Appl Microbiol Biotechnol (1986) 25:14--17 Applied Microbiology

Biotechnology © Springer-Verlag 1986

Microbiological dehydrogenation of polyfunctional AS-3[i-acetoxy steroids by Corynebacterium mediolanum strain B-964

Violeta Datcheva 1, Aleksei Kamernitskii 2, Radoslav Vlahov 1, Natalia Voishvillo 2, Vera Levi 2, Irina Reshetova 2, and Elena Chernoburova 2

1 Institute for Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 2 Institute of Organic Chemistry "N.D. Zelinskii", Academy of Sciences USSR, Moscow, Leninskii pr. 47, USSR

Summary. A high-yield microbiological transfor- mation of polyfunctional AS-3fl-acetoxy steroids, containing an additional ring E, by Corynebacter- ium mediolanum strain B-964 was carried out, re- sulting in the corresponding A4-3-ketones. It was shown that the type of transformation and the yield of the reaction depend on the degree of sa- turation of the ring E and on the position of the oxygen-containing substituents in it.

Introduction

In the course of our investigations of the synthesis and biological activity of a new class of modified polyfunctional steroidal compounds with an addi- tional ring E (Kamernitskii 1984), a microbiologi- cal dehydrogenation of a series of AS-3fl-acetoxy compounds was performed and their Aa-3-ke - tones were obtained.

It is known that the presence of such a conju- gated system in ring A radically affects the physi- ological activity of the compounds. On the other hand, a chemical pathway of selective oxidation is unsuitable for conversion of polyfunctional ste- roids. Microbiological transformation of this type of compound have not yet been investigated.

Corynebacterium mediolanum was chosen as dehydrogenating organism. Its ability to convert As-3fl-hydroxy steroids into A4-3-ketones has long been established (Mamoli 1939). Though the sub- strate specificity of C. mediolanum has not been studied thoroughly, it has been shown to trans- form the 3fl-acetates of steroids more efficiently than free alcohols (Spalla et al. 1962). Therefore, besides obtaining Aa-3-ketones, it was rather in-

Offj~rint requests to: R. Vlahov

teresting to determine the influence of the substi- tuents in ring E and of its structure on the dehy- drogenating ability of C. mediolanum.

Three types of steroidal compounds were chosen as substrates, differing in the structure of the ring E; these were the derivatives of tetrahy- dropyranone-l,4; (2a--4a) (including furanones la- -5a) ; tetrahydropyranol-l,4; 7a--9a, 1Ia, 12a (including furanols 6a, lOa); 'tetrahydropyra- none- 1,2; 13a-- 14a, "dihydropyranone- 1,2; 15a-- 16a, '" pyranone-l,2, 17a, as well as compound 18a, which is an intermediate in the synthesis of steroids 13a, 15a (Scheme 1).

Material and methods

Organism and culture conditions. Corynebacterium mediolanum B-964, used in this work, was maintained and stored at 4°C on agar slants (yeast extract 3 g, caseine hydrolysate 4 g, meat ex- tract 150 ml, glucose 1 g, agar 30 g, distilled water 1 1, pH 6.9-- 7.0). Periodic transfer (every 1--2 months) preserved the cul- ture.

Shaken cultures for biotransformation experiments were generated by a three-stage fermentation procedure in medium 1 (for stage I and stage II), consisting of (per liter of tap water) yeast autolysate 6g, glucose 3g, K2HPO4.3H20 2.4g, Na2HPO4.12H20 4 g, NaC1 2 g; pH 7.2--7.3, and in medium 2, consisting of (per liter of tap water) corn-steep extract 7.8 g; Na2HPO4.12HzO 4.5 g, KH2PO4 1 g; pH 7.0--7.1.

Stage I cultures were initiated by suspending the surface growth from a 7-day-old slant in 50ml of medium 1 in a 250-ml Erlenmeyer flask. The stage I culture was incubated for 65 h with shaking (220 strokes/min, 28°C); 2 ml of culture were then withdrawn to inoculate 50 ml of medium 1 for a stage II culture.

