Microbiological transformation of prostaglandins

Biochimicu et Biophysics Acta, 348 (1974) 263-268 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

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BBA

$427

MICROBIOLOGICAL

TRANSFORMATION

II. STEREOSPECIFIC PROSTAGLANDJN

REDUCTION

W. J. MARSHECK

OF PROSTAGLANDINS

OF A (+)-d*“%5-DEHYDRO-

and M. MfYANO

Searle Laboratories, P.O. Box

5110,

Chicago, Ill. (U.S.A.)

(Received November 6th, 1973)

SUMMARY

Flavobacterium sp. NRRL B-5641 was found to reduce the rg-ketone of (+)_@W r g-dehydroprostaglandin El to produce a cis racemic mixture of 6*(’ 2)prostaglandin E, and I 1,Is-epi-d *(12)-prostaglandin El, The efficiency of the conversion is dependent upon the fermentation medium used and the method of preparation of the inoculum. Pseudomonas sp. NRRL B-3875 reduced (f)-d8”‘)-~5dehydroprostaglandin E, to give optically active I I-epi-d’(‘*‘-prostaglandin El in good yield. Optically active rg-epi-d8”2’ -prostaglandin E, was isolated when Flavobacterium sp. NRRL B-3874 or Arthrobacter sp. NRRL B-3873 reduced (-t-)-d8(‘2)15-dehydroprostaglandin E,. The reduction of the Is-ketone or the 13, r4-double bond of ( 2 )-d *(’*)-I~-dehydroprostaglandin Et was found to be a reaction produced by many bacteria.

INTRODUCTION

The stereospecific microbiological reduction of ketone functions in certain organic compounds has been a very useful method to resolve racemic mixtures. The reduction of the r7-ketone of synthetically prepared racemic steroids to give either the tl- or P-alcohol has been reviewed [I]. Optically active lactones were made by the microbiological reduction of y- and b-keto acids [2]. In this reduction of &ketocapric acid yeasts of the genus Ca~did~ gave the dextrorotatory lactone while both the fungus CIadosporium butyri and the bacterium Sarcina lutea produced the ievorotatory enantiomer. Recent work toward the total synthesis of prostaglandins [3-51 has produced compounds suitable. for microbial reduction and resolution. Sih et al. [6] have recently reported on the asymmetric reduction of (~)-2-(6~carbomethoxyhexyl)cyclo~ntane-1,3,4-trione using the fungus ~i~oda~cu~ ~nin~c~eutus. The q(R)-r,3-dione alcohol was isolated exclusively. Schneider et al. [7] employed fermenting baker’s yeast to reduce the 9-ketone of prostaglandin E, to ptostaglandin F,a and prostaglandin E, to prostaglandin [email protected] natural 9(s) configuration was obtained in both cases.

264

Fig.

I

NRRL

Racemic B-5641.

cis reduction Conditions:

of

( ! )-A*“” -I 5-dehydroprostaglandin

outlined

in Materials

E, with

~/~r~,o-hrr~.rcrirr/rr sp.

and Methods.

( +)-A8”2’ -15-Dehydroprostaglandin E, has been reported by Miyano et al. (refs. 8, 9) as a new prostaglandin analogue and an intermediate in the total synthesis of racemic A’(“) -prostaglandin E,. Chemical reduction of the I 5-ketone of ( +)-A8”2’15-dehydroprostaglandin E, affords a pair of cis and a pair of t~ans isomers with respect to the C-I I and c-15 hydroxyls. A screening program was undertaken to look for microorganisms capable of producing a stereospecific reduction at c-15. In a previous communication [IO] we reported two bacterial reductions of ( f )-AR”2)- I sdehydroprostaglandin E,. This paper will describe further investigations into these microbial reactions. MATERIALS

AND

METHODS

Organisms Arthrobacter sp. NRRL B-3873, Flavobacterium sp. NRRL B-3874. Pseur/omonas sp. NRRL B-3875 and Flavobacterium sp. NRRL B-5641 were isolated from the soil. The organisms are deposited with the North Central Utilization Research and Development Division, United States Department of Agriculture, Peoria, III., U.S.A. Media Medium A, g/l: 10.0 dextrose; I .o yeast extract (Difco Laboratories, Detroit, Mich. 48232, U.S.A.): 5.0 meat peptone No. 70 (General Biochemicals, Chagrin Falls, Ohio 44022, [J.S.A.): 3.0 meat extract (Difco): 2.0 NaCI. The pH was adjusted to 7.0 with KOH. Medium B, g/l: 10.0 dextrose; 10.0 yeast extract: 5.0 meat peptone: 2.0 K,HPO,. The pH was adjusted to 7.0 Medium C, g/l: 10.0 dextrose; 5.0 casein hydrolysate (Sheffield Chemical, Union, N.J. 07083, U.S.A.); 5.0 soy peptone (Sheffield); 2.0 NaCl: 2.0 K,HPO,. The pH was adjusted to 7.0. Medium D, g/l: 5.0 meat peptone No. 70; 3.0 meat extract; 2.0 NaCI. The pH was adjusted to 7.0. Substrate The synthesis

