Regiospecific reduction of adrenosterone to 11-ketotestosterone by microalga T76 Scenedesmus quadricauda

BIOTECHNOLOGY LETTERS Volume 18 No.6 (June 1996) p.639-642 Received as revised 1st April.

REGIOSPECIFIC REDUCTION OF ADRENOSTERONE TO II-KETOTESTOSTERONE BY MICROALGA T76

Scenedesmus quadricauda

Marina Della Greca 1, Antonio Fiorentino 1, Gabriele Pinto 2, Antonino Pollio 2 and Lucio Previtera 1. 1Dipartimento di Chimica Organica e Biologica, Universi~ Federico II, Via Mezzocannone 16, 1-80134 Napoli, Italy 2Dipartimento di Biologia Vegetale, Universi~ Federico II, Via Foria 223, 1-80139, Napoli, Italy

SUMMARY Adrenosterone is reduced to 1J-ketotestosterone by cultures of microalga Scenedesmus quadricauda. The best bioconversion yields (62%) were obtained in cultures containing 3x106 cells/mL of alga and 100 mg of adrenosterone in 400 mL of medium after 10 days at 24°C with a photoperiod light - dark 16 : 8 h.

INTRODUCTION 11-Ketotestosterone (1) is an important androgen which seems to inhibit the mechanisms of human decidual cell growth (Zhao and Li, 1994). Apart some more complex syntheses (Van Royen et al., 1983), the easier via to this steroid is based on the partial reduction of adrenosterone (2) by chemical or biochemical methods. OH

1

O

2

Norymberski and Woods (1955) reported that reduction of 2 by NaBH4 at 0°C in MeOH afforded 1 in 45 % (w/w) yields and Zhao and Li described the obtainment of the same product in 57 % (w/w) yields in a modified procedure in presence of pyridine.

639

Bioreduction of 2 by Saccharomyces cerevisiae (Herzog et al, 1953) or Trichornonas

gallinae (Sebek and Michaels, 1959) gave I in 62% and 94% amounts respectively. Freshwater microalgae have been proved to be useful bioreactors in modification of steroidal material (Fiorentino et al., 1991; Pollio et al., 1994; Della Greca et al., 1996) and in this paper we describe the bioconversion of 2 into 1 by the green alga T76

Scenedesmus quadricauda. E X P E R I M E N T A L METHODS

Bioconversion ofadrenosterone (2).The strains of I76 S. quadricauda were supplied by The Algal Collection of Texas at Austin, USA. i) In a preliminary experiment adrenosterone (2) (100 mg sterilized at 100 °C for 1 h) dissolved in dioxane (0.8 mL) was added to an Erlenmeyer flask (2 L) containing the axenic culture of S. quadricauda in Bold basal medium (BBM) (800 mL) (Nichols and Bold, 1965) during the exponential phase of growth of the strain with algal concentration 6 x 105 cells/mL. The suspension was stirred at 24 °C, irradiated with a fluorescent lamp (irradiance of 120 mmol photons.m2s J) with a photoperiod of 16 h light - 8 h dark and after 50 days was extracted with ethyl acetate (2 x 100 mL). The residue (98 mg) was chromatographed on preparative silica gel TLC (chloroform - ethyl acetate 17 : 3) to give unreacted 2 (21 mg), ll-ketotestosterone (I) (27 mg) and a mixture of dihydroxyderivatives (3) (46 rag). l l-Ketotestosterone (1)had: m.p. 187 - 188 °C; 1HNMR ~ (CDC13) 5.72 (1H, s, H-4), 3.87 (1H, t, J = 8.1 Hz, H-17), 0.76 (3H, s, H18), 1.43 (3H, s, H-19); 13C-NMR 8 (CDC13) 34.7 (C-l), 33.7 (C-2), 199.7 (C-3), 124.5 (C-4), 168.8 (C-5), 32.2 (C-6), 31.7 (C-7), 37.5 (C-8), 62.8 (C-9), 38.2 (C-10), 208.8 (C-11), 54.7 (C-12), 46.9 (C-13), 49.6 (C-14), 22.8 (C-15), 30.6 (C-16), 79.7 (C-17), 11.7 (C-18), 17.2 (C-19). In the next experiment adrenosterone (50 mg) in dioxane (0.4 mL) was added to suspensions of algal cultures (400 ml, 6 x 105 cells/mL) and the reactions were run for 10, 20, 30, 40 and 50 days. ii) In the experiments with twice the amount of adrenosterone, (2) (100 mg) was added to the cultures (400 mL, 6 x 105 cells/mL). The cultures were incubated for 10 days as above reported. The ethyl acetate extract was checked by ~3C-NMR and GC analysis. iii) In the five-fold algal inoculum experiment adrenosterone (2) (100 rag) was added to a suspension of the strain in BBM (400 mL) with an algal concentration 3 × 106 cells/mL. After 10 days as above reported the ethyl acetate extract was monitored by 13C-NMR and GC analysis. iv) The apparatus for continuous algal culture consisted in a glass bioreactor (1 L) equipped with an inlet way, regulated by a peristaltic pump and an outlet overflow tube. Both the ways were collegated to a reservoir containing BBM (2 L). The continuos culture was obtained growing axenically at 24 °C the strain in the bioreactor filled with BBM (900 mL). The medium was air bubbled, shaked by a magnetic stirrer and irradiated with a light - dark period of 16 to 8 h. When the algal concentration was 3 x 106 cells/mL, BBM was continuosly added from the reservoir by 64.28 mL/h flow with the overflow tube ensuring a constant volume in the bioreactor. In these conditions the algal concentration was unchanged. A filter (11 ktm, Wathman 1) was put on the way-in of the reservoir to avoid the recircling of algae. Adrenosterone (250 mg) dissolved in dioxane (6 mL) was added during 12 h by a peristaltic pump (0.5 mL/h) and the