Thick, 24-h stage II culture was used to inoculate stage III fermentation; the inoculum represented 10% of the volume of medium 2, held in a stage III culture flask. The stage III cul- ture was grown in 20 ml medium in a 100 ml Erlenmeyer flask, or 150 ml medium in 750 ml conical flasks, and was used for steroid biotransformation.

Steroids were added to a 6 h stage III culture as a 2% so- lution in dimethylformamide to a final medium concentration of 0.5 mg/ml. Incubation was continued for 15 h after steroid

V. Datcheva et al.: Dehydrogenation of steroids by C. mediolanum 15

¸

a b

( ~ A c ~ H 2 O A ¢ A co.,[__~ H:~O H '- X,Y: ~ ,o X=~6 y : ~

A r,.O'-.[~,,.O C H a

3. X,Y: 12.X,y= ~ . ' o

9Ac OH

4 . × , ~ ¥= ~ 13.X.y= ~ .

8.X,Y : Ac~ 17. X,Y

qAc OH

a.X, y= 18. X: ~,OAc Y:~-oAc

(Kamernitskii 1984). Compounds 6a, 9a and 10a were ob- tained from their 20 ketones.

Lithium aluminium hydride (86 mg, 2.25 mmol) in 10 ml dry tetrahydrofuran was added to the ketone la (100 rag, 0.225 mmol) and the mixture was stirred at ambient tempera- ture for 2 h. After the usual treatment, the resulting solid was acetylated in pyridine-acetic anhydride and chromatographed on a silica gel column. The product 6a (80 rag, 0.164 mmol) was eluted with ether-heptane (1:2).

Sodium borohydride (50 mg, 0.160 mmol) in 1.5 ml H20 was added to solutions of the ketone 4a (100 mg, 0.240 retool) and the ketone 5a (120 rag, 0.260 mmol) in 8 ml DMF at am- bient temperature. After 20 h at this temperature, the mixtures were treated with diluted hydrochloric acid. The products were isolated with ethyl acetate and acetylated in pyridine- acetic anhydride with dimethylaminopyridine as catalyst. Chromatography on silica gel in ether-heptane (1:2) yielded 9a (60 mg, 0.120 mml) and 10a (100 mg, 0.198 retool), respec- tively.

The acetate 14a, with a natural configuration of the lac- tone ring at C-16, was prepared by the route described in Scheme 2. Using the same method as for shiogralactone syn- thesis (Kamernitskii et al. 1983), we obtained 6-oxy-i-steroid A, which was converted into the final product under the fol- lowing conditions: the mixture of the lactone A (30rag, 0.838 mmol), acetic acid (3 ml) and concentrated sulphuric acid (0.07 ml) was kept at room temperature for 18 h. Then water was added and the mixture was extracted with ethyl ace- tate. The organic layer was separated, washed with 10% Nai l - CO3, followed by water, dried (MgSO4), and evaporated. The resulting solid was chromatographed on a silica gel column. Elution with ether-hexane (1:0.2) afforded the actate 14a (20 mg, 0.561 mmol).

13 a A 14a

addition, by which time the steroid had been completely con- vetted.

Quantitative analytical experiments. The entire culture was ex- tracted with an equal volume of chloroform. The solvent was removed under vacuum. Samples of extract were examined by thin layer chromatography (tic).

Chromatography. Analytical thin-layer chromatography (tic) was performed on Silufol UV-254" plates. Tic plates were de- veloped with an eluent of either-heptane: No. 1 (1:3) for com- pounds lb - -3b ; No. 2 (1:2) for compounds 6a, 4b--7b; No. 3 (1:1) for compounds 9a, 10a, 13b--18b; and No. 4 (2:1) -- for compounds 11, 12, 14a, 8b--10b (see Scheme 1). Developed chromatographs were examined under 254 nm UV light and by spraying with a solution of Ce(SO4)z, followed by warming on a hot plate.

