The Microbiological Transformation of Steroids

The Microbiological Transformation of Steroids

T.H. STOUDT Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. General Techniques A. Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................ B. Analysis and Isolation of Products.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Transformations of Steroids A. Hydroxylation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Dehydrogenation and Hydrogenation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Miscellaneous and Mixed Transformations. . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Transformation Mechanisms.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183 187 189 190 203 210 215 217 218

1. Introduction Mamoli and Vercellone (1937), in applying the hydrogenating and dehydrogenating activity of microorganisms to steroids, must be cited as having carried out the first microbiological steroid conversions. The preparation of testosterone from dehydroepiandrosterone, as worked out by Mamoli, was of commercial importance for some time.

Cor ynebacterium

HO

//

/

0

Dehydroepiandrosterone

I

Testosterone

Subsequently, reports of steroid conversions were made by Turfitt (1948) on the transformation of cholesterol to 4-dehydroetiocholanic acid, using 183

184

T. H. STOUDT

Nocardia sp., and by KrBmli and HorvBth (1948, 1949) , using Nocardia roseus, to produce the 76-hydroxylation of cholesterol.

Cholesterol

1

un

'I.$-Hydroxycholesterol

Nocerdiosp.

COiH I

4Dehydroetiocholanic acid

However, the remarkable potentials of the microbes in the transformation of steroids had to await the development of the antiarthritic steroids. With the announcement of Hench eC al. (1949) on the dramatic therapeutic effect of cortisone on rheumatoid arthritis, a tremendous research effort was touched off to find practical methods of producing commercial quantities of this steroid hormone. The cortisone used in this initial work was prepared in a partial chemical synthesis by Sarett (1946), utilizing deoxycholic acid, one of the more abundant of the bile acids, as the starting material.

;qOzH 0y,/Ky

CHzOAc

I

+

32 chemical steps

H Oi

-c'3

Deoxycholic acid

C'7

0//

Cortisone acetate

This synthesis required over 2 years to complete and resulted in 11 mg. of cortisone acetate. The complexity of the synthesis is perhaps best illustrated by the difficulties faced in the initial scale-up of the Sarett synthesis. I n March 1946,

185

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

a development group in the Merck Laboratories, headed by J. van de Kamp,

set out to produce additional material by charging 1270 pounds of deoxycholic acid in the first step. I n April 1948, the synthesis was completed, with a yield of 938 gm. of cortisone acetate. This represents better than 2 years to go through the 32-step synthesis, with an over-all yield of 0.15%. Adding to the complexity of the problem of commercial production was the limited availability of the starting material, as well as some essential steps which appeared to be only laboratory curiosities. The widespread research effort which followed took four main courses: ( 1 ) the improvement of the original Sarett synthesis, with appropriate modifications for commercial production ; ( 2 ) the development of syntheses from other available steroid starting materials, as well as the search for still unknown potentially useful steroids of plant origin; (3) the total chemical synthesis from relatively simple starting materials; and ( 4 ) the use of biological transformations to effect the crucial 11-oxygenation of readily available steroidal starting materials. The first two approaches were quite successful and played an important role in supplying the useful antiarthritic steroids in the intervening 10 years. The total chemical synthesis, although successful, has remained up t o now only an academic accomplishment. The last of these approaches probably was the most speculative and thus has appeared most dramatic. The observation of Hechter e t al. (1949) that adrenal homogenates are capable of introducing the 11-oxygen function into deoxycorticosterone to form corticosterone was a demonstration of a novel biosynthetic reaction, the direct enzymatic introduction of oxygen into the steroid nucleus. CHzOH

CHzOH

I

c=o I

I

Deoxycorticosterone(D0C)

HO

L o

(

Corticosterone

The later observation of 17a-hydroxylation and 21-hydroxylation by adrenal enzymes further supported the theory that the more highly oxygenated cortical hormones arise, a t least in part, from the direct enzymatic oxygenation of a relatively unoxygenated steroid such as progesterone. A classical demonstration of the unity of biology was made with the dramatic announcement by Peterson and Murray (1952) of the lla-hy-

186

T. H. STOUDT

droxylation of progesterone with the fungus Rhizopus nigncans to form 1la-hydroxyprogesterone. CHs

C Ha

L o

C=O

PI+

(1 ‘ 3-’

I

HO....& .) Rhizopus nigricans

/ / / 0

’

Progesterone

r‘3’--

/ / /

0

lla-Hydroxyprogesterone

This introduction of the oxygen function into the 11 position in high yields made available a large number of potential starting materials which could not be used in the existing chemical syntheses. Subsequently, the microbial llp-, 1 7 ~ and , 21-hydroxylations were also observed, which paralleled the route used by the mammalian enzyme system in the biosynthesis of cortisol (Compound F, hydrocortisone). The search for other hydroxylating systems in both mammalian and microbial systems has now produced many additional hydroxylations. Figure 1 is a comparison of the status of hydroxylations by enzymes from these two types of systems.

C=O

HO

C=O

HO OH

Mammalian Enzymes 2a, 28, 6a, 6j3, 7, 118, 16a, 17a, 18, 19, 21

OH

OH

OH Microbial Enzymes la, 28, 68, 7a, 78,9a, lop, l l a , 118, 128, 14a, 15a, 158, 16a, 17a, 19, 21

FIG.1. Comparison of status of hydroxylations by enzymes from mammalian and microbial systems.

The only mammalian hydroxylations which have not yet been reproduced microbially are the 2a, 6~t,and 18. The versatility and effectiveness of the microbial systems has permitted the preparation of many new steroids difficult to obtain by chemical synthesis. Some of these new steroids have actually displaced, in part, the natural steroid hormones, cortisone and cortisol, in therapeutic use. The microbial transformations, on the other

187

THE MICROBIOLOGICAL TRANSFORMATION O F STEROIDS

hand, are frequently beset by a serious difficulty. Multiplicity of steroid products is rather common, seriously affecting the yield of the desired product, as well as increasing greatly the difficulty of isolation. Vischer and Wettstein (1953) and Fried et al. (1953) independently reported the microbial 1-dehydrogenation of progesterone, accompanied by the degradation of the acetyl side chain. Nobile et al. (1955) in applying the 1-dehydrogenation to cortisone and cortisol using Corynebacterium simplex, reported the resulting 1-dehydro derivatives possess three to four times the antiinflammatory activity of the natural hormones. This represents a rather dramatic example of microbial transformations leading to thc discovery of analogs of the normal steroid hormones which are therapeutically more active. CHzOH

CHzOH

HO

Cortisol

1

Prednisolone

This biological dehydrogenation is not known in any but microbial systems, and the effectiveness of the new series is, in part, due to the lessened ability of mammalian systems to inactivate through the reduction of ring A. The author does not intend this to be a complete review paper on the microbiological conversions of steroids. All types of transformations are discussed, citing in some cases only some of the more pertinent references on the matter. Complete coverage can be obtained in a number of excellent review papers by Vischer and Wettstein (1958), Peterson (1958), Shull (1956a), Eppstein et ul. (1956), and Fried et al. (1955).

II. General Techniques A. FERMENTATION The microbial transformation of steroids represents, in type, an example of the application of fermentation to bring about relatively minor structural changes to a complex molecule, referred to as the substrate. This is in contrast to the complete synthesis of antibiotics, vitamin BIZ, etc., from medium ingredients. The steroid transformations occupy a rather unique position, in that the economic value of the substrate is generally very high, frequently in excess of $1.00 per gram. As a consequence of this

188

T. H. STOUDT

and the relatively low aqueous solubility, fermentation techniques differ somewhat from normal to insure the maximum yield of product. The fermentation cycle is normally carried out in two phases. The first phase consists of the growth period, which usually employs environmental conditions capable of supplying a luxurious growth of culture. The nutritional requirements of the transforming cultures are so variable that the medium may be as simple as a sucrose-mineral medium or an exceedingly complex type. The temperature selected is usually optimum for the growth of the culture along with highly aerobic conditions. I n some few cases the medium is quite important in directing the course of the transformation. For example, Sutter et al. (1957) reported the 1dehydrogenation of cortisone with very little side reactions with Corynebacterium simplex, using a yeast-extract medium, whereas other types of media yielded predominantly the 20p-dihydrocortisone and 20p-dihydrol-dehydrocortisone. The length of time of the growth phase is dependent on the culture and environmental conditions, bacteria usually requiring 12-24 hours and fungi 24-72 hours to attain full growth. To facilitate the isolation of product, the cells in some cases can be filtered or centrifuged from the broth and resuspended in water before steroid addition. The addition of steroid is generally made a t the end of the growth phase, although, occasionally, favorable yields are also realized by making the addition simultaneously with the inoculation. The addition can be made as finely divided crystalline material, but, more frequently, solutions in appropriate solvents are more conveniently made. Acetone, ethanol, and methanol generally meet the requirement of fair solubility for steroids and relatively low toxicity for the conversion enzymes. Dimethylformamide possesses exceptional steroid solubility properties (10-20%) and is relatively nontoxic below levels of 2% in the medium. The level of steroid added is quite variable, being dependent on the transforming capacity of the culture, as well as the potential toxicity of substrate or product ; e.g., deoxycorticosterone possesses rather significant antifungal activity (Tarbet et al., 1953). Normally 200-800 mg. per liter are effectively converted, but a charge up to 4 gm. per liter was reported by Karow and Petsiavas (1956) in the conversion of progesterone to Ilahydroxyprogesterone in 5-liter fermentors. These authors also utilized periodic or continuous addition of substrate to alleviate the toxicity problem of substrate and carrier. The conversion period normally ranges from 12 t o 72 hours, being largely dependent on the charge of the substrate and the conversion capacity of the culture. Highly aerobic conditions are always required for rapid transformations which produce steroid oxygenation or dehydrogenation.

