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Reduction of steroid a ring double bonds by Penicillium decumbens
BIOCHIMICA ET BIOPHYSICA ACTA
354 BBA 55664
REDUCTION
OF STEROID
A RING
DOUBLE
BONDS
BY
PENICILLIZJM
DECUMBENS
T. L. MILLER AND E. J. HESSLER The Ufijohn Company,Kalamazoo, Mich.
(U.S.A.)
(Received September 4th, 1969)
SUMMARY
Penicilli~m decumbens (NRRL-74~) of some steroids.
was found to reduce the 4,s double bond
The 44 bonds of progesterone
and androst-4-ene-3,17-dione
were
efficiently reduced to the 5x-H isomer. The reduction was less efficient with androstar,4-diene-3,r7-dione as substrate; however, with androsta-r,4-diene-3,r7-dione some reduction
of the I,Z double bond also occurred,
and either the 3- or r7-keto
group was
partially reduced forming the alcohol. Either reduced aeration of the addition of an alcohol such as ethanol to the fermentation beer resulted in an increase in the amount of reduced steroid formed.
INTRODUCTION Cleavage of the progesterone been reported’.
Interest
side chain by a number of Penicillium
in this bioconversion
species has
led to studies using Penicillium
decum-
bens (NRRL-742), an organism that reportedly converts progesterone to testosterone (refs. I, 2). However, initial studies indicated that no detectable cleavage of the steroid side chain by this culture had occurred. Instead, preliminary data suggested that progesterone was converted to a steroid product that did not absorb ultraviolet radiation. It appeared likely that the C-4 double bond in the steroid A ring had been reduced perhaps with the concomitant reduction of the 3-keto group. Reduction of steroid A ring double bonds by bacteria3-7, yeasts and Streptomyces9 has been reported. In many instances where the 3-keto group was present in the steroid substrate it was reduced to a hydroxyl group; the hydrogen in the 5 position that resulted Only Mycobacterium from the ,4” reduction was generally of the /!l configuration. smegmatis, of the cultures reported, reduced 3-keto-A4-steroids to their 3-keto 5a-H derivative9. In this bioconversion it was necessary to have a 6-hydroxy group on the substrate in order to block steroid degradation. Reductions of steroid double bonds in the A6, 117 and Ale positions by Nocardialo and by Mycobacteriumll species have also been studied. This report describes for the first time the reduction of steroid A ring double B&him.
Biophys.
Acta,
202 (1970) 354-360
A DOUBLE
STEROID
bonds occurring
BOND
without
355
REDUCTION
reduction
of the 3-keto group by a mold, P. decumbens. The
84 double bond was stereospecifically degradation
apparently
reduced
to the 5~H
derivative,
did not occur. Double bonds in the A1 position
to a lesser degree by P. decumbens.
Conditions
for optimum
reduction
and steroid were reduced were investi-
gated. MATERIALS
AND
METHODS
Fermentatiova Medium. g/l: 10.0 Cerelose (dextrose), 5.0 Torula yeast, 5.0 Kaysoy 200 C (Archer Daniels Midland Co., Minneapolis, Minn. 55440, U.S.A.), 3.0 cornsteep liquor; PH 5.5 Sterilization.
IOO ml of the above
5oo-ml erlenmeyer
flasks closed at the mouth with filter discs.
Inoculum.
medium was sterilized
Shake flasks were inoculated
with 1%
at 121' for 20 min in
vegetative
culture grown on
the above medium. Incubation. Shake flasks were incubated at 28" on either a rotary shaker (e-inch throw, 257 rev./min) or a reciprocal shaker (4-inch throw, g2 cycles/min) Substrates. Progesterone was added to shake flasks either as a dry micronized powder or in ethanol solution; 4-androstene-3,17-dione, testosterone, and androstar,4-diene-3,r7-dione after inoculation. purity
were added dissolved in ethanol. Substrates were added 23-24 h The substrates were all obtained from The Upjohn Co. and the
of each substrate
was in excess of 98%.
Reference compounds. 5u-Androstan-3,17-dione was purchased from Sigma Chemical Co., 3500 DeKalb St., St. Louis, MO. ; 5/I-androstan-3,r7-dione was purchased from Mann Research Laboratories, Inc., 136 Liberty St., New York, N.Y. 10006.The purities
of these
obtained
from The Upjohn
compounds
was g5-98%.
The
Co. steroid collection
Bioconversion assays 5-ml beer samples were withdrawn with an equal volume of methylene
other
reference
compounds
and their purities exceeded
from duplicate
chloride. The extracts
flasks and extracted
were
98%.
twice
were assayed by thin-layer
chromatography and gas-liquid phase chromatography. Thin-layer chromatography: Solvent system, chloroform-acetone-water (95 : 4: I, v/v); detection, ultraviolet absorbance and H,SO, char.; absorbent, silica gel GP,,,. Gas-liquid phase chromatography : An internal standard was used for quantitation of steroid substrates and products; the determination of steroid distribution in a sample was accomplished by integration of the areas under the peaks plotted on a strip-chart recorder. The column used was 3% OV-I on gas chrom Q, 100/120 mesh, 220°, 6 ft x3 mm. The instrument ionization detector.
was a Hewlett
Biochim.