of ( &)-A8”2’- I 5-dehydroprostaglandin

E, has been described

265

previously [8, 91. The compound which existed as a glass (purity approximately 99 %) was dissolved in acetone for addition to the fermentations. Shake $ask Arthrobacter sp. NRRL B-3873, Flavobacterium sp. NRRL B-3874, and Pseudomonas sp. NRRL B-3875 were grown for 15 h in Medium C. A I % inoculum was

transferred to r-l Erlenmeyer flasks containing 300 ml Medium A or C and incubated on a rotary shaker (t-inch orbit, zoo rev./min) at 27 “C for 24 h. At this time the prostaglandin (0.1% w/v) was added and the bioconversion allowed to proceed at the growth conditions. After 48-72 h the cultures were adjusted to pH 4.0 with citric acid and extracted with methylene chloride. Stirredj&rmentor

A 7.5-l glass stirred-jar fermentor (New Brunswick Scientific Co., New Brunswick, N. J. 08903, U.S.A.) was charged with 5 1 of Medium A or B and sterilized by autoclaving. Four 72-h growth slants of Flavobacterium sp. NRRL B-5641 on Medium C (I .5 % agar added) were washed with IO ml each sterile distilled water. The cell suspension was added to the fermentor. The agitation was controlled at 160 rev./ min, aeration at 20% (by vol.) and temperature at 34 “C. At 24 h incubation 1.5 g of (+)_&J’rX I 5-dehydroprostaglandin E, was added to the fermentor. After 44 additional hours the fermentor was extracted with methylene chloride at pH 4.0. Thin-layer chromatographic analysis

Evaluation of the methylene chloride extracts was made by thin-layer chromatography run on 20 cm x 20 cm glass plates coated with 0.25 mm silica gel HF,,, (E. Merck, Darmstadt, Germany). The plates were previously activated for 1 h at I 20 “C. The plates were developed twice in a solvent system of benzene-ethyl acetateacetic acid (50: 50: 2, by vol.) Visualization of the compounds was made under ultraviolet light followed by spraying with IO% phosphomolybdic acid (ethanol) and heated at I I 5 “C for several minutes. Column chromatograph~c isolation

The methylene chloride was removed in vacua. The extract from the bioconversion of ( * )-d 8(’2,-15-dehydroprostaglandin E, with Flavobacterium sp. NRRL ~-5641 was dissolved in 30 % ethyl acetate-o.5 % acetic acid in benzene and chromatographed on a a-inch (diameter) glass column containing 450 g of silica gel (E. Merck). The extracts from the conversion of (~)-~8(12)-~5-dehydroprostaglandin E, with B-3873, B-3874, and B-3875 were submitted to partition chromatography using “Magic Column” procedure [II]. The stationary phase consisted of SilicAR CC-4 silicic acid (Mallinckrodt Chemical Works, St. Louis, MO. 63160) and the lower phase of benzene-methanol-water (I 5: 5: 2, by vol.) mixture. The upper phase of the system was used as the moving phase. RESULTS Flavobacteriunt sp. NRRL B-5641

Thin-layer chromatographic

analysis of the solvent extract indicated the pre-

266

sence of two major components; one compound with the same R, (0.52) as the fermentation substrate (2 J-d@‘*) -I 5-dehydroprostaglandin E, and another more polar compound (RF 0.28). The thin-layer chrolnatography mobility of the polar material corresponded to that shown by an authentic mixture of A*“*‘-prostaglandin E, (Compound 2, Fig. I) and I t,t5-epi-d*““) -prostaglandin E, (Compound 3, Fig. I ). The oily extract (3.0 g) was chromatographed on silicagel. Compounds having an R, of 0.28 were eluted with 40 y;; ethyl acetate-o.5 y$ acetic acid in benzene. Fractions were combined to give 349 mg of glassy material. This represents a yield of 23 I’;, {by wt) from (-~)-LI*‘~*~-I 5-dehydroprostaglandin E,. The nuclear magnetic resonance (NMR), infrared, and ultraviolet spectra of the material was identical with a synthetic mixture of I I, I 5-epi-d”” 2’-prostaglandin E, and disc’ 2’-prostaglandin E,. The lack of an optical rotation [XX];’0.00 (I .OI I “,;, methanol) indicated that the material was 50: 50 mixture of the two cis diastereoisomers.