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

189

B. ANALYSISAND ISOLATION OF PRODUCTS The analysis or isolation requires the extraction of steroids from the broth with appropriate immiscible organic solvents; e.g., the use of methylene chloride, chloroform, ethyl acetate, and methyl isobutylketone have been reported. The solvent ratios, of course, are dependent on the distribution ratio of the steroid product between the extracting solvent and the broth. Whole broth may yield unmanageable emulsions, which can be prevented by prior centrifugation or filtration of cells. Normally, transformation products are largely contained in the filtered broths, but the cell material, if it contains steroidal product or substrate, can be extracted separately. Concentration of the extract produces material suitable for chromatography or, a t times, direct isolation by fractional crystallization. A major factor in the rapid progress in microbiological transformation of steroids since 1949 undoubtedly has been the application of paper chromatography, particularly the methods of Zaffaroni and Burton (1951) and Bush (1952). Reinke (1956) described the use of these basic systems, including a number of modifications, as applied to the qualitative and quantitative determinations of steroids. The mobility relationships of a large number of compounds are tabulated and a description given of the detection methods used. Zaffaroni (1953), Savard (1954), and Neher (1958) discuss in great detail the use of chromatography as applied to steroids. A very rapid assay procedure, particularly in the case of ultraviolet absorbing steroids, can be applied to paper strips cut into channels, utilizing an automatic recording densitometer. The use of paper chromatography and the appropriate detection tests can, of course, give only tentative identification. Further characterization may include the preparation of appropriate derivatives, e.g., esters, oxidation products, reduction products, etc., which can frequently be prepared directly on the paper chromatograms and then be developed in a second dimension. The ultraviolet spectrum, as well as the spectrum of the reaction products with 95% sulfuric acid (Bernstein and Lenhard, 1953, 1954), are useful measurements which can be performed on approximately 100 pg. of material eluted from a paper chromatogram. I n the case of biologically active steroids, microanimal tests can be extremely useful. For example, the antiinflammatory steroids cause a marked drop in circulating eosinophiles, and a mouse responds to less than 10 pg. of material in the testing of the more active compounds. The degree of difficulty of isolation of substantial quantities of product is quite variable, being largely influenced by the yield of the sought-after product, as well as the similarity of products produced by side reactions. I n the better conversions, the product can generally be crystallized directly

190

T. H. STOUDT

from a concentrate of the extract. Mixtures which resist separation by fractional recrystallization require column chromatography.

111. Transformations of Steroids

A. HYDROXYLATION 1. 11-Hydroxylation

a. Ila-Hydroxylation. The occurrence of the lla-hydroxylation enzyme systems in microorganisms is quite widespread, judging from the large number of reports which have appeared, covering a wide range of types of cultures and substrates transformed. Fungi, particularly of the genera R hizopus and Aspergillus, apparently are most adaptable for commercial applications. The lla-form is apparently novel for microbial systems and is inactive in the antiinflammatory tests. However, chemical conversion to the 11-keto or the llp-hydroxyl is readily performed as outlined below. CHzOH

CHzOH I

L o microbial transformntion

’ 11-Epicortisol

11-Deoxycortisol (Compound S)

1

chromic acid

CHiOH

CHzOH

C=O

C=O

I

I

Hoy,,/pjOH

( ‘ 1 3

0/ / /

Cortisol

&OH

hydrogenation

/

0

0

\\

\/

/ Cortisone

The lla-hydroxylation is a high-yielding transformation as are the subsequent chemical steps. I n commercial production, the microbial step is frequently preferred earlier in the synthesis, particularly on the quite available starting material,

191

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

progesterone. Progesterone now has been made quite plentiful and inexpensive as derived from diosgenin and stigmasterol, both abundant plant steroids.

Progesterone

1la-Hydroxyproges terone

I

chemical oxidation

CHzOH

c=o I

CHa I

Cortisone CHzOH I

Cortisol (Compound F)

The lla-hydroxylation also lends itself well to the synthesis of 9a-fluoro steroids which are the most active class of antiinflammatory steroids known. The discovery of the marked influence of the 9a-flUoro group on the antiinflammatory activity of corticosteroids was made by Fried and Sabo (1953) while attempting to convert chemically the microbially produced lla-hydroxyl to the llp-form. The dehydration of the ll-hydroxyl, either the a- or p-form, yields the Ag911-derivative, which is converted chemicalIy to the 9a-fluoro-1 lp-hydroxy steroid.

192

T. H. STOUDT

CHzOH I

c=o

Ho....,.,,f',,),OH

CH20H

I

C=O

("3'-

/ / / 0

11-Epicortisol

CHzOH I

CHzOH

I

c=o

9a-Fluorocortisol

The Sa-fluorocortisol, despite its high activity, is limited to topical use because of its extremely high salt-retaining properties. However, the 16ahydroxyl and 16a-methyl derivatives are considerably less toxic and are extensively used as antiinflammatory agents in the 9a-fluoroprednisolone series. Peterson and Murray (1952) made the original discovery of the microbial ll-hydroxylation with the observation of the lla-hydroxylation of progesterone with R h i z o p s nigricans. This is a high-yielding reaction, with reported yields of 88-92% a t charge levels of 3 gm. per liter. Shortly thereafter, Fried and co-workers (1952) reported the same conversion with Aspergillus niger. Subsequently, many other reports of lla-hydroxylation have been made, being fully covered in the review papers cited previously. Besides being high yielding, this transformation is generally quite versatile in having been applied to a wide range of steroidal substrates, but usually limited to A4 ,3-keto steroids. Table I summarizes the versatility of the lla-hydroxylation as to substrates converted, as well as the wide range of microbes capable of the transformation. b. Ilp-Hydroxylation. The less common llp-hydroxylation was first observed by Hanson et al. (1953) with Cunninghamella blakesleeana and Streptomyces fradiae (Colingsworth et al., 1952,1953). The yields are apparently not feasible for commercial application. One interesting and unique facet of the transformation by Cunninghamella was the production of the 11-keto derivative, cortisone. The presence of alcohols, fatty acids,

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

193

TABLE I MICROBIAL ~~~-HYDROXYLATION Substrate Progesterone

Culture

Rhizopus nigricans Aspergillus niger Bacillus cereus Aspergillus ochraceus Dactylium dendroides Aspergillus niger Cephalothecium roseum

Compound S

1-Dehydro -Compound S Allopregnane-3,20dione 19-Nortestosterone 3-Ketobisnor-4-cholen-22-a1

-OH

1la lla 1la lla 1la 1la

+ 6s + 6s

Reference Peterson and Murray (1952) Fried et al. (1952) McAleer et al. (1958~) Dulaney et al. (1955b)

+ 68 Dulaney et al. (1955a) lla + 17< lla Weisa et al. (1956) lla + 21 1la Meister et al. (1954a) lla + 17q lla 1la

1la

Hanson et al. (1953)

Schmidt-Thorn6 (1957)

Delacroixia coronata

1la 11s 1la

Testa (1957)

Rhizopus nigricans

1la

Eppstein et al. (1953)

Rhizopus nigricans Rhizopus nigricans

1Iff 1Iff

Pederson et al. (1956) Meister et al. (1954b)

Cunninghamella blakesleeana Absidia glauca

11s

and polysaccharides were found to favor the production of cortisol, while 2,4-dinitrophenol improved the yield of cortisone (Mann e t al., 1955a, 1955b). Shull and Kita (1955) reported the use of Curvularia lunata for the conversion of 11-deoxycortisol to cortisol. As is the case in most of the llphydroxylating systems, some 1la-hydroxylation also takes place. The 14a-

11-Deoxycortisol (Compound S)

Cortisol (Compound F)

194

T. H. STOUDT

CHIOH I

11-Epicortisol

CHzOH

14a-Hydroxycortisol

CHzOH

O ,)lH ,,,,

c=o

,,,']3i-

/\A

0

14~-Hydroxy-ll-deoxycortisol

hydroxylation of the substrate and the cortisol also complicate the conversion. The only report of a bacterial llp-hydroxylation utilizes a Pseudomonas sp. (Nawa et at., 1958) with Compound S as the substrate. Rather intriguing is the fact that the 1-dehydrogenation is also performed by this culture. CHzOH I

CHzOH

c=o I

11-Deoxycortisol (Compound S)

Cortisol

+

CHzOH

I

HC-OH

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

195

TABLE I1 MICROBIAL 118-HYDROXYLATION Culture

Substrate Progesterone Compound S

l-DehydroCompound S

Curvularia lunata Curvularia lunata Streptomyces fradiae

-OH

Reference Shull and Kita (1955) Rubin et al. (1956) Colingsworth et al. (1952, 1953) Hanson et al. (1953)

Cunninghamella blakesleeana Coniothyrium sp. Pycnosporium sp. Rhodoseptoria sp. Dothichizia sp. Trichothecium sp. Pseudomonas sp. Corticium sasaki Sclerotina sp.