Packard
Biophys.
No. 402 with a flame
Acta,
202
(1970)
354-360
T. L. MILLER, E. J. HESSLER
356 RESULTS AND DISCUSSION
Isolation
and identijcation
of the product
from
the fermentation
of progesterone
with
P. decumbens
From the whole beer of 4 roe-ml shake flasks of P. decumbens containing a total of zoo mg of progesterone, the steroid was extracted 4 times with 200 ml of methylene chloride. This extract gave 229 mg of solid after concentration. Thin-layer chromatography (silica gel GF,,,, 250,~~~cyclohexane-ethylacetate (I: I, v/v) revealed two major components; the more polar one had the same RF (0.47) as progesterone and the less polar spot (RF 0.65) was not visible by ultraviolet light inspection. This mixture was separated by preparative thin-layer chromatography. The ultraviolet light visible material was eluted to give 47 mg (23%) an d i t s infrared spectrum was identical to the infrared spectrum of progesterone. (Infrared spectra were measured on a Perkin Elmer Infracord No. 137 in a 5% chloroform solution.) The ultraviolet light non-visible material was eluted to give 89 mg (45%). The NMR spectrum of the product failed to reveal any olefinic hydrogen signal but did show 3 singlet methyl peaks ascribed to C-18, C-19 and C-21 methyl groups. The implication from the NMR spectrum was that the C-4 carbon-carbon double bond had been reduced. The material was crystallized from ethyl acetate-hexane to give 49 mg of colorless crystals, m.p. 199-201.5”. (All melting points were determined on a Thomas melting point apparatus and are uncorrected.) Recrystallization afforded an analytical sample, calculated for C,,H,,O,: C, 79.70; H, 10.19; found: C, 79.38; H, 9.88. This material was compared to the two possible dihydroprogesterone isomers: (a) 5apregnane-3,2o-dione, m.p. 199-201’; (b) 5/3-pregnane-3,2o-dione, m.p. 121-124~. The infrared spectrum of the product was identical to that of (a). The mixed
o@p. .& z;;bl”s_
Ii
PROGESTERONE
So-PREGNANE-3,20-DIONE (ALLO)
H 5a-ANOROSTANE-3,17-OIONE
ANOROST-4-ENE-3,17-DIONE
ANOROSTA-1.4-OIENE-3,l7-OlONE
H 5o-ANOROSTANE-3,17-OIONE
Fig. I. Summary of conversion reactions by P. decumbens. The substrate was dissolved in ethanol (50 mg/ml) for addition to the fermentation beers. Biochim.
Biophys.
Ada.
202 (1970)
354-360
STEROID
A RINGDOUBLE
BOND
REDUCTION
357
m.p. of the product with (a) was undepressed. The gas chromatography of the product gave a peak identical in retention time to that for (a); the retention time of (b) was different. These data prove that our product is identical to 5a-pregnane-3,ao-dione and therefore the reaction may be written as indicated in Fig. I. Isolation alzd identijcation &one with P. decumbens
of the product
from
the fermentation
of androst-4-ene-3,q
The whole beer of IO roe-ml shake flasks of P. decumbelzs, to which had been added 50 mg of androst-4-ene-3,r7-dione per flask, was extracted 4 times with 500 ml of methylene chloride. This extract gave 570 mg of a glassy solid after concentration. a50 ,LE,cyclohexane~thyl acetate (3 : 2, Thin-layer chromatography (silica gel GF,, v/v)) revealed a major component and a minor component; the major one had the same RF (0.40) as the starting material and the minor component had the same RF (0.51) as 5cr-androstane-3,r7-dione. The product was isolated by chromatography over neutral alumina, Brockman activity II with cyclohexane-ether to give 90 mg (18%) of crude solid. This was crystallized from hexane by cooling to -IO” to give material identical to authentic 5u-androstane3,17_dione by infrared spectral comparison, mixed melting point, and elemental analysis. Calculated for C,,H,,O,; C, 79.12; H, 9.78; found: C, 78.96; H, 9.68.
