-14-eudesm-11-en-3-one (2) by Rhizopus nigricans. The biotransformation of Rhizopus nigricans was observed to be regio and stereoselective, obtaining two compounds: a monohydroxylated derivative (3) and a dihydroxylated (4) derivative (Scheme 1). The derivatives 3 and 4 are useful as intermediates in the formation of synthetic agarofurans since it is possible to reduce the total agarofuran synthesis into only two steps12,15-16.

Biotechnological techniques were selected because several approaches for hydroxylated agarofuran synthesis have been reported in the literature12, with in general unsatisfactory yields. In addition, a major problem is the control of the stereochemistry of the hydroxyl groups, which is overcome using the biotransformation approach.

MATERIALS AND METHODS

Compound Characterizations:

1H and 13C NMR (250 and 62 MHz, respectively) spectra were acquired on Bruker AC-250 spectrometer; solvent: CDCl3. The 13C chemical shifts were assigned using a DEPT experiment with a flip angle of 135° and 13C-1H correlation spectra (XHCORR). The stereochemistry of biotransformation products was performed using NOESY experiment.

Analytical plates (silica gel, Merck 60 G) were rendered visible by spraying them with H2SO4 and subsequently heated to 120°.

Synthesis of 5a-hydroxy-14-eudesm-11-en-3-one (2)

Compound 2 was prepared from (+)-dihydrocarvone by a Robinson annelation reaction with ethyl vinyl ketone according to Alarcón et al.16 (Scheme 1). The structure of compound 2 was determined by NMR spectroscopy.


Scheme 1: Chemical synthesis of 11-hydroxy-14-noreudesman-3-one (2) and modification by microbiological biotransformation.

Organism, media and culture conditions

Rhizopus nigricans LSBPN025 was obtained from Colección de Cultivos, Laboratorio de Síntesis y Biotransformación de Productos Naturales, Facultad de Ciencias, Universidad del Bío-Bío, Chillán, Chile, and was kept in Hagen medium containing CaCl2 (0.005 %), KH2PO4 (0.0025 %), (NH4)2HPO4 (0.025 %), MgSO4 × 7 H2O (0.015%), FeCl3 (0.0012 %), malt extract (0.3 %) and glucose (1 %) in H2O at pH 6.5. Erlenmeyer flasks (250 ml) containing 125 ml of medium were inoculated with a dense suspension of R. nigricans. The cultures were incubated with shaking (100 rpm) at 25 °C for 7 days, after which a solution of the substrate in ethanol was added.

Biotransformation of 2.

The substrate 2 (300 mg) was dissolved in 3 ml ethanol and the solution was added in three equal portions to the three Erlenmeyer flask cultures, each containing 125 ml of medium. After incubation for 7 days in a shaker at room temperature, the cultures were filtered over filter paper, the cells were washed thoroughly with EtOAc, and the filtrates were pooled and extracted with EtOAc. The combined extracts were dried over anhydrous Na2SO4 and evaporated under reduced pressure to give a mixture of compound (300 mg). Chromatography on silica gel using n-hexane: EtOAc mixes produced 246 mg (82 %) of the starting material 2, 30 mg (10 %) of 3, 24 mg (8 %) of 4.

Spectroscopy data of compound 2:

[a]D15= +50º IR (thin film) n 3506, 2931, 2869, 1701, 1643, 1454, 1438, 1377, 1138, 1056, 1022, 991, 960, 887, 794 cm-1. MS: (C15H24O2, 236.3), m/z (relative intensity) = 236 (M+, 25), 218 (8), 203 (4), 175 (2), 161 (4), 152 (80), 137 (39), 123 (35), 109 (100), 95 (35), 81 (24), 55 (73). 1H NMR (250 MHz, CDCl3, d in ppm, respect to TMS). d= 4.68-4.65 (d, 2H, J= 8.0 Hz), 2.86 (q, 1H, J = 6.4 Hz), 2.65-2.5 (m, 1H), 2.37-2.28 (m, 1H), 2.26-2.15 (m, 1H), 2.14-2.00 (m, 1H), 1.95-1.80 (m, 1H), 1.68 (s, 3H), 1.63-1.51 (m, 3H), 1.49-1.35 (m, 3H), 1.23 (s, 3H), 1.13-1.06 (d, 1H, J = 13.1 Hz) and 1.03 (d, 3H, J = 6.7 Hz). 13C NMR (62.4 MHz, CDCl3, d in ppm, respect to TMS). d = 210.62, 149.26, 109.03, 77.94, 51.73, 39.70, 37.64, 37.54, 35.38, 33.39, 31.56, 25.76, 21.67, 20.79, 6.52.