Fried et al. (1955) Shull (1956a) Shull (1956a) Shull (1956a) Shull (1956b) Nawa et al. (1958) Hasegawa et al. (1957) Shirasaka et al. (1958)

Absidia glauca

Schmidt-Thorn6 (1957)

Corticium sasaki

Hasegawa et al. (1957)

These same workers have recently applied mutation techniques to the Pseudomonas sp. to produce a culture which is capable of transforming Compound S to prednisolone in 30% yield (Nawa et al., 1959). This transformation has obvious commercial potentialities, as well as demonstrating, for the first time, that remarkable improvements can be made in steroid transformations by the same mutation techniques applied so successfully to other microbial processes. The reported Ilp-hydroxylations are listed in Table 11. 2. 17a-Hydroxylation

The microbial introduction of the tertiary 17a-hydroxyl group is relatively uncommon, as indicated by the infrequency of reports of the transformation. Also, the usefulness of the reported transformations is generally rather limited because of low yields, limited versatility as to substrate transformed, or the simultaneous hydroxylations in other positions. Meystre et at. (1954) reported the 17~~-hydroxylation of progesterone, deoxycorticosterone, and 11-dehydrocorticosterone with Tricothecium roseum. The 17a-hydroxylation of progesterone by Trichoderma viride was demonstrated by McAleer and Dulaney (1956b). Cephabthecium roseum (Meis-

196

T. H. STOUDT

ter et al. (1954a) (synonymous with Tricothecium roseum) and Dactylium dendroides (Dulaney et al., 1955a) were reported to 17a-hydroxylate progesterone with the simultaneous introduction of the lla-hydroxyl. The evidence presented in the latter paper indicated the l7a-hydroxylation could occur only on 1la-hydroxyprogesterone. Table I11 lists the reported 17a-hydroxylations.

3. 21-Hydroxylation Since the introduction of the 21-hydroxy function of most steroids is performed quite effectively with established chemical procedures, the importance of the microbial transformation has been generally reduced. Ophiobolus herpotrichus was observed to give rather good yields (80%) in the conversion of progesterone to dcoxycorticosteronc (Meystre et al., 1954), and subsequently was put to use in a rather ingenious way to accomplish the synthesis of aldosterone (Vischer et al., 1956). The methyl ketone side chain of a racemic lactone (I) was hydroxylated selectively so that only the a-ketol (11) was formed. This accomplished an important synthetic step as well as the resolution of the racemic mixture. The a-ketol (11) was then reduced chemically to produce the naturally occurring aldosterone.

OphioboEua herpotrichus

11 (4 chemiarl

reduction

d-Aldosterone

The 21-hydroxyIation was found coupled with the lla-hydroxylation with Aspergillus niger (Weisz et al., 1956) , while Curvularia lunata simultaneously introduces the 21- and llp-hydroxyls in the conversion of pro-

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

197

TABLE I11 MICROBIAL 17-IIYDROXYLATION Substrate Progesterone

Deoxycorticosterone 1I -Dehydrocorticosterone

Culture

-OH

Trichoderma viride

17a

Cephalothecium roseum Dactylium dendroides

17a 17a lla 17a 6fl 17a

Tricothecium roseum Tricothecium roseum

Reference

+ 110 + lla

McAleer and Dulaney (195613) Meister et al. (1954s) Dulaney et al. (1955a) Meystre et al. (1954) Meystre et al. (1954)

TABLE IV MICROBIAL 21-HYDROXYLATION Substrate Progesterone

l7a-Hydroxypro-

gesterone

Culture

-OH

Ophiobolus herpotrichus Aspergillus niger Wojnowicia graminis

21 21 21

Aspergillus niger Curvularia lunata Ophiobolus herpotrichus

21 21 21

+ +

Reference

Meystre el al. (1954) Zaffaroni et al. (1955) McAleer and Dulaney (1956a) lla Weisz et al. (1956) 11s Rubin et al. (1956) Meystre et al. (1954)

gesterone to corticosterone (Rubin et al., 1956). Table I V lists the reported 21-hydroxylations. 4. 16a-Hydro~ylution

The report of the 16a-hydroxylation of progesterone by Streptomyces sp. (Perlman, 1952) followed closely the announcement of the lla-hydroxylation of steroids from the Upj ohn Laboratories. This hydroxylation appears to be the most common among the actinomycetes, although several fungi have subsequently been reported. The transformation had no practical implication until the announcement of the therapeutic usefulness of triamcinolone (Bernstein et d., 1956b). The 16~hydroxy1,although reducing substantially the over-all antiinflammatory activity of the extremely active 9a-fluorocortisol and 9afluoroprednisolone, eliminates the high salt-retaining properties of the parent compounds. Thoma et al. (1957) reported the 16a-hydroxylation of gafluorocortisol and ga-fluoroprednisone by Streptornyces roseochronaogenus,

198

T. H. STOUDT

HO

CHzOH I C=O

CHzOH

S . roseoehromogenus

0 9a-Fluorocortisol

16~-Hydroxy-9a-fluorocortisol

1

Mgcohacterium rhodocbous

Corynebacten'um simplex

CH20H

I

9a-Fluoroprednisolone

CHzOH I

Triamcinolone

which, coupled to the microbial 1-dehydrogenation, gives two alternate microbial routes to triamcinolone from 9a-fluorocortisol. The reported 16-hydroxylations are shown in Table V.

5. Other Hydroxylations a. 1 - and 6-Hydroxylation. Hydroxylations in the 1and 2 positions have only recently been reported with a limited number of substrates. Some degree of interest existed in obtaining these hydroxylations because of their TABLE V

MICROBIAL 16-NYDROXYL AT ION ~~

Substrate Testosterone Progesterone Deoxycorticosterone 9a-Fluorocortisol 9a-Fluoroprednisolone

1

Culture Streptomyces roseochromogenus Pastalotia fumera Streptomyces s p . Streptomyces sp. Didymella vodakii Streptomyces roseochromogenus Streptomyces roseochromogenus

-OH

Reference

16a

Fried et al. (1955)

16a

16a 16a

Thoma et al. (1955) Perlman et al. (1952a) Vischer et al. (1954) Wettstein (1955) Thoma et al. (1957)

16a

Thoma et al. (1957)

16a 16a

T H E MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

199

TABLE VI MICROBIAL 1- AND 8-HYDROXYLATION Substrate 4-Androstene-3,17dione Compound S

Sol-Fluorocortisol

1

Culture

-OH

1

Reference

Penicillium sp.

Dodson et al. (1957)

Streptomyces s p . Rhizoctonia ferrugena

Herzog et aZ. (1957) Greenspan et al. (1957)

Sclerotina sp.

Shirasaka el al. (1958)

Streptomyces s p .

McAleer et al. (1958a)

structural relationship to the l-dehydro steroids. However, no reports of any significant physiological activity have appeared. Dodson et al. (1957) reported the la- and 2P-hydroxylation of 4-androstene-3,17-dione by Penicillium sp., while Rhizoctonia ferrugena (Greenspan et al., 1957) hydroxylated Compound S in the 2 position and the 16 position. Streptomyces sp. affected the 2p-hydroxylation of Compound S (Herzog et al., 1957) as well as the 16-hydroxylation of 9a-fluorocortisol (McAleer et al., 1958a). Reported 1- and 2-hydroxylations are listed in Table VI. b. 6p-Hydroxylation. Reports of the microbial hydroxylation of the 6 position are extremely common but are confined to the 6p-hydroxylation. A wide range of substrates has been reported, but generally the hydroxylation is accompanied by hydroxylations in the l l a , 14a, or 17a positions, providing they are not already substituted. I n the conversion of progesterone by Aspergillus ochraceus (Dulaney et al., 1955a), the 6p-hydroxylation proceeds only after the 11a-hydroxylation has been essentially complete. Also, the use of Zn++-deficient medium to prevent 6p-hydroxylation was reported, which implies a significant difference in enzyme systems. Table VII lists reported 6p-hydroxylations. c. 7-Hydroxylation. The first report of a microbial hydroxylation of a steroid was made by KrAmli and HorvAth (1948, 1949) with the 76hydroxylation of cholesterol with Nocardia roseus. Subsequently, the 7aand 7p-hydroxylation have been reported with a number of substrates. One interesting aspect of 7-hydroxylat8ionis the frequency of this transformation with steroids which do not possess the A4-3-keto group. For example, a Rhizopus sp. 1la-hydroxylates deoxycorticosterone but 7phydroxylates allopregnane-3p,21-diol-20-one(Kahnt et al., 1952). Rhizopus arrhizus also was found to 7P-hydroxylate pregnenolone (Eppstein et al., 1956). Curvularia lunata very commonly hydroxylates in the 7p and 14a

200

T. H. STOUDT

~

Substrate 4-Androstene-3,17dione Deoxycorticosterone l7a-Hydroxyprogesterone Compound S

I

TABLE VII MICROBIAL 6-HYDROXYLATION Culture Rhizopus arrhizus

I

-OH

I

68 1la 68

Eppstein et al. (1954)

+ 14a + lla

Mucor cornymbifer Aspergillus ochraceus roCephalothecium seum Rhizopus arrhizus Rhizopus arrhizus

6p 1701 6p 6p

Rhizopus arrhizus

6p

68

Reference

Camerino et al. (1953a) Dulaney et al. (195513) Meister et al. (1954a) Eppstein et al. (1953) Meister et al. (1953b) Peterson (1952)

and

Murray

TABLE V I I I MICROBIAL 7-HYDROXYLATION Substrate

Culture

-OH

Reference

Cholesterol

Nocardia roseus

7€

Progesterone

Helminthosporium sp. Cladosporium sp. Rhizopus arrhizus

7a 7P

78

Krhmli and Horvhth (1948) McAleer et al. (1958b) McAleer et al. (1958b) Eppstein et al. (1956)

Curvularia sp. Rhizopus sp.

