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Steroid epoxide cleavage by Cylindrocarpon radicicola
402
BIOCHIMICA ET BIOPI-[YSICA ACTA
BBA 2 5 1 4 i
STEROID
CLEAVAGE
EPOXIDE
BY C YLINDROCA RPON (). E L - T A Y E B , S. (;. K N I G H T
RA D I C I C O L A *
AND C H A R L E S
J. S i l t ' *
Departmenl of Bacteriology a~,d School of Pharmacy, Umversil), of liqsconsin, Madison, Wise. (U.S.A.) ( R e c e i v e d March 17th, I964)
SUMMAIC.Y
When I6a,I7a-oxido-pregn-4-ene-3,2o-dione (I) was incubated with Cylindrocarpon radicicola four products were obtained. These have been characterized as 2oa-hydroxyI6~,I7~-oxido-pregn-4-ene-3-one (II), 2o~-hydroxy-I6a,I7~-oxido-pregna-I,4-diene3-one (III), I6~-hydroxy-I7a-oxa-androsta-I,4-diene-3,I7-dione (IV) and i6a,i7a-dihydroxy-androsta-I,4-diene-3-one (V). When iI was exposed to this organism it was readily transformed into I I I which in turn was converted into IV and V. V was resistant to further a t t a c k by this organism. Ibe{-hydroxy-pregn-4-ene-3,eo-dione (XVI) was converted into V by C. radicicola but no IV was detected. The relative rates of metabolism of I and lI were found to be similar whereas I 6 a - h y d r o x y - a n d r o s t - 4ene-3,I7-dione (XV) was metabolized at a much slower rate into IV and V. A possible mechanism of epoxide opening by C. radicicola has been proposed.
I NTROI)UCTION
Although microbial epooxidations of steroids have been well documented 1, only two examples of microbial opening of steroid epoxides have been recorded. CAMr:mNO et al. 2 noted the conversi(m of x6~,IT~-oxido-pregn-4-ene-3,xT-dione into I6c~,2o~-dihydroxy-I7fl-methyl-xS-nor-I7~-pregna-4,I3-die.ne-3-one by yeast via a retropinacoline type rearrangement and Ih~OCHAZKA et al. 3 reported the conversion of 3flhydroxy-5,b-oxido-B-nor-androstan-IT-one into 3fl,5~,6fl-trihydroxy-B-nor-andro stan-x7-one in low yields by Rhizopus nigricans. C3,lindrocarpon radicicola is an organism capable of oxidizing pregn-4-ene-3,zodionc into x7a-oxa-androsta-I,4-diene-3,I7-dione via the following sequence of reactions : pregn-4-ene-3,2o-dione - ---, pregna-I,4-diene-3,eo-dione --> x7fl-hydroxy-androsta-i,4-diene-3-one .- ~ androsta-i,4-diene-3,i7-dione - ~ i7a-oxa-androsta-x,4diene- 3, IT-dione 4. In an a t t e m p t to gain an insight as to the mechanism of side-chain degradation, i6c~,I7a-oxidopregn-4-cne- 3, IT-dione wa.~ incubated with C. radicicola and it was found that its epoxide had becn cleaved. In view of the uniqueness of opoxide cleavage bv microorganisms in general, we wish to record our observations which are " I ' r c s e n t e d a t the 03rd m e e t i n g of the A m e r i c a n Soc i e t y for Microbiology, C l e ve l a nd, Ohio, May 5 0 , ]9()Y * * .Xll r e q u e s t s for r e p r i n t s should be a d d r e s s e d to t h e l a s t a u t h o r .