Preparative separations of steroid metabolites were con- ducted on a column, packed with silica gel 40/100 It, with the solvent systems described above. Metabolites were recrystal- lized from methanol.

Compounds. The starting steroids la--5a, 7a, 8a, l l a - -13a , 15a--18a, were synthesized by previously described methods

Analytical methods. Melting points were determined with a Kofler apparatus. Infrared spectra were recorded in CHC13 on an UR-20 spectrometer. Nuclear magnetic resonance spectra were recorded in CDC13 at 250 MHz on a Bruker WM-250 spectrometer (TMS as internal standard). Low-resolution mass spectra were obtained on a JEOL JMS D-300 mass spectrom- eter with chemical ionisation (C4Hlo-i). All prepared A4-3-ke - tones showed UV absorption at 240 nm, e ~ 10,000. The physi- cal data obtained are listed in Tables 1 and 2.

The transformation products lb, 2b, 18b were compared with authentic samples (Kamernitskii et al. 1975; Griffths et al. 1963).

Results and discussion

Microbiological dehydrogenation of A S-3fl-acetates of tetrahydropyranone-l,4 and tetrahydropyranol- l , 4 (including furanones and furanols)

T h e d a t a i n T a b l e 2 s h o w t h a t t h e t r a n s f o r m a t i o n

o f s t e r o i d s l a - - 5 a b y Corynebacterium medio-

16

Table 1. Physical and chemical data of AS-3~-acetates

V. Datcheva et al.: Dehydrogenation of steroids by C. mediolanum

Corn- Yield mp. MS a IR b lH-nmrC (6, ppm) pound (%) (°C) (m/z) (cm- 1, CHCI3)

6a 60 210--212 428 1260 (M-60) + 1723 386 368 326

9a 72 188--190 504 1260 (M)+ 1730 486 3460 426

10a 50 118--120 504 1216 (M) + 1729 486 3500 426 384

14a 76 167--168 400 1260 (M)+ 1725

1.01 s (3H, 18-CHa), 1.07 s (3H, 19-CH3), 2.01 s, 2.03 s, 2.13 s (9H, acetoxy), 3.45 m (1H, 16-H), 3.68 m (2H, 22- OCH2), 5.38 br.s (1H, 6-H)

1.04 s (3H, 18-CH3), 1.13 s (3H, 19-CH3), 2.05 s, 2.12 s, 2.15s (9H, acetoxy), 4.12--4.38m (3H, 16-H, 23- OCHz), 4.53 dd (J=3.5 and 8 Hz, 1H, 22-H), 4.58 m (1H, 3-H), 5.38 m, d 0 = 8 Hz, 2H, 6-H, 20-H) 101 s (3H, 18-CH3), 1.04s (3H, 19-CH3), 2.01 s, 2.11 s, 2.14s (9H, acetoxy), 4.07--4.26m (2H, 23-OCH2), 4.42m (2H, 16-H, 22-H), 4.57m (1H, 3-H), 5.11 d 0 = 9 Hz, 1H, 20-H), 5.36 br.s (1H, 6-H)

0.80s (3H, 18-CH3), 1.05s (3H, 19-CH3), 1.12d (J=6.5 Hz, 3H, 21-CH3), 2.04 s (3H, acetoxy), 4.68 m (2H, 3-H, 16-H), 5.38 br.s (1H, 6-H)

a MS, mass spectra b IR, infrared spectra c 1H-nmr, proton magnetic resonance spectra

lanum into A4-3-ketones affords a very high yield and seems to be independent of the type of substi- tuent in ring E. It was found that the primary and secondary acetate groups in ring E are hydrolysed (4b--5b). The same hydrolysis of the accessible acetoxy groups is also observed in the case of te- trahydropyranols-l,4 and furanols 6a--12a (Table 2, see next page). The highest A4-3-ketone yield was obtained from the compound 3a. It was es- tablished that the oxygen-containing substituents at C-17, C-20 and C-22 in ring E do not affect the conversion, while the methoxy groups at C-23, re- gardless of their configuration, inhibit microbial dehydrogenation ( l la, 12a). Instead of A4-3-ke - tones, hydrolysis of the acetate groups was ob- served, and the AS-bond was preserved.