7a 78

Meystre et al. (1955) Kahnt et al. (1952)

Curvularia lunata Cephalosporium sp.

7a

5-Pregnenolone Deoxycorticosterone Allopregnane-3p ,21diol-20-one Compound S

lla

7a

+ 14a

Shull (1956a) Bernstein et a l . (1959)

positions simultaneously (Shull, 1956a). McAleer et al. (1958b) reported the 7a-hydroxylation of progesterone with a Helminthosporium sp., while a Cladosporium sp. introduced the 7P-hydroxyl. Table VIII lists reported 7-hydroxylations. d. 8- and 9-Hydroxylation. A number of reports of 8- or 9-hydroxylation have appeared, based on the demonstration of the introduction of a tertiary hydroxyl and, subsequently, eliminating the 14a-hydroxy as a possibility. Dodson and Muir (1958a, b) and Schubert e t al. (1958) presented the first structural assignments of an 8- or 9-hydroxylation. The former demonstrated the 9a-hydroxylation with a Nocardia sp. and a Pseudomonas sp. on 4-androstene-3, l7-dione1 while the latter established

201

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

TABLE IX MICROBIAL 8- A N D 9-HYDROXYLATION Substrate

Culture

4-Androstene-3,17dione 1,4-Androstadiene3,17-dione Progesterone

Deoxycorticosterone

Nocardia sp.

9a

Pseudomonas sp.

9a

Streptomyces aureofaciens Circinella sp.

8- or 9-

Helicostylum piriforme

Mucor parasiticus Neurospora crassa Curvularia pallescens (I

-OH

9a 14a 9a

Reference Dodson and Muir (1958b) Dodson and Muir (1958a) Fried et al. (1955) et al. Schubert (19%)

+ 14c

al. 8- or 9-0 Eppstein et (1956) 8- or 9-0 Stone el al. (1955) 8- or 9-a Stone el al. (1955) 8- or 9-" Wettstein (1955)

Probably all identical.

the 9a-hydroxylation of progesterone, using a Circinella sp. Table I X gives the reported 8- and 9-hydroxylations. e. lop-Hydroxylation. In the transformation of 19-nortestosterone (I) by Rhizopus nigricans, some lop-hydroxyl-19-nortestosterone (11) was isolated (Pederson et al., 1956). However, the main course of the transformation of this 19-norsteroid was the expected lla- and 6p-hydroxylations. OH

OH

I

I1

f . 12p-Hydroxylation. The microbial 12-hydroxylation has been reported in several recent papers. Progesterone was converted by Calonectria decora to the 12p, 15p-dihydroxyprogesterone (Schubert et al., 1957). Fusarium lini was found to 12p-hydroxylate digitoxigenin, while other substrates devoid of the 14p-hydroxyl are hydroxylated in the 15a position (Gubler and Tamm, 1958). Tamm and Gubler (1959) reported the 12phydroxylation of bufalin, a steroid toad poison, with F. lini. g . l4a-Hydroxylation. The 14a-hydroxylation is a frequently observed

T. H. STOUDT

TABLE X MICROBIAL14-HYDROXYLATION

Progesterone

Mucor hiemalis Bacillus cereus Curvularia sp.

Deoxycorticosterone

Cunninghamella esleeana

Compound S

Reference

-OH

Culture

Substrate

blak-

Curvularia lunata Curvularia lunata

1401 14a 14a

Meister el al. (1953~) Fried et al. (1955) Wettstein (1955)

14a 118

Mann et al. (1955b)

+

1401 14a 118

Shull (1956a) Shull and K i t a (1955)

701

transformation carried out by a wide range of microorganisms. The 14ahydroxylation has been reported to occur frequently along with the llp-, 7p-, or the 9a-hydroxylation. Reported 14-hydroxylations are listed in Table X. h. 15-Hydroxylation. Vischer and Wettstein (1958) reported a Fusarium sp. capable of both the 1 5 ~and - 15p-hydroxylation of deoxycorticosterone. A considerable number of other reports cite the introduction of either a 1 5 ~ or - 15p-hydroxyl including the 15p-hydroxylation by a bacterium, Bacillus megaterium (McAleer et al., 1 9 5 8 ~ )Fried . et al. (1955) established the basis for assigning configuration to the 15-hydroxyls, since both were unknown prior to the microbial synthesis. Table XI lists the reported 15-hydroxylations.

Substrate Progesterone

Deoxycorticosterone

Compound S

I

TABLE XI MICROBIAL~ ~ H Y D R O X Y L A T I O N Culture Penicillium notatum Colletotrichum antirrhini Fusarium culmorum Phycomyces blakesleeana Bacillus megaterium Gibberella baccata Lenzites abientina Fusarium sp. Spicaria sp. Hormodendrum viride

1

-OH 1501

1

-1

Reference

Camerino and Modelli (1956) 15a Fried et al. (1955) 15a Kluger et al. (1957) 1 5 ~ Fried el al. (1955) 1 5 ~ MeAleer et al. (1958~) Meystre et al. (1955) 150 1 5 ~ Meystre et al. (1955) 15a Vischer and Wettstein (1958) 15P Bernstein et al. (1956a) 158 Bernstein el al. (1956a) 15lY

203

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

i. 19-Hydroxylation. The only existing report of the microbial hydroxylation of the angular methyl groups is the 19-hydroxylation of Compound S (I) by Corticium sasakii (Nishikawa and Hagiwara, 1958) to 19-hydroxy Compound S (11). CHZOH

CHzOH

c=o

C=O

I

I

Corticium saaakii ___f

/v

0

1

I1

The 19-hydroxylation is of importance in mammalian systems as the key intermediate in the biological conversion of steroids belonging to the androstane series to steroids of the estrogen series. The 19-hydroxylation apparently facilitates the elimination of C-19. The 19-hydroxylated androstanes are also readily converted to estrogens by microorganisms, e.g., Nocardia sp. and a Pseudomonas sp. (Dodson and Muir, 1958a, 1958b).

B. DEHYDROGENATION AND HYDROGENATION 1. Ring Dehydrogenation

a. A1-Dehydrogenation. The 1-dehydrogenation of steroids was first reported accompanied by the degrading of the 17-acetyl side chain. Fusarium solani (Vischer and Wettstein, 1953) and Streptomyces lavendulae Fried et aE., 1953) were reported to convert progesterone (I)to A1s4-androstadiene-3,l’l-dione (11), with the latter also producing some l-dehydrotestosterone (111). CH,

I c.=o

I

f

204

T. H. STOUDT

The conversion is high yielding and produces an intermediate which can be utilized in the synthesis of estrone. 0

0

~l’*~-Androstadiene-3 ,17-dione

Estrone

The discovery that the 1-dehydro analogs of cortisone (prednisone) and cortisol (prednisolone) possessed increased antiinflammatory activity (Bunim et al., 1955) stimulated the search for efficient microbiological methods of preparing these compounds. Nobile et al. (1955) reported the use of Corynebacterium simplex in the preparation of prednisone and prednisolone. CHzOH

CHzOH

I

I

c=o

PIT’“\p,+oH

c=o Corynebacterium simplex

/\/ 0

Cortisone

0 Prednisone CHzOH

CHzOH I

c=o Ho

\

f

\

(1 ‘ 3-’

/\/

Cortisol

\

I

H0

c=o

?\‘a’-*ol+<\

0/\/

Prednisolone

The only side reaction, which seems to be medium-dependent, is the reduction of the 20-carbonyl to the 2Op-01. Subsequently, additional bacteria and fungi have been reported to 1-dehydrogenate selectively a wide range of A4 ,3-keto steroids: Mycobacterium laticola (Sutter, 1957), Bacterium subtilis (Lindner et al., 1956), Septomyxa afinis (Sutter et al., 1957), Bacillus sphaericus (Stoudt et al., 1955) , Didymella lycopersiri (Vischer et al., 1955a), Ophiobolus sp., Calonectria decora, and Alternaria sp. (Vischer et al., 1955b). Fusarium solani (Vischer et al., 195513) is applicable only to 17-hydroxysteroids.

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

205

The microbial 1-dehydrogenation can be developed into a high-yielding process, attested to by its use in the commercial production of the l-dehydro steroids. The low order of substrate specificity has also contributed to its wide application in the preparation of a large number of 1-dehydro steroids which were hitherto unknown. b. A*-Dehydrogenation. The 4-dehydrogenation was reported by Vischer and Wettstein (1953) in the conversion of androstane-3 ,17-dione (I) and allopregnane-3 ,20-dione (11) to 1,4-androstadiene-3 ,17-dione (111) by Fusarium sp. 0

I

CHI

I

c=o

-

Fuearium sp

0

111

Levy and Talalay (1957) also demonstrated the 1,4-dehydrogenation with soluble enzyme of Pseudomonas testosteroni, using androstane-3 ,17dione or etiocholane-3 ,17-dione as substrate. This latter substrate represented the first example of a 4-dehydrogenation involving a 5p-hydrogen. Stoudt et al. (1958) applied the 1 , 4 dehydrogenation by Nocardia blackwellii to produce prednisone (I)and prednisolone (11) from the corresponding 3-hydroxypregnanes and 3-hydroxyallopregnanes. CH20H

Pregnane-3a,l l , ~ , 17a, 2l-tetrol-2O-one

I1

206

T. H. STOUDT

YHz OH

CHzOH I

Allopregnene-.h, 17a,21triol-ll,20-dione

I

The transformation apparently occurs with the 3-keto and the A4,3keto as intermediates, since they are readily isolated after relatively short incubation periods. Tamaki (1958) reported the 4-dehydrogenation of cholic acid (I) by Streptomyces rubescens and a Corynebacterium sp. Also, this work makes the only report of a 6-dehydrogenation, with the isolation of 3,la-dioxy4,6-choladienic acid (11) from the cholic acid transformation with S. rubescens.