Biochim. t3iophys..4eta, 93 (1904) 4 o z - 4 n o
STEROID EPOXIDE CLEAVAGEBY C. radiciccda
403
significant in establishing the opening of the oxide of I6a,I7~-oxidopregn-4-ene3,2o-dione by C. radicicola. EXPERIMENTAL
Materials and methods Stock cultures of C. radicicola (ATCC I I O I I ) were maintained on nutrient agar slants supplemented with 1% glucose and 1%, yeast extract. The fermentation medium used throughout this work has been described previously 5. Small-scale fermentations were carried out in 25o-ml erlenmeyer flasks containing 5° ml of medium; large-scale fermentations were carried out in 2-1 erlenmeyer flasks containing 400 ml of medium. The flasks were incubated at 27 ° on a rotary shaker. I6~,I7~-Oxido-pregn-4-ene-3,2odione was a product of Nutritional Biochemicals, Cleveland, Ohio. I6~-Hydroxyandrost-4-ene-3,IT-dione was prepared by incubation of androst-4-ene-3,I7-dione with Streptomyces roseochromogenes (ATCC 1340o ). All melting points were uncorrected and were determined on a T h o m a s - Hoover capillary melting point apparatus. Ultraviolet absorption spectra were determined in 95 % ethanol on a Cary recording spectrophotometer (Model I i MS). Infrared spectra were recorded on a Beckman IR 5 A double beam infrared recording spectrophotometer. Microanalyses were carried out by Mr. J. ALIClXO of Metuchen, N.J. The procedure for quantitative estimation of steroids by paper chromatography has been previously described ~.
Fermentation of I6o~,z7o~-oxido-pregn-4-ene-3,2o-dione (I) C. radicicola was grown in 9.6 1 of the fermentation medium (24 2-1 erlenmeyer flasks). After 24 h of incubation, 4.8 g of I, dissolved in 38 ml of dimethylformamide was added equally to the flasks. After 17 h, the culture broth was filtered and the filtrate was extracted three times with 3-1 portions of chloroform. An aliquot of the chloroform extract was spotted on Whatman No. i paper and developed in a toluene.propylcne glycol system n for 3 h. Four products were observed as viewed under an ultraviolet scanneff ; the RF values were 0.47, 0.34, o.15 and o.oo.The combined chh)roform extract was dried over NaoSO 4 and taken down to dryness to give 5.12 g of residue. 2.56 g of this residue was chromatographed over a cellulose-powder column ~ (3.4 cm .. 54 cm), using propylene glycol as the stationary phase. The mobile phase eonsistt.d of toluene--cvclohexane (75: 25, v/v), saturated with propylene glycoh IO ml fractions were collected. After Fraction I2o, the mobile phase was changed to toluene, saturated with propylene glycol. After analysis of the fractions by paper chromatography, homogeneous fractions were pooled, evaporated down to dryness and taken tip in chloroform. The chloroform solution was washed three times with water, dried over Na~S04 and again taken down to dryness. Fractions 62-74 gave 1.21 g of solids. Recrvstallization from acetone afforded 0.65 g of the starting material, 1, m.p. 206-208 ° , identical in all respects to an authentic sample. Fractions 127-131 afforded 132 nag of residue. Two reerystallizations from acetone yielded lO6 mg of I I, m.p. 246-247 ~' '~cdJ~ 6 ! - I O 2 ° in chloroform (c, 0.5) ; light-absorption max. in ethanol 24o m/~ (e 1570o) and in chloroform 2.92, 6.06 and 6.20/~. Calc. for C2~H3oO~: C, 76.32; H, 9.15. Found: C, 76.15; H, 9.13°.3 . Fractions 139-148 yielded 320 mg of solids. Two recrystallizations from acetone, followed by one recrystallization from acetone-light petroleum (b.p. 60-.80 °) afforded Biochim. Bioph.vs..4cla, 93 (I{}64) 4°2 4 to
404
O. EL-TAYEI:~, S. G. KNIGHT,
C. J. SIH