The whole beer of IO roe-mi shake flasks of P. decumbens, to which had been added 50 mg of androsta-r,4-diene-3,r7-dione per flask, was extracted 4 times with 500 ml of methylene chloride. This extract gave 560 mg of a glassy solid after concentration. Thin-layer chromatography revealed a major and a minor component; the major one had the same RF (0.29) as the starting material and the minor component had the same RF (0.51) as 5c+androstane-3,17-dione. Gas chromatography revealed four peaks: A (IO%), B (14%), C (67%) and D (9%) in order of increasing retention time. By use of the LKB-9000 gas chromatography -mass spectrometer the following data were obtained: Peak A, M+=zgo; Peak B, Mf = 288 and identical to the mass spectrum of 5~-androstane-3,17-dione~ Peak C, M+ = 284 and identical to the mass spectrum of the starting material. The trimethylsilyl ether derivativization of the reaction mixture was carried out and the mass spectra measured. Peak A, (shifted) to new position M+ = 362; Peak R, M+ = 288; Peak C, M+ = 284. Peak B (14% of the mixture) was assigned the structure of 5a-androstane-3,17dione. Its mass spectrum was identical to that of either 5~ or @androstane-3,r7dione, but its gas chromatography retention time was identical to only the 5~ compound. Peak C (679/o o f the mixture) was assigned r,4-androstadiene-3,17-dione, the starting material of the bioconversion, by its mass spectrum and retention time. Peak A (ro"/o of the mixture) was unidenti~ed but contains a hydroxyl group as evidenced by its conversion to a trimethylsilyl derivative. Biochim.
B&plays.
Acta,
202
(1970) 354-360
T. L. MILLER, E. J. HESSLER
358
During early experiments on the bioconversion of progesterone by P. deczdmhens, it was observed by thin-layer chromatography that the only detectable product was slightly less polar than the substrate and was not ultraviolet light absorbing. This product was isolated and identified as ga-pregnane-3,zo-dione as described in the A 0 0 ----
RECIPROCAL SHAKER ROTARY SHAKER + CUP TOP PLUS ROTARY SHAKER 3 SAME AERATION MHUS ETHANOL
CONtVERSION
2 TIME (DAYS)
ETHANOL
3
Fig. 2. Conversion of progesterone to see-pregnane-3,zo-dione by P. decw&ens. The substrate, either dry or in ethanol (so m&ml), was added 23.5 h after inoculation. The substrate level was 0.5 g/I.Other conditions are described in the text.
analytical section. It was found that the yield of this product could be increased by the addition of 0.5-2.0% ethanol to the fermentation medium. The effect of 1% ethanol on the bioconversion of progesterone is shown in Fig. 2. Microscopic examination of the fermentation beer indicated that the conversion was not caused by a contaminant. An experiment was conducted to determine whether alcohols in addition to ethanol would increase the bioconversion of progesterone to get-pregnane-3,2o-dione. The results obtained with fermentation beers containing 1.5% methanol, ethanol, n-propanol, n-butanol or glycerol are shown in Table I. Methanol and glycerol as well as ethanol appeared to stimulate steroid reduction while n-propanol and n-butanol did not. In this experiment the 72-h conversion in the control appeared good, but the steroid recovery was low, only about 40% ; normally bioconversion in such a control was close to 509/o. Bioconversion rates appeared to be faster with ethanol or glycerol than with methanol or in the controls. The mechanism of stimulation of progesterone reduction by methanol, ethanol and glycerol was not determined. Since the conversion is a reductive reaction it would very likely be favored by limited aeration during the bioconversion period. In fact, previously, reported conversions of this type were often carried out by anaerobic cultures6*‘. Therefore, the effect of aeration on bioconversion was studied. In shake flasks the aeration was varied by incubation either on a rotary shaker (higher aeration) or a reciprocal shaker (lower aeration). In addition, aeration was reduced in some cases by securing plastic CUPS containing two 1/32 in. diameter holes in the bottom over the mouth of the flasks. Biochim. Bin,bltys. Acta,
202
(1970) 354-360
A
STEROID
TABLE THE
RING DOUBLE
BOND REDUCTION
359
I
EFFECTS
OF
VARIOUS
ALCOHOLS
0~
THE
REDUCTION
0F
PRocxsTERoNE
BY
P. decumbens
The growth phase was on the rotary shaker at 7.8’ ; 23.5 h after inoculation 0.5 g/l of progesterone dissolved in the appropriate alcohol or dry as a micronized powder was added to each flask and the incubation continued on the reciprocal shaker at the same temperature. 5-ml aliquots were taken daily from each flask; the results are the averages from duplicate flasks. 0/Oof total steroid* 5a-Pregnane-3,2o-dione
Alcohol
at
24 h
48 h
72 h
Methanol
26 37 26
54 81 43
80
Ethanol n-Propanol n-Butanol Glycerol Control (no alcohol)
0
0
22 39
78 43
83 47 tr 85 82**
* Total steroid was considered to be progesterone plus 5a-pregnane-3,zo-dione. * * The total steroid recovery from these samples at 72 h was only about 40% compared with 90IOO~/~ on all of the other samples. Thus, steroid degradation apparently occurred after 48hperhaps as the result of contamination. The usual reduction of progesterone in control flasks was 40-50% in 72 h.
Thus air flow into the flask was restricted.