Spectroscopy data of compound 3:

IR (thin film) n 3440, 2927, 2866, 1647, 1458, 1377, 1184, 1088, 929, 752 cm-1. MS: (C15H24O2, 236.3), m/z (relative intensity) = 236 (M+, 25), 218 (8), 203 (4), 175 (2), 161 (4), 152 (80), 137 (39), 123 (35), 109 (100), 95 (35), 81 (24), 55 (73). 1H NMR (250 MHz, CDCl3, d in ppm) d = 2.67-2.45 (m, 2H), 2.44-2.24 (m, 2H), 2.20-1.91 (m, 1H), 1.80 (s, 3H), 1.76-1.60 (m, 2H), 1.58-1.50 (m, 3H), 1.41-1.25 (m, 2H) 1.23 (s, 3H), 1.21 (s, 3H), 1.17 (s, 3H). 13C NMR (62.4 MHz, CDCl3, d = 198.63, 164.87, 129.80, 73.43, 44.47, 37.29, 36.36, 35.89, 34.05, 28.75, 27.45, 26.83, 24.64, 21.30, 11.26.

Spectroscopy data of compound 4:

IR (thin film) n 3417, 2931, 2866, 1651, 1454, 1377, 1326, 1184, 1018, 921, 860, 818 cm-1. MS: (C15H24O3, 252.3), m/z (rel. int.) = 251 (M+, 3), 234 (95), 221 (69), 203 (100), 177 (79), 175 (59), 163 (73), 161 (55), 147 (18), 133 (31), 119 (29), 107 (39), 105 (47), 91 (59), 75 (53), 57 (42), 55 (44). 1H NMR (250 MHz, CDCl3, d in ppm) d = 4.99 (d, 1H, J = 5 Hz ), 2.89-2.51 (m, 4H), 1.92 (s, 3H), 1.89-1.70 (m, 2H), 1.65-1.57 (m, 3H), 1.55-1.42 (m, 2H), 1.39 (s, 3H), 1.32 (s, 3H), 1.14 (s, 3H). 13C NMR (62.4 MHz, CDCl3, d in ppm), 199.92, 161.13, 133.15, 73.52, 69.60, 53.81, 37.92, 37.75, 35.09, 34.26, 30.05, 28.00, 25.10, 21.02, 11.18.

RESULTS AND DISCUSSION

Following a published procedure12, the 5a-hydroxy-14-eudesm-11-en-3-one (2) was prepared from (+)-dihydrocarvone (1) and ethyl vinyl ketone. The structure of compound (2) was confirmed by 1H and 13C NMR spectroscopy.

In a typical aerobic fermentation, substrate 2 was incubated with Rhizopus nigricans for 7 days. Extraction of the reaction mixture followed with EtOAc and purified by High Pressure Liquid Chromatography gave 82 % of unconverted 2, 10 % of 3 and 8 % of 4.

The stability of 2 toward the incubation medium was investigated as a blank experiment to test the possibly of non-enzymatic reactions. Thus, compound 2 was incubated in a culture medium without R. nigricans, 99.9 % of unaltered substrate (2) was recovered. The Rhizopus nigricans participated in two biotransformation processes, the dehydration of the C5 position and the hydroxylation of the C6 and C11 positions, producing 3 and 4 compound (Scheme 1).

The double-bond of isopropenyl group elimination performed by Rhizopus nigricans, which produces the hydroxylated product at the C11 position, could occur via an intermediate epoxide although this species was not observed. The formation of intermediate epoxide has also been reported in the limonene17 and other sesquitepenes18 biotransformations. Furthermore, the intermediate epoxide product was not observed since these epoxides are very instable in aqueous systems19-21.

Table 1 displays the 13C-NMR spectra of compounds 2, 3 and 4. It can be observed that the double-bond of isopropenyl group of 2 was transformed and C11 y C12 resonance signals in the compound 3 were displaced at 73.43 and 26.83 ppm, respectively (see Table 1). Analogously, in the derivative 4 the C11 and C12 NMR signals appear at 73.52 and 28.00 ppm, respectively. Therefore, it can be inferred that C11 was hydroxylated and C12 was modified at methyl group, as was confirmed from DEPT and HETCORR experiments. The hydroxyl group at C11 position produces a g-effect, attributed to the steric compression of gauche interactions, which induces a shielding of approximately 4.5 ppm at C8 signal in the compounds 3 and 4.

Table 1. 13C-NMR of compounds 2, 3, 4. Chemical shifts in ppm.