Microbiological dehydrogenation of A 5-3fl-acetates of tetrahydropyranone-l,2

It was established that the yield of final A4-3-ke - tones closely depends on the degree of the ring E saturation. This dependence is illustrated by the yields of the compounds given in Table 2: the sa- turated lactones 13a and 14a give a quantitative yield of Aa-3-ketones; compounds 15a and 16a, having a double bond in ring E, were transformed in a 60% yield; compound 17a, being the least sa- turated, was converted in a 45% yield. However, the position of the double bond and the stereo-

chemistry of the additional ring E at C-16 do not affect the transformation efficiency.

Clearly, in all investigated compounds, ring E is very resistant to the action of C. mediolanum; Aa-3-ketones are formed with a high yield. How- ever, dehydrogenation of the 3fl,16a-diacetoxy- 20-hydroxy-24-norchol-5-ene-23-oic acid ethyl es- ter 18a leads both to the formation of a A4-3-ke - tone group and to degradation of the side chain, resulting in 3fl,16a-diacetoxypregn-4-ene-20- dione (18b). It should be noted that the C-C bond was split for the first time with C. mediolanum cells.

References

Griffiths K, Grant J, Whyte W (1963) Steroid biosynthesis in vitro by cryptorchid testes from a case of testicular femini- zation. J Clin Endocrinol Metab 23/10:1044--1055

Kamernitskii A (1984) The purposed modification of steroid hormones. Izv Akad Nauk SSSR Ser Khim: 650--661

Kamernitskii A, Olgina N, Reshetova I, Chernjuk K (1975) 21- Methoxymethyl-20-oxo- 16a,17a-epoxysteroids in reactions of cis- and trans-opening of the epoxide cycle. Izv Akad Nauk SSSR Ser Khim:411--414

Kamernitskii A, Reshetova I, Chernoburova E (1983) Synthe- sis of naturally-occurring shiogralactone. Khim Prir Soedin (2): 190--197

Mamoli L (1939) Uber biochemische Dehydrierungen in der Cortingruppe. Ber 72B: 1863 -- 1865

Spalla C, Modelli R, Amici A (1962) Production of Hydrocor- tisone by multiple fermentation. USA Patent 3030278

Received December 16, 1985

V. Datcheva et al.: Dehydrogenation of steroids by C. mediolanum 17

Table 2. Physical and chemical data of Ag-3-ketones obtained by Corynebacterium mediolanum B-964

Corn- Yield mp MS a IR b 1H-nmrC (6, ppm) pound (%) (°C) (m/z) (cm - 1, CHC13)