The effects of reducing
also shown in Fig. 2. The results demonstrate bond is increased by restricted aeration. tion by P. decumbens were incubation
that reduction
the aeration
are
of the progesterone
LIP
Conditions most favorable to steroid reducon the reciprocal shaker with 1% ethanol
present in the fermentation beer. Under these conditions the conversion to 5apregnane-3,ao-dione usually exceeded 80% in 3-4 days and additional products were not detected. The reduction of several steroids lacking the C-17 pregnane side chain was also studied. Initially, testosterone, androst-4-ene-x,17-dione and androsta-r,4-diene-x,17dione were tested for reduction by P. decumbens. It was observed by thin-layer chromatography and gas chromatography that each of these substrates several products, one of which was common to all three substrates. subsequently
identified
(see analytical
either androst-4-ene-x,17-dione bioconversion results obtained TABLE
section)
as gee-androstane-x,17-dione
with
or androsta-1,4-diene-x,17-dione as substrate. The are shown in Table II. Identification of the product
II
CONVERSION
OFANDROST-~-ENE-~,I~-DIONE,ANDROSTA-I,~-DIENE-~,I~-DIONE
TO
DERIVATIVES
REDUCED
was converted to This product was
BY
AND
TESTOSTERONE
P. decunzbens
The conditions were the same as in Table I. The product from testosterone was not identified unequivocally. The results are the average of duplicate flasks; the substrate level was 0.5 g/l and the ethanol level was I yO in the beer. The assays are from 4-day conversion samples. ~~ Substrate Incubation Steroid
Adrost-4-ene-3,r7-dione Androsta-r,4-diene-3,r7-dione Testosterone
Reciprocal shaker Reciprocal shaker Rotary shaker
Residual substrate I”/, of total)
5u-Androstane3,r7-dione f % of total J
16.6 73.1 71.7
83.4 26.8* IO.0
-
* The gas chromatography peak showed a small shoulder that represents a product with a reduced keto function in either the 3 or r7 position. Biochim. Biophys. Acta, 202 (1970) 354-360
360
T. L. MILLER,
E. J. HESSLER
of testosterone reduction was only tentative based on thin-layer chromatography and gas chromatography analyses. With both androsta-r,4-diene-3,r7-dione and testosterone, androst-4-ene-3,x7-dione appeared to be an intermediate in the reduction to ga-androstane-3,r7-dione. It was interesting that in most cases the A.%bond of androst-4-ene-3,17-dione, like that of progesterone, was more than 80% reduced, The 3-keto group of both of these substrates was not reduced. With androsta-r,4diene-3,x7-dione as substrate both the A1 and 44 bonds were reduced; however, the overall conversion was low (cu 27%). It is possible that the same enzyme was responsible for the reduction of d1 and na bonds. With androsta-r,4-diene-3,x7-dione, in contrast to the other steroids examined, some reduction of either the 3- or r7-keto group appeared to occur. With testosterone as substrate the reduced product appeared by thin-layer chromatography and gas chromatography to be either androstane-3-one-x7-01 or androstane-3,x7-dione, depending on the aeration level; higher aeration favored the latter product. The steroid reductions by P. decumbens that have been demonstrated are shown in Fig. I. It is significant that with progesterone or androst+ene-3,r7-dione as substrates the reaction is stereospecific, giving only the SK-hydrogen isomer. Thus, microbial steroid A* bond reduction offers a useful alternative to the chemical reaction that gives mixtures of the gee and Q isomers.
The authors are indebted to Duane James and John Wiersma for technical assistance. REFERENCES I 0. HAX~. AND .A. CAPEK, Arzneimittel-forsch., 7 (1957) 175, 2 A. CAPEK AXD 0. HANC, Folia Microbial. Prague, I (1961) 121. 3 L. MAMOLI AND G. SCHRAMM, Chem. Bev., 71 (1938) 2698.
4 L. MAMOLI, K. KOCH AND H. TESCHEK, J. Physiol. Chem., 261 (‘939) 287. 5 S. H. EPPSTEIN, I?.D. MEISTER, H. C. MURRAY ANU D. H. PETERSEN, Vitamins Horm.onrs, 14 (‘956) 395. 6 K. SCHUBXRT, J. SCHLEGEL, K.-H. BBHNE AND C. H~RHOLD, Biochim. Biophys. Acfa, 144 (1967) 132. 7 K. SCIIUBERT, J. SCHLEGEL AND C..HGRHOLD,J. ~~y~~~Z.Che~~., 332 (1963) 3x0. 8 A. BUTENANDT, Ii.DANNENBERG AK;D L. A. SURANYI, ChemBer., 73(194oj 818. 9 H. R. BARICEMEYER,T.H.STOUDT, J.M.CHE~ERDA, M. A. KOZLA~SKI AN~D W.J. %~ALEER, A#. Microbid., 8 (1960) 237. IO Y. Z. TSONG, I<.C. WANG AND C. J. SIH, Biochim. Biophys. Acta, 93 (1964) 398. II G. AEVIBRUS,I?.TBMGRK~NZ AN\'I) K. G. B~KI, Experientia, 24(1968) 432.