Carbon
2 3 4

C1

31.56

37.29 37.75
C2 37.64 34.05 34.26
C3 210.62 198.63 199.92
C4 51.73 129.80 133.15
C5 77.94 164.87 161.13
C6 33.39 27.45 69.60
C7 39.70 44.47 53.81
C8 25.76 21.30 21.02
C9 35.38 36.36 37.92
C10 37.54 35.89 35.09
C11 149.26 73.43 73.52
C12 109.03 26.83 28.00
C13 20.79 24.64 25.10
C14 6.52 28.75 30.05
C15 21.67 11.26 11.18

Hydroxylation at C6 in the compound 4 can be directly inferred from its chemical shift (69.60 ppm). The hydroxylation configuration at C6 by Rhizopus nigricans was spectroscopically determinated. The stereochemistry of 4 was established by using 2D-NOESY experiment. The most relevant dipolar coupling was observed between H6 and H15 (Figure 1). This feature shows that the hydroxylation occurred in the b-space configuration, showing the stereoselectivity of the biotransformation process. Furthermore, the proton-proton coupling constant between H6 and H7 is 5 Hz. This value is greater than the J value (1.3 Hz) reported for a-hydroxylated sesquiterpen eusdemane derivatives previously synthesized22.


Fig. 1: Computer-generated drawing of compound 4 which showed NOE between H-6 and H-15

In conclusion, we have shown that the microbiological hydroxylation of 5a-hydroxy-14-eudesm-11-en-3-one eudesmane 2 provides an acceptable yield of 3 and 4. This procedure may be a convenient new route to the synthesis of polyhydroxyagarofurans since the products are useful as intermediates in the formation of synthetic agarofurans.

ACKNOWLEDGEMENTS

We are grateful to Departamento de Ciencias Básicas de la Universidad del Bío-Bío and Dirección de Investigación de la Universidad de Concepción.

 

REFERENCES

1. Kuo Y. H., King M. L. Chen C. F., Chen C. H., Chen K. and Lee K. H. (1994) J. Nat. Prod., 57, 263-269.         [ Links ]

2. Takishi Y., Ujita K., Tokuda H., Nishino H., Iwashima A. and Fujita T. (1992) Cancer Lett., 65, 19-26.         [ Links ]

3. Zheng Y. L., Xu L. and Liu J. F. (1989) Acta Pharm. Sin., 24, 568- 572.         [ Links ]

4. González A. G., Jiménez I. A., Ravelo Coll, J., González J. A., Lloria J., Biochem System. Ecol., 25, 513-519 (1997).         [ Links ]

5. González A. G., Jiménez I. A., Ravelo A. G., Bellés X. and Piulachs M. D., Biochem. System. Ecol., 20, 311-315 (1992).         [ Links ]

6. Alarcón J., Becerra J. and Silva M., Bol. Soc. Chil. Quím., 43, 65-71 (1998).         [ Links ]

7. Boyer F., Prangé T., Ducrot P., Tetrahedron asymm., 14, 1153- 1159 (2003).         [ Links ]

8. Boyer F., Descoins C.L., Descoins C., Prangé T., Ducrot P., Tetrahedron lett., 43, 8277-8279 (2002).         [ Links ]

9. Spivey A., Weston M., Woodhead S., Chem. Soc. Rev., 31, 43-59 (2002).         [ Links ]

10. Lee C., Floreancig P., Tetrahedron lett., 45, 7193-7196 (2004).         [ Links ]

11. Chen X., Shao S.C., Li T. S. and Li Y. L., Synthesis, 1061-1062 (1992).         [ Links ]

12. Alarcón J. and Águila S., Z. Naturforsch, 59c, 215-217 (2004).         [ Links ]

13. Wu-Jiong X., Liang-Dong S., Lei S., Shu-Yu Z., Yong-Qiang T., Chin. J. Chem., 22, 377-383 (2004).         [ Links ]

14. White J., Shin H., Kim T. and Cutshall N., J. Am. Chem. Soc., 119, 2004-2419 (1997).         [ Links ]

15. Huffman J. W. and Desai R. C., J. Org. Chem., 47, 3254-3258 (1982).         [ Links ]

16. Alarcón J., Alderete J., Meter M. G., Becerra J., Silva M., Bol. Soc. Chil. Quím., 43, 325-327 (1998).         [ Links ]

17. Kieslich K., Abraham W. R., Stumpf B., Thede B. and Washausen P., Transformation of Terpenoids. In Progress in Essential Oil Research, ed. E. J. Walter de Gruyter and Co., Berlin, 1986, pp. 367-394.         [ Links ]

18. García-Granados A., Gutiérrez M., Parra A., Rivas F., J. Nat. Prod., 65, 1011-1015 (2002).         [ Links ]

19. Li Z., Beilen J., Duetz W., Schimid A., Current Opinion in Chemical Biology, 6, 136-144 (2002).         [ Links ]

20. Prichanont S., Leak D., Stuckey D., Enz. Microb. Technol., 27, 134- 142 (2000).         [ Links ]

21. Steinig G., Livingston A., Stuckey D., Biotech. and Bioeng., 70, 553- 563 (2000).         [ Links ]

22. Aladro F., Guerra F., Moreno-Dorado F., Bustamante J., Jorge Z., Massanet G., Tetrahedron, 57, 2171-2178 (2001).         [ Links ]

 

E-mail: jalderet@udec.cl

 

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