I

3-Oxy-4-cholenic acid

-I-

I1

2. Ring Hydrogenation

a. Al-Hydrogenation. Butenandt et al. (1940) reduced l-androstane17p-ol-3-one (I) with yeast to yield the corresponding androstane analog. Herzog and co-workers (1959) reported the conversion of prednisone to cortisone, and prednisolone to cortisol, with Bacillus megaterium.

207

T H E MICROBIOLOGICAL TRANSFORMATION O F STEROIDS

OH

I

l

OH

l

I

l

l

Androstane-17@-01-3-0ne

CH20H

YH20H

I

Bacillus megaterium _9

Prednisone (prednisolone)

Cortisone (cortisol)

b. A4-Hydrogenation. Mamoli et al. (1939) reported the conversion of 4-androstene-3,17-dione to androstane-3,17-dione and etiocholane-3,17dione with Bacillus putrificus.l I n the 1la-hydroxylation of progesterone by Rhizopus nigricans (Peterson and Murray, 1952), some allopregnanella-oI-3,20-dione was isolated. Perlman et al. (195213) reported pregnane16a-ol-3,20-dione as a transformation product of progesterone by a Streptomyces sp. Thus the reduction of the A4-steroids has led to saturated steroids with 5a- and 5p-hydrogens. c. hl6-Hydrogenution. Meister et al. (1953a) reported the transformation of A16-progesterone (I) by Rhizopus nigricans to lla-hydroxy-17aprogesterone (11). CHs

CH3 I

c=o I

C=O Rhizopus nzgncans

0 I

11

This unusual a-Configuration of the side chain is less stable than the pconfiguration and, therefore, is readily transformed to the natural form. The identity of which is discussed by Eppstein et al. (1956).

208

T. H. STOUDT

3. Dehydrogenation of Hydrozyl Groups

A wide variety of microorganisms is capable of dehydrogenating the secondary alcohol groups of steroids to produce the corresponding carbonyl derivative. Bacteria and actinomycetes have been most frequently reported, but many fungi, also, are capable of performing these dehydrogenations. The positions reported are 3, 7, 11, 12, 17, and 20, with the following examples typifying the conversions : CHs

CHI

I

I

c=o

c=o

5-Pregnene-3p-ol-20-one

ci.p'

Progesterone

(Perlman, 1952)

0

OH

/\ HO

Estradiol

Streptomgees albus

(Welsch, 1948)

Flauobacterium sg.

+

/

HO 5-Androstene-3@,17@-diol

//

0

Testosterone

;;qcOzH ,,....C13' - c!3<(Ercoli, 1941)

Alcaligenesz faecales

HO"

a

cus.

___ "OH

Cholic acid

0//

(Hoehn et al., 1944)

3,7,12-Triketocholanic acid

This culture has been characterized in the Merck Laboratories as Bacillzis sphaeri-

209

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

VH20H

CHZOH

I

I

C=O

C=O

Cortisol

Cortisone

(Meystre et al., 1954) CHa

CHI

I

Flavobacterium dehrdrogennns

/

HO OH 7a-Methyl-5-pregnene3&7&20-trio1

0 (Robinson et at., 1959)

OH 7a-Methyl-4-pregnene7@-01-3,20-dione

4. Hgdrogenation of Carbonyls The microbial reduction of carbonyl groups has been reported for carbonyls a t 3,7 (Fukui, 1937),17,20, and 22. The reduction of 3-carbonyls produces either 3a- or 3p-hydroxylsJ while the reduction of 17- and 20carbonyls has resulted only in the p-orientation. These reductions are extremely common side reactions in hydroxylations and the 1-dehydrogenation conversions. CH,

I

\,qA-,

0

A WJ/A

c;-15° CHa

I c=o yeast

___f

0 H H 0.'" H Allopregnane-3,11,2O-trione Allopregnane-3a-01-11,20-trione (Camerino et al., 1953b)

0 HO H Gndro~tane-3~17-dione Androstane-3B,17B-diol (Vercellone, and Mamoli, 1937)

210

T. H. STOUDT

CHI

CH, I

1

HLOH

C=O I

Progesterone

(Fried et al., 1953)

4-Pregnene-20P-ol-3-one

Bisnor-4-cholen-22-al-3-one 1 l a ,22-Dihydroxybisnor-4-cholen-3-one (Meister et al., 1954b)

C. MISCELLANEOUS AND MIXEDTRANSFORMATIONS 1. Miscellaneous Transformations

a. Epoxidation. The epoxidation of steroidal double bonds is an extremely rare example of biological epoxidation. Bloom and Shull (1955) demonstrated the Sfi,ll-epoxidation of 9(11)-dehydro Compound S ( I ) , and the 14a,15-epoxidation of 14 (15)-dehydro Compound S (11), using CHQOH I C=O

I

CHzOH I C=O

Curualaria

9p,11-Oxido-CompoundS

CHgOH

CH2 OH

L

c=o

I

O

A

0

11

0

Id.\

OH

14a,15-Oxldo Compound 6

211

THE MICROBIOLOGICAL TRAKSFORMATION OF STEROIDS

Curvalaria lunata and Cunninghamella blakesleeana. These cultures normally introduce the 1lp-hydroxyl and 14a-hydroxyl into compound S. Cultures which normally introduce the lla-hydroxy failed to form the 9,ll-epoxide from the corresponding olefin. 16-Dehydroprogesterone, also, was not epoxidized by an actinomycete capable of 16a-hydroxylation. On the basis of these findings, Bloom has postulated that microorganisms which normally hydroxylate a saturated steroid will epoxidiee the unsaturated analog only if the hydroxyl group to be introduced is axial in conformation. I n this case, the epoxide introduced will also be axial. Some speculation now exists as to the possibility that very similar mechanisms exist for hydroxylation and epoxidation, although epoxides are not intermediates for hydroxylation, as evidenced by the fact they are not further transformed. 6. Ring A Aromatization. The arorriatization of the ring A of 19-hydroxy-4-andro~tene-3~17-dione to form estrone, as demonstrated by Meyer (1955) with mammalian systems, established a biological route to estrogens from the androstane series. The same transformation was observed recently by Dodson and Muir (1958b) with a Nocardia sp. 0 Nocardia sp.

/-

4

HO

0

Estrone

19-hydroxy-4-androstene3,17-dione

The microbial aromatization appears to go through the 1-dehydro intermediate, although this intermediate has been largely ruled out in mammalian systems. Levy and Talalay (1957) demonstrated the aromatization of 19-nortestosterone to yield estrone and estradiol-3,17a, using Pseudomonas testosteroni. A novel aromatization was observed by Dodson and Muir (1958a, b) with l14-androstadiene-9a-ol-3, 17-dione (I), including the cleavage of the C9-Clo bond, to form 9,10-seco-3-hydroxy-l,3,5 (10)-androstatriene-9,17-dione (11). 0

0 ocardia sp.

I

I1

212

T. H. STOUDT

This is undoubtedly an important microbial route in the degrading of steroids to serve as a source of carbon. c. Side Chain Degradation. The oxidation of the side chain of steroids of the pregnene series yielding a 17-keto androstene, appears to be an extremely common transformation, frequently accompanied by a hydroxylation or 1-dehydrogenation. Peterson et al. (1953) described the conversion of progesterone (I) to 4-androstene-3 ,17-dione (11)with Penicillium lilacinum and Gliocladium catenulatuna.

<\,,gs (fp CHB

I c=o

0

’

/ / ,

0

/ / ’

0

I

I1

The degradation of cholesterol to 4-dehydroetiocholanic acid by Nocardia sp. was cited in the Introduction. This rare observation of the microbial degradation of the side chain of cholesterol is in contrast to the ability of mammalian systems to utilize cholesterol in the biosynthesis of adrenal hormones. d. Steroid Esterases. The hydrolysis of estrone acetate, propionate, and butyrate to estradiol-3,17~with yeast was reported in the early work of Mamoli (1938). The widespread occurrence of esterases capable of hydrolyzing steroid esters is indicated by the fact that 21- and 3-acetates are nearly always hydrolyzed prior to hydroxylation or dehydrogenation. A notable exception is the 1-dehydrogenation of cortisone-21-acetate to prednisone-21-acetate with Corynebacterium simplex (Nobile et al., 1955). The chemically inert llp-acetate was hydrolyzed by Flavobacteriuin dehydrogenans in the conversion of cortisol-ll,21-diacetate to cortisol (Charney et al., 1959). The reaction is reported to be high yielding and, therefore, could be of some importance as a final step in a synthesis of cortisol, utilizing the llp-acetate as an intermediate. This esterase is not known in mammalian systems, as cortisol-11-acetate is inactive. The microbial hydrolysis of steroid saponins to their corresponding aglycones was observed in the conversion of diosin to diosogenin with Aspergillus and Penicillium (Krider et al., 1954; Rothrock et al., 1955). e. Ring D Rearrangements. Fried et al. (1953) reported the transformation of progesterone (I)to 1-dehydrotestolactone (11) with Cylindrocarpon radicicola. 1-Dehydrogenation, side-chain cleavage, and a ring D rearrangement are realized in this conversion.