Biochiwz. Biophys. Acta, 202 (1970) 354-.360
354 BBA 55664
REDUCTION
OF STEROID
A RING
DOUBLE
BONDS
BY
PENICILLIZJM
DECUMBENS
T. L. MILLER AND E. J. HESSLER The Ufijohn Company,Kalamazoo, Mich.
(U.S.A.)
(Received September 4th, 1969)
SUMMARY
Penicilli~m decumbens (NRRL-74~) of some steroids.
was found to reduce the 4,s double bond
The 44 bonds of progesterone
and androst-4-ene-3,17-dione
were
efficiently reduced to the 5x-H isomer. The reduction was less efficient with androstar,4-diene-3,r7-dione as substrate; however, with androsta-r,4-diene-3,r7-dione some reduction
of the I,Z double bond also occurred,
and either the 3- or r7-keto
group was
partially reduced forming the alcohol. Either reduced aeration of the addition of an alcohol such as ethanol to the fermentation beer resulted in an increase in the amount of reduced steroid formed.
INTRODUCTION Cleavage of the progesterone been reported’.
Interest
side chain by a number of Penicillium
in this bioconversion
species has
led to studies using Penicillium
decum-
bens (NRRL-742), an organism that reportedly converts progesterone to testosterone (refs. I, 2). However, initial studies indicated that no detectable cleavage of the steroid side chain by this culture had occurred. Instead, preliminary data suggested that progesterone was converted to a steroid product that did not absorb ultraviolet radiation. It appeared likely that the C-4 double bond in the steroid A ring had been reduced perhaps with the concomitant reduction of the 3-keto group. Reduction of steroid A ring double bonds by bacteria3-7, yeasts and Streptomyces9 has been reported. In many instances where the 3-keto group was present in the steroid substrate it was reduced to a hydroxyl group; the hydrogen in the 5 position that resulted Only Mycobacterium from the ,4” reduction was generally of the /!l configuration. smegmatis, of the cultures reported, reduced 3-keto-A4-steroids to their 3-keto 5a-H derivative9. In this bioconversion it was necessary to have a 6-hydroxy group on the substrate in order to block steroid degradation. Reductions of steroid double bonds in the A6, 117 and Ale positions by Nocardialo and by Mycobacteriumll species have also been studied. This report describes for the first time the reduction of steroid A ring double B&him.
Biophys.
Acta,
202 (1970) 354-360
A DOUBLE
STEROID
bonds occurring
BOND
without
355
REDUCTION
reduction
of the 3-keto group by a mold, P. decumbens. The
84 double bond was stereospecifically degradation
apparently
reduced
to the 5~H
derivative,
did not occur. Double bonds in the A1 position
to a lesser degree by P. decumbens.
Conditions
for optimum
reduction
and steroid were reduced were investi-
gated. MATERIALS
AND
METHODS
Fermentatiova Medium. g/l: 10.0 Cerelose (dextrose), 5.0 Torula yeast, 5.0 Kaysoy 200 C (Archer Daniels Midland Co., Minneapolis, Minn. 55440, U.S.A.), 3.0 cornsteep liquor; PH 5.5 Sterilization.
IOO ml of the above
5oo-ml erlenmeyer
flasks closed at the mouth with filter discs.
Inoculum.
medium was sterilized
Shake flasks were inoculated
with 1%
at 121' for 20 min in
vegetative
culture grown on
the above medium. Incubation. Shake flasks were incubated at 28" on either a rotary shaker (e-inch throw, 257 rev./min) or a reciprocal shaker (4-inch throw, g2 cycles/min) Substrates. Progesterone was added to shake flasks either as a dry micronized powder or in ethanol solution; 4-androstene-3,17-dione, testosterone, and androstar,4-diene-3,r7-dione after inoculation. purity
were added dissolved in ethanol. Substrates were added 23-24 h The substrates were all obtained from The Upjohn Co. and the
of each substrate
was in excess of 98%.
Reference compounds. 5u-Androstan-3,17-dione was purchased from Sigma Chemical Co., 3500 DeKalb St., St. Louis, MO. ; 5/I-androstan-3,r7-dione was purchased from Mann Research Laboratories, Inc., 136 Liberty St., New York, N.Y. 10006.The purities
of these
obtained
from The Upjohn
compounds
was g5-98%.
The
Co. steroid collection
Bioconversion assays 5-ml beer samples were withdrawn with an equal volume of methylene
other
reference
compounds
and their purities exceeded
from duplicate
chloride. The extracts
flasks and extracted
were
98%.
twice
were assayed by thin-layer
chromatography and gas-liquid phase chromatography. Thin-layer chromatography: Solvent system, chloroform-acetone-water (95 : 4: I, v/v); detection, ultraviolet absorbance and H,SO, char.; absorbent, silica gel GP,,,. Gas-liquid phase chromatography : An internal standard was used for quantitation of steroid substrates and products; the determination of steroid distribution in a sample was accomplished by integration of the areas under the peaks plotted on a strip-chart recorder. The column used was 3% OV-I on gas chrom Q, 100/120 mesh, 220°, 6 ft x3 mm. The instrument ionization detector.
was a Hewlett
Biochim.