205 mg of I I I , m.p. 2 2 7 - 2 2 8 " [~7~)6 -,-420 in chloroform (c, I.O)' light-absorption max. in ethanol 244 m/x (,: 153o0 ), and in chloroform 2.92, 6.05, 6.20 and 6.28/x. Calc. for C2xlI.,~O3: C, 7~).79; H, 8.59. F o u n d : C, 76.86 • H, 8.6!)"o. The dr\" weight obtained from Fractions I6O-I8~ was 670 nag. Two recrvstallizations from a c e t o n e - l i g h t petroleum (h.p. (;0--80") gave 48o nag of IX,.',m.p. 206-208°; [ c ~ ---(~6° in chloroform (c, I.O)" light-absorption max. in ethanol 240 mr* (~: I~)7oo), and in nujol 2.94, 5.60, 6.o6, 6.2o and 6.25/~. Calc. for C19H24() ~" (', 72.17; H, 7.65, F o u n d : C, 71.96; I-I, 7.72 %. After 2oo fractions, the mobile phase was changed to claloroform-methanol (I "I, v/v). When Fractions I99-2IO were pooled, I24 mg of residue was obtained. After two recrvstallizations from acetone, followed bv one recrystallization in a c e t o n e - l i g h t r -.2s 4- 2 = in chloropetroleum (b.p. 60-80':) 88 mg of V was obtained, m.p. 2oo--2o1"; '_~i~ form (c, i.o); light-ahsorption m a x i m u m in ethanol 241 m/x (e I57OO), and ill nujol 2.92, 6.06, 6.20 and 6.26 ~. Cale. for C~9H260 ~" C, 75.46; H, 8.67. F o u n d : C, 75.31;
H, 8.61%. 2oe¢-Hydroxv-r6~,z7~-oxido-pregn-4-ene-3-one pregn-4-ene-3-one (XI)
(11) and 2ofL~,droxy-z6~,z7,-oxido-
173 mg of NaBH4 in 5 ml of absolute methanol was added dropwise with stirring to a solution containing 500 nag of I, in 5 ml of absolute methanol. The mixture was stirred overnight at room temperature. 20 ml of water was then added, followed hv a few drops of o.I N HC1. The mixture was extracted with chloroform; the combined chloroform extract was washed successively with 5 % NaHCO:3 and water, dried over Na2S() 4 and the volume of chloroform was brought down to IOO ml. 5 g of Mn() 2 (ref. 9) was added to the chloroform solution and the mixture was stirred for 24 h. Upon filtration and evaporation of the solvent, 44 ° mg of a white powder was obtained. Repeated fractional crystallizations from hot acetone afforded two fractions: 267 m g o f X I , m.p. 199-2o2 ° and 75 mg of II, m.p. 242-246° . Repeated crystallizations from acetone afforded 46 mg of X I , m.p. 2o2-2o3 °" .~aD :26 ,_ 94 ° in chloroform (c, o.5); light-absorption max. in ethanol 240 m/~ (e 19000 ), and in chloroform 2.92 , 6.06 and 6.20 /x. Calc. for C2~Ha0()a: C, 76.32; H, 9.15. F o u n d : (', 75.94; H, 9.15 %. Three recrystallizations of II from acetone gave pure II, m.p. 246-247 ', identical in all respects (mixed melting point, infrared spectrum and mixed paper chromatography) to I I ohtained by fermentation.
Chromic acid oxidation of 2oa-hydroxy-I6a,z7a-oxido-pregn-4-ene-3-one (II) and 2o~hydro.~v-i6~,I7c~-oxido-pregn-4-ene- 3-one (X I) To 46 mg of X I in 1 ml of pyridine was added 46 mg of Cr203 in I ml of pyridinO °, and the mixture was left standing overnight at room temperature for 16 h. 20 ml of water was then added and the mixture was extracted with three 2o-ml portions of ether. The ethereal laver was washed three times with water, dried over Na2SO , and taken down to dryness to give 45 mg of a white powder. Two recrystallizations from acetone gave 37 mg of I, m.p. 2o~-2o8L identical in all respects (mixed melting point and infrared spectrum) to an authentic sample of I. The procedure was repeated with II, l was obtained in approximately the same yield. Biochim. t¢mph)'s...1 cla, 93 (t 904) 4o2 -4 m
STEROID EPOXIDE CLEAVAGE
2oc~-Acetoxy- I6cc,r7~-oxido-pregn-4-ene-3-one
BY C. radicicola
405
(X I I)
IOO mg of II was dissolved in 3 ml of an acetic anhydride-pyridine mixture ( I : I , v/v) and was left standing at room temperature for 16 h. After removing the solvents the residue was crystallized from acetone. Two recrvstallizations from acetone gave 67 mg of X I I , m.p. 228-229 °. r~-25. :r~ ~-63 ° in chloroform (c, I.O); light-absorption max. in ethanol 240 m/z (e I78OO), and in chloroform 5.72, 6.02, 6.2o and 8.04 V~Calc. for C~3H~2Oa: C, 74.16; H, 8.66. Found: C, 73.99; H, 8.41 o.o.