640

experiment was carded out for 3 and for 10 days. The overall suspensions (bioreactor plus reservoir) were extracted with ethyl acetate (2 x 300 mL) and the organic layers were checked as above reported. Evaluation of the bioconversion yields. The yields of the bioconversion of 2 into 1 were evaluated by integrated 13C-NMR analysis of the signals of the Co18 methyls (11.7 ppm in 1 and 14.6 ppm in 2) in the bioreaction mixture. GC analysis was run on OV-1 capillary column (285 °C, flow 1 mL/min) using 21-hydroxyprogesterone as internal standard. The data from the two methods differed in + 1% range. R E S U L T S AND D I S C U S S I O N In a preliminary experiment, adrenosterone (2) was incubated in culture of Scenedesmus

quadricauda in axenic conditions during the exponential phase of growth of the strain. The initial algal concentration was 6 x 105 cell/ml and after 50 days the main bioproduct formed was 11-ketotestosterone (1) identified by comparison of its physical features with those of an authentic commercial sample. It was isolated in about 28 % amounts from the suspension by ethyl acetate extraction and TLC chromatography along with unreacted adrenosterone (2) and a complex mixture of minor components 3.1H-NMR and t3CNMR analysis of 3 showed it was a mixture of dihydroxyderivatives, To verify the influence of the reaction time on the course of the bioreaction, adrenosterone (2) was cultured in the same conditions for 10, 20, 30, 40 and 50 days. Noteworthy, the best result was obtained after 10 days with 1 formed in about 50% yields and absence of coproducts. In the longer runs, even if further adrenosterone reacted, the amounts of I were lower owing to formation of the mixture 3 (Table 1). In the next experiment the influence of ratio between adrenosterone amount and algal concentration was examined: in a first case two-fold adrenosterone amount was cultured at the same algal concentration and after 10 days only testerone (1) and adrenosterone (2) in 11 : 9 ratio were detected in the reaction mixture (Table 1). When the bioreaction was run with two fold adrenosterone (2) amounts and five fold algal concentration, testosterone (1) was formed in 62 % amounts but, along with unreacted adrenosterone (2) (24 %), the mixture 3 was also present in 13 % amounts (Table 1). Finally, we tested the bioreduction under continous algal culture. Adrenosterone (2) (250 mg) dissolved in dioxane was slowly added by a peristaltic pump to the bioreactor containing the algal culture (9.8 x 106 cells/ml) The bioreactor was collegated to a reservoir and BBM was added to the bioreactor by a second peristaltic pump so as to ensure a constant algal concentration in the bioreactor. An overflow way kept constant the volume. The results of experiments run for 3, 7 and 10 days are reported in Table 1. In conclusion T76 Scenedesmus quadricauda has been proved to be an interesting microorganism in the regiospecific reduction of adrenosterone. It is able to reduce only the C-17 keto group with stereospecific formation of the 17[3-hydroxy derivative. In the

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best conditions 62% of 11-ketosterone (1) was isolated from the cultures (yield calculated on the unrecovered adrenosterone 82%). This yield is lower than that obtained by Sebek and Michaels but is fairly comparable to that obtained by Herzog et al. and higher than those obtained by chemical reduction. However further work is in progress to improve the bioreduction yields. Tablel. Bioproducts (%) in the reduction of adrenosterone (2). days

1

2

3

10

50.3

49.6

-

20

49.4

42.1

10.2

30

40.7

39.0

20.0

40

35.2

31.3

32.9

50

28.1

23.4

48.0

10a

54.8

44.9

-

l0 b

62.1

23.9

13.3

3c

18.5

73.3

7.3

7c

39.4

50.9

9.8

10c 57.3 30.1 11.6 atwo-fold amountof 2; btwo-fold amountof 2 and five -fold algal concentration;Ccontinuosculture

REFERENCES Della

Greca, M., Fiorentino, A., Pinto, G., Pollio, A., Previtera, L. (1996)

Phytochemistry, in press. Fiorentino, A., Pinto, G., Pollio, A., Previtera, L. (1991) Biomed. Chem. Letters, 1, 673-674. Herzog, H.L., Jevnik, M.A., Perlman, P.L., Nobile, A, Hershberg, E.B. (1953) J.

Am. Chem. Soc., 75, 266-269. Nichols, H.W. (1973). Growth media-freshwater in: Handbook of Phycological Methods, J.R. Stein ed. pp 7-24, Cambridge: Cambridge University Press. Norymberski, J.K., Woods, G;F. (1955) J. Chem. Soc., 3426-3430. Pollio, A., Pinto, G., Della Greca, M., DeMaio, A., Fiorentino, A., Previtera, L. (1994)

Phytochemistry, 37, 1269-1272. Sebek, O.K., Michaels, R.M. (1959) C.A., 53, 16285. Van Royen, L.A., Mijngheer, R, De Clercq, P.J. (1985) Tetrahedron, 41, 4667-4680. Zhao, Q., Li, Z. (1994) Steroids, 59, 190-195.

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