Packard
Biophys.
No. 402 with a flame
Acta,
202
(1970)
354-360
T. L. MILLER, E. J. HESSLER
356 RESULTS AND DISCUSSION
Isolation
and identijcation
of the product
from
the fermentation
of progesterone
with
P. decumbens
From the whole beer of 4 roe-ml shake flasks of P. decumbens containing a total of zoo mg of progesterone, the steroid was extracted 4 times with 200 ml of methylene chloride. This extract gave 229 mg of solid after concentration. Thin-layer chromatography (silica gel GF,,,, 250,~~~cyclohexane-ethylacetate (I: I, v/v) revealed two major components; the more polar one had the same RF (0.47) as progesterone and the less polar spot (RF 0.65) was not visible by ultraviolet light inspection. This mixture was separated by preparative thin-layer chromatography. The ultraviolet light visible material was eluted to give 47 mg (23%) an d i t s infrared spectrum was identical to the infrared spectrum of progesterone. (Infrared spectra were measured on a Perkin Elmer Infracord No. 137 in a 5% chloroform solution.) The ultraviolet light non-visible material was eluted to give 89 mg (45%). The NMR spectrum of the product failed to reveal any olefinic hydrogen signal but did show 3 singlet methyl peaks ascribed to C-18, C-19 and C-21 methyl groups. The implication from the NMR spectrum was that the C-4 carbon-carbon double bond had been reduced. The material was crystallized from ethyl acetate-hexane to give 49 mg of colorless crystals, m.p. 199-201.5”. (All melting points were determined on a Thomas melting point apparatus and are uncorrected.) Recrystallization afforded an analytical sample, calculated for C,,H,,O,: C, 79.70; H, 10.19; found: C, 79.38; H, 9.88. This material was compared to the two possible dihydroprogesterone isomers: (a) 5apregnane-3,2o-dione, m.p. 199-201’; (b) 5/3-pregnane-3,2o-dione, m.p. 121-124~. The infrared spectrum of the product was identical to that of (a). The mixed
o@p. .& z;;bl”s_
Ii
PROGESTERONE
So-PREGNANE-3,20-DIONE (ALLO)
H 5a-ANOROSTANE-3,17-OIONE
ANOROST-4-ENE-3,17-DIONE
ANOROSTA-1.4-OIENE-3,l7-OlONE
H 5o-ANOROSTANE-3,17-OIONE
Fig. I. Summary of conversion reactions by P. decumbens. The substrate was dissolved in ethanol (50 mg/ml) for addition to the fermentation beers. Biochim.
Biophys.
Ada.
202 (1970)
354-360
STEROID
A RINGDOUBLE
BOND
REDUCTION
357
m.p. of the product with (a) was undepressed. The gas chromatography of the product gave a peak identical in retention time to that for (a); the retention time of (b) was different. These data prove that our product is identical to 5a-pregnane-3,ao-dione and therefore the reaction may be written as indicated in Fig. I. Isolation alzd identijcation &one with P. decumbens
of the product
from
the fermentation
of androst-4-ene-3,q
The whole beer of IO roe-ml shake flasks of P. decumbelzs, to which had been added 50 mg of androst-4-ene-3,r7-dione per flask, was extracted 4 times with 500 ml of methylene chloride. This extract gave 570 mg of a glassy solid after concentration. a50 ,LE,cyclohexane~thyl acetate (3 : 2, Thin-layer chromatography (silica gel GF,, v/v)) revealed a major component and a minor component; the major one had the same RF (0.40) as the starting material and the minor component had the same RF (0.51) as 5cr-androstane-3,r7-dione. The product was isolated by chromatography over neutral alumina, Brockman activity II with cyclohexane-ether to give 90 mg (18%) of crude solid. This was crystallized from hexane by cooling to -IO” to give material identical to authentic 5u-androstane3,17_dione by infrared spectral comparison, mixed melting point, and elemental analysis. Calculated for C,,H,,O,; C, 79.12; H, 9.78; found: C, 78.96; H, 9.68.