2o~-A ceto.Lv-z6~,17~-oxido-pregn- 4-ene-3-one (X I I I) 60 mg of XI was dissolved in 2 ml of an acetic anhydride-pyridine mixture (1 : i, v/v) and it was left standing at room temperature for 16 h. After removing the solv,?nts, the residue was crystallized from acetone to yield 56 mg of crystals, m.p. 18o -192:'. Repeated crystallization from hot acetone gave pure X I I I , m.p. 193 -194 ° ; Zc~22s :- 1560 in chloroform (c, 0.8); light-absorption max. in ethanol 240 roll (,: I64OO), and in chloroform 5.74, 6.02, 6.20 and 8.06 ~. Reported ~t m.p. I9O-I93°; !c~o + 165 °.
2oe-Acetoxy-x6~,r7cc-oxido-pregna-z,4-diene-3-one (XIV) 45 mg of I I I was dissolved in 1.5 ml of an acetic anhydride-pyridine (I : I, v/v) and was left standing at room temperature for 16 h. After removing the solvents the residue was crystallized from acetone to yield 38 mg of crystals, m.p. 222-225 °. Recrystallization from acetone gave a sample, m.p. 226-227 °; ~ c ~ - - 5 ° in chloroform (c, 1.21) ; light-absorption max. 241 rap, (e I46OO), and in chloroform 5.78, 6.02, 6.20, 6.24 and 8.05/z. Calc. for C2sHs00,: C, 74.56; H, 8.16. Found: C, 74.17; H, 8.41%.
Fermentation of z6~-hydroxv-androst-4-ene-3,r7-dione (XV) C. radicicola was grown in 400 ml of the fermentation medium (one 2-1 erlenmeyer flask). After 24 tx of incubation 200 mg of XV, dissolved in 16 ml of dimethylformamide was added and the fermentation was continued for 72 h. After removing the mveelia by filtration, the filtrate was extracted with three 2oo-ml portions of chloroform. The combined chloroform extract was dried over Na2SO 4 and evaporated down to dryness to give 18o nag of a crude powder. The residue was chromatographed over 15 g of silicic acid containing IO % celite. The mobile phase consisted of 2 % me.thanol in chloroform and io-ml fractions were collected. Fractions 73-89 were pooled and afforded 65 mg of residue after evaporation of the solvents. Two crystallizations from acetone-light petroleum (b.p. 60-80 °) gave 53 mg of IV, m.p. 206-208 °, identical to an authentic sample of I6~-hydroxy-I7a-oxa-androsta-I,4-diene-3,I7-dione. Fractions 189-215 afforded 45 mg of residue after remoxdng the solvent. Two crystallizations from acetone gave 32 mg of V, m.p. 199.5--2oo.5 °.
Fermentation of I6c~-hydroxy-pr egn-4-ene-3,2o-dione (X V I) C. radicicola was grown in 1.2 1 of the fermentation medium (three 2-1 erlenmever flasks). After 24 h of growth, 500 mg of XVI in 4.8 ml of dimethylformamide was added and the fermentation was continued for 72 h. After filtration, the filtrate was extracted with three 4oo-ml portions of chloroform, dried over Na,,SO 4 and taken down to dryness to give 385 mg of residue. This residue was chromatographed over 15 g of silicic acid with IO % Celite. The mobile phase consisted of 2 o~ /o methanol in chloroform and Io-ml fractions were collected.
Biochim. Biophys. Acta, 93 (I9~')4) 4o'-4 lo
406
O. E L - T A Y E B ,
S. (;. K N I G H T ,
C. J . S I H
Fractions 54---69 afforded 36 nag of crude material. Recrystallizations from acetone-light petroleum (b.p. 6o--80 °) gave 12 mg of a compound, m.p. 162-163°; lightabsorption max. in chloroform 2.92, 5.72, 6.Ol, 6.I 5 and 6.22/,. Calc. for C2~H2804: C, 73.22; H, 8.19 . Found: C, 73.66; H, 8.47%. Fractions 79-86 gave I I nag of crude material. Many attempts to crystallize this fiaction have not met with success. Fractions 123-152 yielded 285 mg of crystals. Two recrystallizations from acetone afforded 242 nag of V, m.p. 2oo-2ol ~, identical in all respects (mixed melting point and infrared spectruna) to an authentic sample of V.
