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Eugene J. Volker Shepherd College Shepherdstown. West Virginia 25443
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The Microbial Hydroxylation Of Progesterone An organic-biological experiment involving a reaction important to the pharmaceutical industry
A number of recent articles in this Journal have recognized the need for organic experiments directed to the interests of students in the life sciences; one of these involves the use of microorganisms to bring about a chemical transformation, specifically, the epoxidation of alkenes.1. While epoxidation of alkenes is also readily achieved by conventional chemical reagents, one well known example of the use of microorganisms in organic chemistry, the transformation of progesterone (I) to lln-hydroxyprogesterone (lI) by the mold Rhizopus nigricans, represents a reaction for which no convenient synthetic alternative is availahle. CH3
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(11) This microhial hydroxylation, mentioned in several recent organic texthooks,2 illustrates well the possibilities inherent in the use of microorganisms as "chemical reagents:" a good yield of stereospecifically formed product (90%under suitable conditions), functionalization of the molecule a t a normally unactivated site, and a simple, rapid experimental procedure. The importance of the above reaction to the pharmaceutical industry is great, this hydroxylation being a crucial step in a sequence that allows the conversion of the readily available plant-derived steroid diosgenin to cortisone and its derivatives, which have found use as antiinflammatory medical agenk3 The experiment described here is part of the organic chemistry laboratory program a t Shepherd College. It is based on the original procedure for the microhial synthesis of lla-hydroxyprogesterone published by Peterson and Murray in 1952.' This experiment offers the following principal advantages
1) it combines biology and chemistry 2) it illustrates an important commercial process 3) it is simple to perform, requiring no training in microbiology and only minimal specialized equipment (much uf which can be
improvised) Response to the experiment by the organic chemistry class, composed of students of chemistry, biology, medical technology, premedicine and prepharmacy, has been cousistently favorahle.
Dfscussion The experiment is performed in our laboratory in two ways 1) without the isolation of the reaction product, which ia characterized by thin layer chromatography 2) with the isolation of the reaction product, which is charactmized by its melting point and its infrared spectrum For the former purpose, 10 mg of progesterone in 50 ml of culture medium is found to suffice. Aliquots may he taken with sterile disposable pipets during the course of the reaction, and the disappearance of starting material and the appearance can he convenientlv observed hv of lla-hvdroxvoroeesterone ,. .. tlc of th; dichlon,mt:thane extracts of the aqueous samples. Commerrialls a\,ailable steroid3 (Aldrichl are used as refer.~ a vigorous growthof microorganism, ence m a t e ~ i a i s Given most of the starting material reacts a t room temperature within three hours, although longer reaction times are recommended for complete conversion. In conducting the chromatography, it is necessary to take into account the "hackground" due to the suhstrate-free growth medium; for this reason a small sample of the nutrientmedium should he taken immediately before the addition of the progesterone. The dichloromethane extract of this sample generally shows a single, high Rf spot by tlc, well separated from the spots due to the steroids. If it is desired to isolate and characterize lla-hydroxyprogesterone, it is most practical to conduct the transformation on about 100 mg of starting material either by comhining the products from several smaller scale runs or by scaling up the reaction in a single flask. Under the conditions employed in our laboratory (which are less favorahle for the conversion than those used by industry), 20-25% yields of crude (mp 157-60°C) Ila-hydroxyprogesterone are routinely obtained. Two recrystallizations are usually sufficient to produce a sample comparable to commercial material hy mixed melting point and ir spectrum. The experimental procedure can he interrupted without harmful effects a t several points (for example, a t the end of each section of the Experimental given below) by storing the flask containing the reaction mixture in a refrigerator.
'Kumler, P. L., and De Jong, P. J., J. CHEM. EDUC., 52, 475 (19761~ ,-. .. ,.
'Roberts, J. D., Stewart, R., and Caserio, M. C., "OrganieChemistry," W. A. Benjamin Inc., New York, 1971, p. 788; Allinger, N. L., Cava, M. P., Lebel, N. A., and Stevens, L. C., "Organic Chemistry,.' Worth Publishers, Inc., New York, 1971, p. 793; Brown, R. F., "Organic Chemistry," Wadsworth Publishing Company, Belmont, California, 1975, p. 878. "or an interesting account of the history and the economic significance of the lla-hydroxylation of progesterone, see Gottschalk, E. C. Jr., "The Wall Street Journal," July 9,1968, p. 1. 4Peterson,D. H., Murray, H. C., Eppstein, S. H.,Reineke, L. M., Weintrauh, A., Meister, P. D., and Leigh, H. M., J Amer. Chem. Soc., 74.5933 (1952). %Idrich's Ilu-hydroxmrq~sreronet95A1, m p 165-166°Cwas used i n all experiments.Thtr ammins a rrace of progesterone, recognhshle Volume 54, Number 1, January 1977 / 65
T h e length of t h e experiment can he tailored t o m a t c h t h e time available; s t u d e n t s m a y be assigned all of t h e s t e p s a s described under Experimental, o r t h e instructor m a y provide either t h e n u t r i e n t medium (step 1) o r t h e f u l l s developed culture of t h e microorganism (step 2). I n our situation i t h a s been found advantageous t o have teams of two students work on this experiment; n o difficulties have been encountered b y those who lack previous training in microbiology.