The whole beer of IO roe-mi shake flasks of P. decumbens, to which had been added 50 mg of androsta-r,4-diene-3,r7-dione per flask, was extracted 4 times with 500 ml of methylene chloride. This extract gave 560 mg of a glassy solid after concentration. Thin-layer chromatography revealed a major and a minor component; the major one had the same RF (0.29) as the starting material and the minor component had the same RF (0.51) as 5c+androstane-3,17-dione. Gas chromatography revealed four peaks: A (IO%), B (14%), C (67%) and D (9%) in order of increasing retention time. By use of the LKB-9000 gas chromatography -mass spectrometer the following data were obtained: Peak A, M+=zgo; Peak B, Mf = 288 and identical to the mass spectrum of 5~-androstane-3,17-dione~ Peak C, M+ = 284 and identical to the mass spectrum of the starting material. The trimethylsilyl ether derivativization of the reaction mixture was carried out and the mass spectra measured. Peak A, (shifted) to new position M+ = 362; Peak R, M+ = 288; Peak C, M+ = 284. Peak B (14% of the mixture) was assigned the structure of 5a-androstane-3,17dione. Its mass spectrum was identical to that of either 5~ or @androstane-3,r7dione, but its gas chromatography retention time was identical to only the 5~ compound. Peak C (679/o o f the mixture) was assigned r,4-androstadiene-3,17-dione, the starting material of the bioconversion, by its mass spectrum and retention time. Peak A (ro"/o of the mixture) was unidenti~ed but contains a hydroxyl group as evidenced by its conversion to a trimethylsilyl derivative. Biochim.
B&plays.
Acta,
202
(1970) 354-360
T. L. MILLER, E. J. HESSLER
358
During early experiments on the bioconversion of progesterone by P. deczdmhens, it was observed by thin-layer chromatography that the only detectable product was slightly less polar than the substrate and was not ultraviolet light absorbing. This product was isolated and identified as ga-pregnane-3,zo-dione as described in the A 0 0 ----
RECIPROCAL SHAKER ROTARY SHAKER + CUP TOP PLUS ROTARY SHAKER 3 SAME AERATION MHUS ETHANOL
CONtVERSION
2 TIME (DAYS)
ETHANOL
3
Fig. 2. Conversion of progesterone to see-pregnane-3,zo-dione by P. decw&ens. The substrate, either dry or in ethanol (so m&ml), was added 23.5 h after inoculation. The substrate level was 0.5 g/I.Other conditions are described in the text.
analytical section. It was found that the yield of this product could be increased by the addition of 0.5-2.0% ethanol to the fermentation medium. The effect of 1% ethanol on the bioconversion of progesterone is shown in Fig. 2. Microscopic examination of the fermentation beer indicated that the conversion was not caused by a contaminant. An experiment was conducted to determine whether alcohols in addition to ethanol would increase the bioconversion of progesterone to get-pregnane-3,2o-dione. The results obtained with fermentation beers containing 1.5% methanol, ethanol, n-propanol, n-butanol or glycerol are shown in Table I. Methanol and glycerol as well as ethanol appeared to stimulate steroid reduction while n-propanol and n-butanol did not. In this experiment the 72-h conversion in the control appeared good, but the steroid recovery was low, only about 40% ; normally bioconversion in such a control was close to 509/o. Bioconversion rates appeared to be faster with ethanol or glycerol than with methanol or in the controls. The mechanism of stimulation of progesterone reduction by methanol, ethanol and glycerol was not determined. Since the conversion is a reductive reaction it would very likely be favored by limited aeration during the bioconversion period. In fact, previously, reported conversions of this type were often carried out by anaerobic cultures6*‘. Therefore, the effect of aeration on bioconversion was studied. In shake flasks the aeration was varied by incubation either on a rotary shaker (higher aeration) or a reciprocal shaker (lower aeration). In addition, aeration was reduced in some cases by securing plastic CUPS containing two 1/32 in. diameter holes in the bottom over the mouth of the flasks. Biochim. Bin,bltys. Acta,
202
(1970) 354-360
A
STEROID
TABLE THE
RING DOUBLE
BOND REDUCTION
359
I
EFFECTS
OF
VARIOUS
ALCOHOLS
0~
THE
REDUCTION
0F
PRocxsTERoNE
BY
P. decumbens
The growth phase was on the rotary shaker at 7.8’ ; 23.5 h after inoculation 0.5 g/l of progesterone dissolved in the appropriate alcohol or dry as a micronized powder was added to each flask and the incubation continued on the reciprocal shaker at the same temperature. 5-ml aliquots were taken daily from each flask; the results are the averages from duplicate flasks. 0/Oof total steroid* 5a-Pregnane-3,2o-dione
Alcohol
at
24 h
48 h
72 h
Methanol
26 37 26
54 81 43
80
Ethanol n-Propanol n-Butanol Glycerol Control (no alcohol)
0
0
22 39
78 43
83 47 tr 85 82**
* Total steroid was considered to be progesterone plus 5a-pregnane-3,zo-dione. * * The total steroid recovery from these samples at 72 h was only about 40% compared with 90IOO~/~ on all of the other samples. Thus, steroid degradation apparently occurred after 48hperhaps as the result of contamination. The usual reduction of progesterone in control flasks was 40-50% in 72 h.
Thus air flow into the flask was restricted.