213

T H E MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

I

I1

Camerino and Modelli (1956) and Camerino and Vercellone (1956) observed a rather unique biologically induced rearrangement with the action of yeast on a 16a, 17-oxidopregnene. The 20-carbony1 is reduced and the ring D rearrangement is also effected. This represents the only example of a microbially produced 20a-01. CHa

CH3

I

C=O

I&, 17-Oxidoprogesterone

I

HO-C-H

A4J3-17&Methyl-18-nor20~~17a-pregnadiene-16~~, diol-3 one

Fried et al. (1952) observed a ring D arrangement resulting in the transformation of L7a-hydroxyprogesterone (I) with Aspergillusniger to 17a-diol-3,17a D-homosteroid, 17a-methyl-D-homo-4-androstene-lla, dione (11). CH3

I

c=o

I

I1

f. Hydrolysis of Eporides. Epoxides are relatively resistant to microbial hydrolysis, as indicated in the discussion of epoxide formation. Recently, Camerino and Schiaky (1959) reported an epoxide hydrolysis with fermenting yeast ; 4 p , 5-epoxyprogesterone (I) yielded allopregnane3a ,4p ,5a-triol-2O-one (11).

214

T. H. STOUDT

CH3

CH3

I

I c=o

c=o

I

I

I 2. Mixed Transformations

I1

Mixed transformations are extremely common, and plague, to varying degrees, many attempts to direct a microbial transformation to a highyielding process. The degradation of the side chain accompanied by l-dehydrogenation was discussed under 1-dehydrogenation, while hydroxylations accompanied by hydrogenations also have been exemplified. a. Side Chain Oxidation and Hydroxylation. An extremely common mixed transformation consists of a hydroxylation accompanied by the oxidation of the acetyl side chain. An example of this transformation was made by Peterson and co-workers (1953) in the conversion of progesterone (I) to 4-androstene-6p-ol-3,17-dione (11). CH3

I c=o

0

b. A1-Dehydrogenation and Side Chain Degradation of Saturated Steroids. Meeks and co-workers (1958) reported the side chain oxidation and 1-dehydrogenation of pregnane-3,11,2O-trione,as well a s the corresponding allopregnane, using Septomyxa afinis. The allopregnane transformation is the more rapid and the higher-yielding conversion.

0\(\,3 CH3

I

Scptomyza a@&

( ‘ 1 3

0//VH

PregnaneS,Il, 20-trione

’

J$,c, //

0

JU

0 l-Etiocholene-3,11,17-dione

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

215

CHa I

Allopregnane-3,lI ,2O-trione

l-Androstene-3,11,17-dione

This 1-dehydrogenation is unique in that it proceeds with or without the double bond in the 4 ( 5 ) position. The 1,4-dehydrogenations, cited previously, occur stepwise with the 4-dehydrogenation occurring first. c. Complete Ring A Hydrogenation. Ring A hydrogenation, including the reduction of the 3-carbonyl, has resulted in the 3 ~ h y d r o x yand the 5phydrogen. Mamoli et al. (1939) reported the reduction of 4-androstene3 ,17-dione to etiocholane-3a ,17p-diol. Barkemeyer et al. (1960) observed the reduction of cortisone (I) to tetrahydrocortisone (11) with a Streptomyces sp. The reduction of ring A appears to be less frequent in the presence of a 21-hydroxyl. CHzOH

CHzOH I

I

I

I1

D. TRANSFORMATION MECHANISMS 1. Hydroxylation

The pioneer work on the steroid hydroxylation mechanism was carried out particularly by Dorfman and Hayano with mammalian enzymes (Hayano and Dorfman, 1954; Hayano et al., 1955a, b ). These studies were performed with the soluble llp-hydroxylase systems obtained from beef adrenals, with the supplementation of the cofactor TPN.3 Using tracer studies, these workers established t,hat the oxygen introduced into the steroid originates from the atmospheric oxygen and not from water (D20, H2018, and 0218were used as tracers). Furthermore, the llp-hydroxylation of progesterone-lla, 12a-T (Hayano et al., 1958) resulted in no change * TPN and DPN are used to designate the tri- and diphosphopyridine nucleotides respectively.

216

T. H. STOUDT

of the tritium content, but the chemical oxidation of the introduced l l p hydroxyl to the carbonyl function resulted in a 70% loss of tritium. This evidence strongly supports the theory of direct replacement of the hydrogen by the hydroxyl, with retention of configuration. CH3

CHa I

I

lip-hydroxylase of beef adrenal8

chromic acid

The microbial systems, although not yet reported as cell-free systems, appear to have a similar mechanism; i.e., hydroxylation in an 0218atmosphere with Rhizopus arrhizus (6p-) , Rhizopus nigricans ( l l n - ) , Cunninghamella blakesleeana (11p-), Cephalothecium roseurn (17a-) and Ophiobolus herpotrichus (21-) yielded in each case O18-labeled steroids (Bloom et al., 1956; Dorfman and Hayano, 1956; Hayano et al., 1955a). Furthermore, Hayano et a2. (1958) 1la-hydroxylated progesteronel l a ,12a-T with Rhizopus nigricans and found the introduced hydroxyl displaces the lla-T. Corey et al. (1958), in lla-hydroxylating progesteronellp-D with Rhizopus nigricans, came to the same conclusion. The inability to prepare cell-free systems has prevented convincing experimentation on the cofactor requirements of microbial systems. However, a few reports do exist on the effect of various inhibitors on hydroxylations. The 16a- hydroxylation by Streptomyces sp. (Perlman et al., 1952a; Perlman et al., 1955) is inhibited by selenite, arsenite, and azide, while no inhibition was observed with cyanide and fluoride (Perlman, 1956). Dulaney et al. (1955a) reported a zinc requirement for 6p-hydroxylation. The microbial enzymes have been reported to be, in origin, both constitutive and adaptive. Perlman and co-workers (1957) demonstrated the adaptive nature in two Streptomyces cultures, one capable of 16a-hy-

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

217

droxylation, and the other, the side chain degradation and l-dehydrogenation, with progesterone as substrate. The simultaneous addition of a number of antibiotics with the substrate prevented steroid conversions, while these antibiotics had little effect when added after the conversion had begun. On the basis of these studies, the mechanism appears to involve a series of enzymatic actions with the following sequence: (1) an oxygen-activating system, (2) a steroid-activating system which lends the specificity as to the position hydroxylated, and (3) a system mediating the oxidation of T P N H or DPNH. 2. Hydrogenation and Dehydrogenation

Talalay and co-workers have made a rather extensive study of the steroid dehydrogenating enzymes of P s e u d m o n a s testosteroni. This culture, after repeated transfer on medium in which testosterone is the sole carbon source, was shown to possess ~ c L3p- , (17p) and ring A dehydrogenases (A1,A4-5a, and A4-5p), as well as an isomerase which shifts the double bond from A5 to A4. The purification, kinetics, and specificity studies have been reported in a series of papers (Levy and Talalay, 1957; Marcus and Talalay, 1956; Talalay et al., 1953; Talalay and Marcus, 1954-1956; Talalay and Wang, 1955; Talalay, 1957). The 3a-, and 3/3-(17p) dehydrogenases were shown to be DPN-linked, while the ring A dehydrogenases are probably flavoprotein in nature (Levy and Talalay, 1957). These studies represent rather convincing evidence that a hydroxylation is not an intermediate step in ring dehydrogenations.

E. DISCUSSION The remarkable similarity of microbial and mammalian systems in steroid hydroxylations, as to positions attacked, and mechanism, has been presented in this paper. Our current knowledge indicates only a somewhat greater versatility for the microbes as to positions hydroxylated and substrates which can be converted. Fungi, certainly, dominate the literature on microbial hydroxylations, but bacteria and actinomyces are being cited more frequently in the recent literature. Fungi as a group are apparently the most versatile as to positions hydroxylated, as well as to the yields of a single steroid product which can be realized. Actinomycetes and bacteria are well known for their ability to utilize steroids as a sole source of carbon, and, therefore, we might anticipate the fact that many drastic changes in the steroid are frequently brought about by a single culture. The similarity of microbial and mammalian systems extends to the hydrogenation reactions. The reversibility of this reaction, however, appears to be limited to the microbes, as evidenced by the fact that the mam-

218

T. H. STOUDT

mals inactivate steroids principally through hydrogenation. The degrading of side chains, hydrolysis of esters, and aromization of ring A are common to both types of systems. From a practical standpoint, the microbiological transformations of steroids represent a valuable adjunct to the chemical synthesis of the complex steroid hormones and their analogues. The pharmaceutical industry in recent years has come to be more dependent on the microbial conversions of complex molecules, usually involving complex stereoisomerism. Microbial reactions have also proven invaluable in the preparation of new steroids in the research laboratory. The discovery of the l-dehydro steroids and the 9a-fluoro steroids came about as a result, the latter somewhat indirectly, of microbial conversions of steroids. Frequently raised is the question of function of the steroid conversion in the physiology of the microbe. A number of persons have suggested a possible detoxification mechanism, including CBpek et al. (1957) , on the basis of his fiinding that the steroid substrate, and not the product, had an effect on the respiration rates of the converting culture. The study was rather limited, having been performed on a single example of a hydroxylation, a hydrogenation, and a dehydrogenation. The potential energy gain for the organism is generally of such a small order that this hardly seems to be functional. Rather, it seems more likely that the answer lies in the fact that many microorganisms are capable of utilizing steroids as a sole source of carbon, under special conditions. Obviously, this utilization involves a stepwise oxidation which apparently does not always allow a substantial build-up of intermediates. Perhaps relatively simple transformations are merely expressions of this basic ability of microbes to degrade steroids completely, but, for reason of environmental conditions or genetic deficiencies, the intermediates are largely trapped. The Pseudornonas testosteroni isolated by Talalay is a good example of this phenomenon, in that it is capable of complete degradation of steroids, and yet the isolated enzymes can be shown to 1- and 4-dehydrogenate the steroid ring A, a property also reported for fungi and actinomyces. Dodson’s work, previously cited, with Pseudomonas sp. and Nocardia sp. demonstrates how, through dehydrogenation and hydroxylation, the steroid nucleus can ultimately be broken, presumably on its way to serving as a carbon source for these cultures.