Experimentals Preparation of the Nutrient Medium One gram of Lactalysate (a casein hydrolysate), 2.5 g of dextrose, and 0.1 gof corn steep,7and enough tap water tomake 50ml of solution are stirred together in a beaker, and the p H of the mixture is adjusted to appro;imately 4.5 by the dropwis; addition of concentrated HCI. The solution is filtered into a 250-ml Erlenmeyer flask which is stoppered with a large cotton plug, a part of which should protrude from the neck of the flask for easy removal. The solution is autoclaved for 20 min; a number of pipets, wrapped in paper, are also sterilized at this time. (If an autoclave is not available, a pressure cooker of suitable size may be used.) Growing the Microorganism An inoculating loop is heated to redness in a flame, allowed to cool, then used to transfer a small "wad"of fungal mycelium of thestock culture of Rhizopus nigricanss into the liquid broth in the Erlenmeyer flask. In performing the inoculation, care must be taken to open containers for a minimum length of time only, to keep the interior end of the cotton plug from contamination, and to gently flame the mouth and neck of all containers both before and after opening. Next. the inoculated culture medium is olaced on a reciorocal shaker tar similar aerating device) for 2&48 i r at room temperature. During this time a thick mat of mycelium will form. ~
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start of the reaction. If complete conversion is desired, 24 hr of reaction time is allowed. Isolation a n d Characterization of the Product When reaction is complete, the aqueous reaction medium is decanted into a separatory funnel from the fungal mycelium which is pressed dry with a stirring rod. The aqueous layer is extracted with three portions of dicNoromethane, and the extracts are combined with a dichloromethane wash of the mycelium. The organic layer is first washed with saturated NaHC03, then with distilled water, and finally dried over anhydrous NazSOa and filtered. The solvent is evaporated under reduced pressure, and the residue examined by tlc.'O On preparative scale runs (100 mgor moresubstrate) the product Recrystallization is crystallized from dichloramethane-ether-pentane. from the same solvent system or from methanol-water produces crystals of lla-hydroxwrogesterone which are compared to cammereial material by mixed melting point and ir spectrum.
Acknowledgment T h e a u t h o r wishes t o t h a n k Dr. C a d F. Bell of t h e Biology Department of Shepherd College for t h e stock culture of Rhizopus nigricans, a n d for m a n y helpful discussions. T h e valuable comments o n t h e m a n u s c r i ~ of t Dr. J a m e s A. Moore of t h e Department of Chemistry of'the University of Delaware a r e gratefully acknowledged. technique ior theorganic T o r o prncriral guide fu micrubi~rl~~giral chemisr. w e Foukm G..and .lohnsun, H.."Chemrral Oxidations with DP. ! d h r . ~ > r ~ a n r a mh%r "c d Ihkkrr Inc.. New Y m k . 1972.. . . 213. ' ~ l t e ~ k e l ycorn , steep liquor, prepared hy soaking overnight 5 g of cracked corn in 100 ml of water and filtering, may be used. aThe fungus used in our laboratory was originally obtained by allowing wet squash strips to stand in a humid environment. I t was identified by microbiological examination and is now maintained an potato starch agar in Petri dishes and in the nutrient medium described in the experimental section. A pure culture of Rhizopus stolonifer + (nigricans) can be obtained from a commercial source such as Carolina Biological Supply Co., Burlington, N.C., 27215or Connecticut Valley Biological Supply Co., Inc., Southamptan, Mass.,
The Microbial Hydroxylation of Progesterone Immediately before the addition of the steroid substrate, a small sample of the liquid cultwe medium is withdrawn with a sterile pipet, to be used as a "backaround" samole for tlc, after suitable workuo. n1n72. Ten millierams ofhoeesterone are dissolved in 0.4 ml of N.N. 91t is not necessary to sterilize the DMF solution of progesterdimethvlfarknide. andthis solution is added with ~n ~ aterile ~ . . ~ . ~ ~ ~ . -nikat ~~ - - - ~ one. to the cunlents 191 the Erlenmeyer rlask using sterile transfer rech'°Fluorescent Silica Gel plates, a 1:l mixture of CClr and ethyl acn i q ~ r . ~ T hflask e ii mounted un a reciprocal shaker, and the reaction etate eluent, and uv spot detection are used. The proportions of the is followed by the periodic withdrawal of small portions of liquid with two solvents may need adjustment depending on the activity of the a sterile pipet; suggested sampling times are 1,2, and 3 hr after the stationary phase. ~
66 / Journal of Chemical Education
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