The effects of reducing
also shown in Fig. 2. The results demonstrate bond is increased by restricted aeration. tion by P. decumbens were incubation
that reduction
the aeration
are
of the progesterone
LIP
Conditions most favorable to steroid reducon the reciprocal shaker with 1% ethanol
present in the fermentation beer. Under these conditions the conversion to 5apregnane-3,ao-dione usually exceeded 80% in 3-4 days and additional products were not detected. The reduction of several steroids lacking the C-17 pregnane side chain was also studied. Initially, testosterone, androst-4-ene-x,17-dione and androsta-r,4-diene-x,17dione were tested for reduction by P. decumbens. It was observed by thin-layer chromatography and gas chromatography that each of these substrates several products, one of which was common to all three substrates. subsequently
identified
(see analytical
either androst-4-ene-x,17-dione bioconversion results obtained TABLE
section)
as gee-androstane-x,17-dione
with
or androsta-1,4-diene-x,17-dione as substrate. The are shown in Table II. Identification of the product
II
CONVERSION
OFANDROST-~-ENE-~,I~-DIONE,ANDROSTA-I,~-DIENE-~,I~-DIONE
TO
DERIVATIVES
REDUCED
was converted to This product was
BY
AND
TESTOSTERONE
P. decunzbens
The conditions were the same as in Table I. The product from testosterone was not identified unequivocally. The results are the average of duplicate flasks; the substrate level was 0.5 g/l and the ethanol level was I yO in the beer. The assays are from 4-day conversion samples. ~~ Substrate Incubation Steroid
Adrost-4-ene-3,r7-dione Androsta-r,4-diene-3,r7-dione Testosterone
Reciprocal shaker Reciprocal shaker Rotary shaker
Residual substrate I”/, of total)
5u-Androstane3,r7-dione f % of total J
16.6 73.1 71.7
83.4 26.8* IO.0
-
* The gas chromatography peak showed a small shoulder that represents a product with a reduced keto function in either the 3 or r7 position. Biochim. Biophys. Acta, 202 (1970) 354-360
360
T. L. MILLER,
E. J. HESSLER
of testosterone reduction was only tentative based on thin-layer chromatography and gas chromatography analyses. With both androsta-r,4-diene-3,r7-dione and testosterone, androst-4-ene-3,x7-dione appeared to be an intermediate in the reduction to ga-androstane-3,r7-dione. It was interesting that in most cases the A.%bond of androst-4-ene-3,17-dione, like that of progesterone, was more than 80% reduced, The 3-keto group of both of these substrates was not reduced. With androsta-r,4diene-3,x7-dione as substrate both the A1 and 44 bonds were reduced; however, the overall conversion was low (cu 27%). It is possible that the same enzyme was responsible for the reduction of d1 and na bonds. With androsta-r,4-diene-3,x7-dione, in contrast to the other steroids examined, some reduction of either the 3- or r7-keto group appeared to occur. With testosterone as substrate the reduced product appeared by thin-layer chromatography and gas chromatography to be either androstane-3-one-x7-01 or androstane-3,x7-dione, depending on the aeration level; higher aeration favored the latter product. The steroid reductions by P. decumbens that have been demonstrated are shown in Fig. I. It is significant that with progesterone or androst+ene-3,r7-dione as substrates the reaction is stereospecific, giving only the SK-hydrogen isomer. Thus, microbial steroid A* bond reduction offers a useful alternative to the chemical reaction that gives mixtures of the gee and Q isomers.
The authors are indebted to Duane James and John Wiersma for technical assistance. REFERENCES I 0. HAX~. AND .A. CAPEK, Arzneimittel-forsch., 7 (1957) 175, 2 A. CAPEK AXD 0. HANC, Folia Microbial. Prague, I (1961) 121. 3 L. MAMOLI AND G. SCHRAMM, Chem. Bev., 71 (1938) 2698.
4 L. MAMOLI, K. KOCH AND H. TESCHEK, J. Physiol. Chem., 261 (‘939) 287. 5 S. H. EPPSTEIN, I?.D. MEISTER, H. C. MURRAY ANU D. H. PETERSEN, Vitamins Horm.onrs, 14 (‘956) 395. 6 K. SCHUBXRT, J. SCHLEGEL, K.-H. BBHNE AND C. H~RHOLD, Biochim. Biophys. Acfa, 144 (1967) 132. 7 K. SCIIUBERT, J. SCHLEGEL AND C..HGRHOLD,J. ~~y~~~Z.Che~~., 332 (1963) 3x0. 8 A. BUTENANDT, Ii.DANNENBERG AK;D L. A. SURANYI, ChemBer., 73(194oj 818. 9 H. R. BARICEMEYER,T.H.STOUDT, J.M.CHE~ERDA, M. A. KOZLA~SKI AN~D W.J. %~ALEER, A#. Microbid., 8 (1960) 237. IO Y. Z. TSONG, I<.C. WANG AND C. J. SIH, Biochim. Biophys. Acta, 93 (1964) 398. II G. AEVIBRUS,I?.TBMGRK~NZ AN\'I) K. G. B~KI, Experientia, 24(1968) 432.
Biochiwz. Biophys. Acta, 202 (1970) 354-.360