REFERENCES Barkemeyer, H. R., Stoudt, T. H., Chemerda, J. M., Kozlowski, M. A,, and McAleer, W. J. (1960). J . A p p l . Microbiol. I n press. Bernstein, S., and Lenhard, R. H. (1953). J . Org. Chem. 18, 1146. Bernstein, S., and Lenhard, R. H. (1954). J. Org. Chem. 19, 1269. Bernstein, S., Feldman, L. I., Allen, W. S., Blank, R. H., and Linden, C. E. (1956a). Chem. & Znd. ( L o n d o n ) p. 111.

THE MICROBIOLOGICAL TRANSFOWTION OF STEROIDS

219

Bernstein, S., Lenhard, R. H., Allen, W. S., EIeller, M., Littell, R., Stolar, S. M., Feldman, L. I., and Blank, R. H. (195613). J . Am. Chem. SOC.78, 5693. Bernstein, S., Allen, W. S., Heller, M., Lenhard, R. H., Feldman, L. I., and Blank, R. H. (1959). J . O r g . Chem. 24, 286. Bloom, B. M., and Shull, G. M. (1955). J . Am. Chem. SOC.77, 5767. Bloom, B. M., Hayano, M., Saito, A., Stone, I).,and Dorfman, R. I. (1956). Federation Proc. 15, 222. Bunim, J. J., Pechet, M. M., and Bollet, A. J. (1955). J . Am. Med. Assoc. 157, 311. Bush, I. E. (1952). Biochem. J . 50,370 Butenandt, A,, Dannenberg, H., and Surhye, L. A. (1940). Ber. 71,818. Camerino, B., Alberti, C. G., and Vercellone, A. (1953b). Helu. Chim. Acta 36, 1945. Camerino, B., and Schiaky, R. (1959). Gazz. chim. ital. 89, 654. Camerino, B., and Vercellone, A. (1956). Gazz. chim. ital. 86,260. Camerino, B., Alberti, C. G., and Vercellone, A. (1953a). Gazz. chim. ital. 83, 684. Camerino, B., Modelli, R., and Spalla, C. (1956). Gazz. chim. ital. 86, 1226. Cipek, A., Han6, O., and Malikovi, E. (1957). Ceskoslov. mikrobiol. 2, 286. Charney, W., Weber, L., and Oliveto, E. P. (1959). Arch. Biochem. Biophys. 79, 402. Colingsworth, D. R., Brunner, M. P., and Haines, W. J. (1952). J . Am. Chem. SOC.74, 2381. Colingsworth, D. R., Karnemaat, J. M., Hanson, F. R., Brunner, M. P., Mann, K. M., and Haines, W. J. (1953). J . Biol. Chem. 203, 807. 80, 2338. Corey, E. J., Gregoriou, G. A., and Peterson, D. H. (1958). J . Am. Chem. SOC. Dodson, R. M., and Muir, R. D. (1958a). J . A.m. Chem. SOC.80, 5004. Dodson, R. M., and Muir, R. D. (195813). J . A.m. Chem. SOC.80, 6148. Dodson, R. M., Goldkamp, A. H., and Muir, R. D. (1957). J . Am. Chem. SOC.79, 3921. Dorfman, R. I. (1958). Ann. Rev. Biochem. 26, 523. Dorfman, R. I., and Hayano, M. (1956). Abstr. 130th Meeting Am. Chem. SOC.p. 57c. Dulaney, E. L., McAleer, W. J., Barkemeyer, :El. R., and Hlavae, C. (1955a). J . Appl. Microbiol. 3, 372. Dulaney, E. L., Stapley, E. O., and Hlavac, C. (195513). Mycologia 47,464. Eppstein, S. H., Meister, P. D., Peterson, D. H., Murray, H. C., Leigh, H. M., Lyttle, D. A,, Reineke, L. M., and Weintraub, A. (1953). J . Am. Chem. SOC.75, 408. Eppstein, S. H., Meister, P. D., Leigh, H. M., Peterson, D. H., Murray, H. C., Reinke, L. M., and Weintraub, A. (1954). J . Am. Chem. SOC.76, 3174. Eppstein, S. H., Meister, P. D., Murray, H. C., and Peterson, D. H. (1956). Vitamins and Hormones 14, 359. Ercoli, A. (1941). 2. physiol. Chem., Hoppe-Seyler’s 270, 266. Fried, J., and Sabo, E. F. (1953).J . Am. Chem. SOC.75, 2273. Fried, J., Thoma, R. W., Gerke, J. R., Herz, J. E., Donin, M. V., and Perlman, D. (1952). J . Am. Chem. SOC.74, 3962. Fried, J., Thoma, R. W., and Kingsberg, A. (1953). J . Am. Chem. SOC.75,5764. Fried, J., Thoma, R. W., Perlman, D., Herz, ,J. E., and Borman, A. (1955). Recent Progr. in Hormone Research 11, 149. Fukui, T. (1937). J . Biochem. (Tokyo) 25, 61. Greenspan, G., Schaffner, C. P., Charney, W., Herzog, H. L., and Hershberg, E. B. (1957). J . Am. Chem. Soc. 79, 3922. Gubler, A,, and Tamm, C. (1958). Helv. Chim. Acta 41, 297, 301. Hanson, F. R., Mann, K. M., Nielson, E. D., Anderson, H. V., Brunner, M. P., Karnemaat, J. N., Colingsworth, D. R., and Haines, W. J. (1953). J . Am. Chem. SOC.75, 5369.

220

T. H. STOUDT

Hasegawa, T., Takahashi, T., Nishikawa, T., and Hagiwara, H. (1957). Bull. Agr. Chem. Soc. Japan 21, 390. Hayano, M., and Dorfman, R. I. (1954). J . Biol. Chem. 211,227. Hayano, M., Lindberg, M. C., Dorfman, R. I., Hancock, J. E. H., and von Doering, W. (1955a). Arch. Biochem. Biophys. 59, 529. Hayano, M., Saito, A,, Stone, D., and Dorfman, R. I. (195513). Biochim. et Biophys. Acta 21, 380. Hayano, M., Gut, C., Dorfman, R. I., Sebek, 0. K., and Peterson, D. H . (1958). J . Am. Chem. SOC.80, 2336. Hechter, O., Jacobsen, R. P., Jeanloe, R . W., Levy, H., Marshall, C. W., Pincus, G., and Schenker, V. (1949). J . Am. Chem. SOC.71,3261. Hench, P. S., Kendall, E. C., Slocumb, C. H., and Polley, H. F. (1949). Proc. Staff Meetings Mayo Clinic 24, 181. Herzog, H. L., Gentles, M. J., Hershberg, E. B., Carvajal, F., Sutter, D., Charney, W., and Schaffner, C. P. (1957). J . Am. Chem. SOC.79,3921. Herzog, H . L., Gentles, M. J., Charney, W., Sutter, D., Townley, E., Yudis, M., Kabasakalian, P., and Hershberg, E. B. (1959). J. Org. Chem. 24, 691. Hoehn, W. M., Schmidt, L. H., and Hughes, H . B. (1944). J . Biol. Chem. 152,59. Kahnt, F. W., Meystre, Ch., Neher, R., Vischer, E., Wettstein, A. (1952). Ezperientia 8, 422. Karow, E. O., and Petsiavas, D. W. (1956). Ind. Eng. Chem. 48, 2213. Kluger, B., Siebert, R., and Schubert, A. (1957). Naturwissenschaften44, 40. Krhmli, A., and HorvBth, J. (1948). Nature 162, 619. Krhmli, A,, and Horvhth, J. (1949). Nature 163, 219. Krider, M. M., Cordon, T . C., and Wall, M. E. (1954). J . Am. Chem. Soc. 76, 3515. Levy, H . R., and Talalay, P. (1957). J. Am. Chem. SOC.79, 2658. Lindner, F., Junk, R., Kehl, H., Nesemann, G., and Schmidt-ThomC, J. (1956). Naturwissenschaften 43, 39. McAleer, W. J., and Dulaney, E. L. (1956a). Arch. Biochem. Biophys. 62, 109. McAleer, W. J., and Dulaney, E. L. (1956b). Arch. Biochem. Biophys. 62, 111. McAleer, W. J., Kozlowskj, M. A., Stoudt, T. H., and Chemerda, J. M. (1958a). J . Org. Chem. 23, 508. McAleer, W. J., Kozlowski, M. A., Stoudt, T. H., and Chemerda, J . M. (1958b). J . Org. Chem. 23, 958. McAleer, W. J., Jacob, T. A., Turnbull, L. B., Schoenewaldt, E. F., and Stoudt, T. H . (1958~).Arch. Biochem. Biophys. 73, 127. Mamoli, L. (1938). Ber. 71,2698. Mamoli, L., and Vercellone, A. (1937). Ber. 70,470,2079. Mamoli, L., Koch, R., and Teschen, H. (1939). 2.physiol. Chem., Hoppe-Seyler’s 261, 287. Mann, K. M., Hanson, F. R., O’Connell, P. W., Anderson, H. V., Brunner, M. P., and Karnemaat, J . N. (1955a). Appl. Microbiol. 3, 14, 16. Mann, K. M., Hanson, F. R., and O’Connell, P. W. (1955b). Federation Proc. 14, 251. Marcus, P. J., and Talalay, P. (1956). J . Biol. Chem. 218, 661. Meeks, R. C., Meister, P. D., Eppstein, S. A., Rosselet, J . P., Weintraub, A., Murray, H. C., Sebek, 0. K., Reineke, L. M., and Peterson, D. H. (1958). Chem. di. Ind. (London) p. 391. Meister, P. D., Peterson, D. H., Murray, H . B., Eppstein, S. H., Reineke, L. M., Weintraub, A,, and Leigh, H . M. (1953a). J . Am. Chem. SOC.75, 55. Meister, P. D., Peterson, D. H., Murray, H. C., Spero, G. B., Eppstein, S.H., Weintraub, A., Reineke, L. M., and Leigh, H . M. (1953b). J. Am. Chem. SOC.75, 416.