The thin-layer chromatography analysis of the solvent extract from B-3875 showed the presence of ( -~)-LI~(‘~)- 15-dehydroprostaglandin E, and a more polar component (RF 0.24). This polar component corresponded to the thin-layer chromatography mobility of a mixture of I t-epi- and t5-epi-zl’““‘-prostaglandin E,. Partition chromatography of the 742-mg extract on IOO g SilicAR CC-4 gave 130 mg of a crude crystalline mass (R, 0.25): [CC];’+ 26.24 (I .o’I;,, methanol); m.p. 54-58 ‘C. This yield (theoretical) from ( +)-d8(‘2’- I 5-dehydroprostarepresents a 24?,, conversion glandin E,. Chromatographic fractions consisting of ( ir )-LI~(‘~‘- I 5-dehydroprostaglandin E, were combined to give 400 mg of material enriched in one enantiomer: on 5 p [a];’ - I 6.5 ( I .o “:,, methanol). The I 30 mg (R, 0.25) was rechromatographed SiIicAR CC-4 to give 55 mg of pure compound: [cr]~~+28.89 (I.o@ Y;,, methanols; m.p. 58-61 ,C. The optical rotation (dextrorotatory) and NMR of this compound was identical to synthetically prepared I I-epi-~I*“~’ -prostaglandin E,. (Compound 4. Fig. 2).

frrrns reduction Fig. 2. Microbial in Materials and Methods.

of

( J-~LI~“~‘-IS-dehydroprostaglandin

El.

Conditions:

outlined

Fiavobacferiutn sp. NRRL B-3874 A 2.3-g extract residue was chromatographed on 300 6 of SilicAR CC-4 to give 300 mg of material with R, 0.24. The preparation was rechromatographed on

267 20 g SilicAR CC-4 yielding 196 mg of material: [c(]ff -32.1 (0.964%, methanol); ultraviolet maximum (methanol) 276 nm, (s 26 700); m.p. 55 “C. This represents a E,. Further purific30 % theoretical yield from (+)-A ‘(I 21-I 5-dehydroprostaglandin ation on to g SilicAR CC-4 (not “Magic Column”) in 50% ethyl acetate-benzene produced pure material (m.p. 58.5-59.5 “C). The optical rotation (levorotatory) and NMR spectrum of this conpound were identical to synthetically prepared r5-epi48(‘2)-prostaglandin E, (Compound 5, Fig. 2). E, with ArthroThe bioconversion of (+ )-d *” 2,-15-dehydroprostagIandin bacfer sp. NRRL B-3873 also produced 15-epi-~*(12)-prostaglandin E,. One column chromatographic step yielded material with [x]g9 - 20.6 (I .021x, methanol). Compounds 4 and 5 (Fig. 2) are optical antipodes. Consequently, they exhibit like physical properties except for optical rotation. The racemic compound (I : I mixture of Compounds 2 and 3) is a diastereomeric isomer of Compounds 4 and 5. The spectral properties (NMR in deuteriochloroform, infrared in chloroform, and ultraviolet in methanol) of Compounds 2 and 3 are very similar to those of Compounds 4 and 5. The identity of Compounds 2 and 3 and Compounds 4 and 5 was demonstrated beyond doubt by thin-layer chromatography, melting points, and optical rotation. Our investigations revealed a high degree of stereospecificity of ketonehydrogenase activity of some bacteria toward a 48”2’-r5-dehydroprostaglandin E,. The presence of 8,r2-double bond was a necessary structural feature for this enzymatic activity. Removal of the unsaturation with the resulting configurational change in the molecule blocked ketone reduction.

DISCUSSION

During the screening for microbial 15-ketone reduction several bacteria were isolated which reduced Compound I to one or both of the @ans epimers; however, only one (NRRL B-5641) was found which produced cis compounds. Several yeast, particularly members of the genera Saccharomyces and Rhodotorula, and fungi were also capable of tram reductions. As the reduction of the C-I 5 ketone of d8(’ 2)-t 5-dehydroprostaglandin E, appears to be a common microbial reaction, so also is the reduction of the 13,14double bond. Yeasts, fungi, Actirzomycetes, and bacteria posses di3-reductase activity. The stereochemistry of the dihydro-product from these microorganisms is not known. One unidentified bacterium produced the dihydro-product. The optical rotation of the isolated material indicated the presence of a racemic mixture (Marsheck, W. J. and Miyano, M., unpublished). The reduction of the t3,r4-double bond of natural prostaglandins has been documented [rz]. The allylic alcohol at c-15 is oxidized first to the ketone followed by double bond reduction. As mentioned, Flavobacterium sp. NRRL B-5641 was the only bacterium which caused a cis reduction of Compound I. The fact that the reduction was directed toward both epimers and produced a racemic mixture is in contrast to the production of one enantiomer during tram reduction. The fermentation conditions to effect the cis reduction by B-5641 were more exacting than tram reduction by the other bacteria. Ffavobacterium sp. NRRL B-5641 grew well in Medium C but produced very low levels of the products as determined by analytical thin-layer chromatography, Washed cells of B-5641 seemed to reduce Compound I better than cells in growth medium.

268

Medium C without glucose and Medium D were tried with the idea that the enzyme(s) responsible for the reduction might be glucose repressible. Again, little or no conversion was realized. Media A and B at a elevated temperature (34 ‘C) appeared to give the best conversion. The method of inoculum preparation was found to have a significant effect on the conversion yield. Various per cent inocula from shake flask cultures of B-5641 were tried without success, however, if the inoculum represented cell suspensions prepared by washing agar slants, conversion yield was increased. The stereospecific microbial reductions of I 5-dehydroprostaglandin are shown in Fig. I and Fig. 2. These reactions provided a convenient method for producing analogues of natural prostaglandin E,. REFERENCES I Marsheck,

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