lb d 62 269--271 400 1610 (M)+ 1660 340 1710

1725 2b ~ 85 203--206 342 1620

(M) + 1667 1698

3b 90 180--182 358 1608 (M)+ 1664

1703 4b 86 210--215 374 1618

(M)+ 1673 356 1756

5b 74 243--246 374 1618 (M)+ 1673 356 1756

6b 85 230--232 402 1620 (M)+ 1662 384 1714 342 3500

7b 55 237--240 344 1620 (M)+ 1663 326 3500

8b 65 220--222 402 1620 (M)+ 1662 384 1714 342 3500 324

9b 60 238--240 418 1608 (M)+ 1663 400 1732

3460

10b 61 218--220

13b 90 160--162

14b 90 198--200

15b 56 238--240

16b 56 210--212

17b 45 190--192

18b d 45 252--255

418 1620 (M)+ 1663 400 1725

3460

356 1616 (M)+ 1660

1730 356 1620 (M) + 1660

1730 354 1616 (M) + 1664

1715 354 1616 (M)+ 1663 310 352 1533 (M)+ 1619 326 1664

1710 372 1616 (M)+ 1662 312 1700

0.96 s (3H, 18-CH3), 1.20 s (3H, 19-CH3), 3.74 m (1H, 23-OCH), 3.96 q 0=3.5 Hz, 1H, 16-H), 4.25 m (1H, 23- OCH), 5.74 s (1H, 4-H) 0.93 s (3H, 18-CH3), 1.20 s (3H, 19-CH3), 3.93 m (2H, 23-OCH2), 4.23 m (1H, 22-H), 4.43 m (1H, 16-H), 5.75 s (1H, 4-H) 1.02 s (3H, 18-CH3), 1.22 s (3H, 19-CH3), 2.65 s (1H, OH), 3.90 m (2H, 23-OCH2), 4.27 t 0=4.5 Hz, 1H, 22- H), 4.65 m(IH, 16-H), 5.75 s (1H, 4-H) 1.11 s (3H, 18-CH3), 1.21 s (3H, 19-CH3), 2.15 s (3H, acetoxy), 3.46td 0 = 2 and 11 Hz, 1H, 20-H), 3.71 m (1H, 23-OCH), 4.04 m (1H, 23-OCH), 5.15 m (1H, 16- H), 5.74s (1H, 4-H) 1.14 s (3H, 18-CH3), 1.21 s (3H, 19-CH3), 3.33 td, J = 2 and 11 Hz, 1H, 20-H), 3.98 m (2H, 23-OCH~), 4.27 m (1H, 16-H), 5.73 s (1H, 4-H) 1.09 s (3H, 18-CH3), 1.17 s (3H, 19-CH3), 2.12 s (3H, acetoxy), 3.44 td (J = 2 and 11 Hz, 1 H, 23-OCH), 3.68 q 0=3 .4 Hz, 1H, 16-H), 4.00 m (1H, 23-OCH), 5.14 dd 0=6.8 and 12.3 Hz, 1H, 20-H), 5.78 s (1H, 4-H)

1.14s (3H, 18-CH3), 1.19s (3H, 19-CH3), 2.14s (3H, acetoxy), 3.75 dd 0=3.5 and 11.5 Hz, 1H, 23-OCH), 3.93 dd 0 = 8 and 11.5 Hz, 1H, 23-OCH), 4.15 m (1H, 16-H), 4.42 m (1H, 22-H), 5.34 d 0=8.7 Hz, 1H, 20-H), 5.73 s (1H, 4-H) 1.22s (6H, 18-CH3, 19-CH3), 2.15 s (3H, acetoxy), 3.62dd 0=3 .4 and l l H z , 1H, 23-OCH), 3.78dd ( J= l l . 3H z , 1H, 23-OCH), 4.04s (1H, OH), 4.30m (1H, 22-H), 4.42q (J=3.4Hz, 1H, 16-H), 5.21d 0 = 8 Hz, 1H, 20-H), 5.74s (1H, 4-H) 0.80s (3H, 18-CH3), 1.04d 0=6.5 Hz, 3H, 21-CH3), 1.20s (3H, 19-CHa), 4.48td 0 = 5 and 10Hz, 1H, 16- H), 5.74 br.s (1H, 4-H) 0.85s (3H, 18-CH3), 1.13d (J=6Hz, 3H, 21-CH3), 4.67 m (1H, 16-H), 5.74s (1H, 4-H), 1.20s (3H, 19- CH3) 0.87 s (3H, 18-CH3), 1.20 s (3H, 19-CH3), 2.06 s (3H, 21- CH3), 4.62 m (1H, 16-H), 5.75 br.s (1H, 4-H), 5.79 br.s (1H, 22-H) 0.96 s (3H, 18-CH3), 1.19 s (3H, 19-CH3), 1.84 s (3H, 21- CH3), 5.06 br.s (1H, 16-H), 5.75 s (1H, 4-H)

1.04 s (3H, 18-CH3), 1.24 s (3H, 19-CH3), 2.15 s (3H, 21- CH3), 5.76 s (1H, 4-H), 5.84 s (1H, 22-H)

a MS, mass spectra b IR, infrared spectra c 1H-nmr, proton magnetic resonance spectra