THE MICROBIOLOGICAL TRANSFORMATION OF STEROIDS

221

Meister, P. D., Eppstein, S. H., Peterson, D. H., Murray, H. G., Leigh, H. M., Weintraub, A , and Reineke, L. M. (1953~).Abstr. 125rd Meeting Am. Chem. SOC.p. 5c. Meister, P. D., Reineke, L. M., Meeks, R. C., Murray, H. C., and Peterson, D. H. (1954a). J . Am. Chem. SOC.76, 4050. Meister, P. D., Peterson, D. H., Eppstein, 8. H., Murray, H. C., Reineke, L. M., Weintraub, A,, and Osborn, H. M. L. (1954b). J. Am. Chem. SOC.76, 5679. Meyer, A. S. (1955). Ezperientia 11,99. Meystre, Ch. Vischer, E., and Wettstein, A . (1954). Helv. Chim. Acta 37, 1548. Meystre, Ch., Vischer, E., and Wettstein, A. (1955). Helv. Chim. Acta 38, 381. Nawa, H. et al. (1958). Tetrahedron 4,201. Nawa, H. et aE. (1959). Tetrahedron Letters [18], 17. Neher, R. (1958). J. Chromatography 1, 122, 205. Nishikawa, M., and Hagiwara, H. (1958). Chem. Pharm. Bull. 53, 452. Nobile, A., Charney, W., Perlman, P. L., Herzog, H. L., Payne, C. C., Tulley, M. E., Jevnik, M. A,, and Hershberg, E. B. (1955). J. Am. Chem. SOC.77, 4184. Pederson, R. L., Campbell, J. A,, Babcock, J. C., Eppstein, S. H., Murray, H. C., Weintraub, A,, Meeks, R. C., Meister, P. D., Reineke, L. M., and Peterson, D. H. (1956). J. Am. Chem. SOC.78, 1512. Perlman, D. (1952). Science 115, 529. Perlman, D. (1956).Abstr. 130th Meeting Am. Chem. SOC.p. 33A. Perlman, D., O'Brien, E., Bayan, A. P., and Greenfield,R. B. (1952a). J. Am. Chem. SOC. 74, 2126. Perlman, D., Titus, E., and Fried, J. (195213).J . Am. Chem. SOC.74, 2126. Perlman, D., O'Brien, E., Bayan, A. P., and Greenfield, R. B. (1955). J. Bacteriol. 69, 347. Perlman, D., Weinstein, M. J., and Peterson, G. E. (1957). Can. J. Microbiol. 3, 841. Peterson, D. H. (1958). Proc. Intern. Congr. Biochem., 4th Congr., Vienna, 1958 4, 83. Peterson, D. H., and Murray, H. C. (1952). .I. Am. Chem. SOC.74, 1871,5933. Peterson, D. H., Eppstein, 8. H., Meister, P. D., Murray, H. C., Leigh, H. M., Weintraub, A,, and Reinke, L. M. (1953). J. Am. Chem. SOC.75,5768. Reinke, L. M. (1956). Anal. Chem. 28, 1853. Robinson, C. H., Gnoj, O., Charney, W., Gilmore, M. L., Oliveto, E. P. (1959). J. Am. Chem. SOC.81, 408. Rothrock, J. W., Stoudt, T. H., and Garber, J. D. (1955). Arch. Biochem. Biophys. 57, 151. Rubin, B. A., Casas Campillo, C., Hendrichs, J., Cordoba, F., and Zaffaroni, A. (1956). Bacteriol. Proc. (SOC. Am. Bacteriologists) 56, 33. Sarett, L. (1946). J. Biol. Chem. 162,591. Savard, K. (1954). Recent Progr. in Hormone Research 9, 185. Schmidt-Thorn;, J. (1957). Angeur. Chem. 69,238. Schubert, A,, Langbein, S., and Siebert, R. (1957). Chem. Ber. 90, 2576. Schubert, A., Onken, D., Siebert, R., and Heller, K. (1958). Chem. Ber. 91, 2549. Shirasaka, M., Tsuruta, M., and Nakamura, M. (1958). Bull. Agr. Chem. SOC.Japan 22, 273. Shull, G. M. (1956a). Trans. N . Y . Acad. Sci. [2] 19, 147. Shull, G. M. (1956b). U. S.Patent 2,765,258. Shull, G. M., and Kita, D. A. (1955). J . Am. Chem. SOC.77, 763. Stone, D., Hayano, M., Dorfman, R. I., Hechter, O., Robinson, C. R., and Djerassi, C. (1955). J . Am. Chem. SOC.77,3926. Stoudt, T. H., McAleer, W. J., Chemerda, J. M., Kozlowski, M. A., Hirschmann, R. F., Marlatt, V., and Miller, R. (1955). Arch. Biochem. Biophys. 59, 304.

222

T. H. STOUDT

Stoudt, T. H., McAleer, W. J., Kozlowski, M. A,, and Marlatt, V. (1958). Arch. Biochem. Biophys. 74, 280. Sutter, D., Charney, W., O'Neill, I'. I,., Carvajal, F., Hcrzog, H. L., and Hersliberg, E. B. (1957). J. Org. C'hem. 22, 578. Talalay, P. (1957). Physiol. R e v . 37, 362. Talalay, P., and Marcus, P. J. (1954). Nature 173, 1189. Talalay, P., and Marcus, P. J. (1956). J. Biol. Chem. 218, 675. Talalay, P., and Wang, V. S. (1955). Biochim. et Biophys. Acta 18, 300. Talalay, P., Dobson, M. M., and Tapley, D . F. (1953). J. Biol. Chem. 205, 823. Tamaki, K. (1958). J. Biochem. ( T o k y o ) 45, 693. Tamm, C., and Gubler, A. (1959). Helv. Chim. Acta 42, 473. Tarbet, J. E., Oura, M., and Sternberg, T . H. (1953). Mycologia 45, 625. Testa, E. (1957). Ann. chim. (Rome) 47, 1132. Thoma, R. W., Gerke, J. R., Greenspan, G., Herz, J. G., and Fried, J. (1955). 69th Meeting, N . Y . C. Branch Soc. A m . Bacteriologists; cited by Shull (1956a). Thoma, R. W., Fried, J., Bonanno, S., and Grabowich, P. (1957). J . Am. Chem. Soc. 79, 4818.

Turfitt, G. E. (1948). Biochem. J. 42, 376. Vercellone, A., and Mamoli, L. (1937). Z . physiol. Chem., Hoppe-Seyler's 248, 277. Vischer, E., and Wettstein, A. (1953). Experientia 9, 371. Vischer, E., and Wettstein, A. (1958). Advances in Enzymol. 20, 237. Vischer, E., Schmidlin, J., and Wettstein, A. (1954). Helv. Chim. Acta 37, 321. Vischer, E., Meystre, C., and Wettstein, A. (1955a). Helv. Chim. Acta 38, 1502. Vischer, E., Meystre, C., and Wettstein, A. (195513). Helv. Chim. Acta 38, 835. Vischer, E., Schmidlin, J., and Wettstein, A. (1956). Experientia 12, 50. Weisz, E., Wix, G., and BodBnszky, M. (1956). Naturwissenschaften 43, 39. Welsch, E., and Heusghem, C. (1948). Compt. rend. S O C . biol. 142, 1074. Wettstein, A. (1955). Ezperientia 11, 465. Zaffaroni, A. (1953). Recent Progr. in Hormone Research 8, 51. Zaffaroni, A,, and Burton, R. B. (1951). J. Biol. Chem. 193, 749. Zaffaroni, A., Casas Campillo, C., Cordoba, F., and Rosenkranz, G. (1955). Expem'entiu 11,219.