RESULTS
AND
DISCUSSION
When I was incubated with C. radicicola four products were obtained. The first product II was initially recognized as a 2o-hydroxy derivative of I by virtue of the absence of a band around 5.85 t* in its infrared spectrum and C and H analysis was in good agreement with C2~Ha00 a. The following series of reactions was used to establish its structure: When I was reduced with NaBH 4 and oxidized with MnO.,, a mixture of two compounds was obtained, one of which, m.p. 246--247 °, was identical with I I, obtained from fermentation; the other (XI) melted at 2o2-2o3 °. However, both II and X1 afforded I after chromic acid oxidation, indicating that they are 2o-dihvdro epimers of I. To establish the configuration of the 2o-hydroxyl group in II and XI, the monoacetates of these epimers were prepared.
2 o - h y d r o x v (I1, X l ) _,o-acetates (XlI, XIII)
.MI~
(Chloroform)
Jr- 3 3 7 " + 235 ~
+ 3 tl~ --. 5 8 i 2
From tile molecular rotation (MD) of the monoacetates, II was assigned tile structure 2ox-hydroxy-x6~,I7~-oxido-pregn-4-ene-3-one and XI as 2o/3-hydroxyi6c
407
STEROID EPOXIDE CLEAVAGE BY C. radicicola
structure V is further confirmed by the fact that both XV and XVI were rapidly converted into V after incubation with C. radicicola. These results suggest that I is metabolized by this organism into IV and V with the possible participation of the 2o=-hydroxyl derivatives (II and III) as intermediates. When II was incubated with this organism, I I I was formed; I I I was in turn transformed into IV and V. The latter compounds IV and V were resistant to further attack by this organism, even after prolonged period of incubation.
•
Ioc
IOO
8(2
x
80
60
::L 4o
40
20
"~..~.
o
20
0 0
4
8
12
16
20
4
Z4
8
Time in Hours
x
16
20
24
Time in Hours
Fig. 2. The rate of metabolism of -o~x-hydroxy16~, 17~-oxido-pregn- 4-ene-3-one (I I). The procedure of assay is the same as those in Fig. x. I I, 2ox-hydroxy- i6(x, 17~-oxido-pregn-4-ene-3one; l I I , 2o~-hydroxy-l(~X, I7~-oxitlo-pregna1,4-diene-3-one; IV, i 6 a - h y d r o x y - I 7a-oxa-an drosta-l,4-diene-3,17-dione ; V, i (xz, z 7~-dihyd r o x y a n d r o s t a - 1,4-diene-3-one.
F i g z. The kinetics of 16~,17x-oxido-pregn-4ene-3,2o-dione oxidation. At the indicated time intervals, an aliquot of the f e r m e n t a t i o n b r o t h was removed and analyzed by the q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h i c m e t h o d described u n d e r METHODS. I, 16~, I 7c(-oxido-pregn-4-ene-3,zodione; if, 2o~-hydroxy-16~,x7~(-oxido-pregn-4ene-3-one; llI, 2o~-hydroxy- I ~x, [ 7x-oxidopregna- 1,4-cliene-3-one ; IV, 16a-hydroxy- x7aoxa-androsta-l,4-diene-3,17-dione; V, 16~,17~d i h y d r o x y - a n d r o s t a - 1,4-diene-3-one.
oJ
12
ot \
xv
6O
20
,
0 0
16
32
;~ 48
A 64
80
96
Time m Hours
1;ig. 3. The rate of metabolism of i6~-hydroxyandrost-4-ene-3, x7-dione. The conditions and proced u r e for q u a n t i t a t i o n are the same as those in Fig. I. IV, I 6 ~ - h y d r o x y - I T a - o x a - a n d r o s t a - I , 4 diene-3, [ 7-dione ; V, 162, [ 7~-dihydroxy-androsta- 1,4-diene-3-one.
Biochim. Biophys. Acta, 93 (I964) 4 o 2 - 4 I o
4o8
o. EL-TAYEB, S. G. KNIGHT, (. J. SIH
To gain further information as to the reaction sequence of these compounds, the relative rates of m e t a b o l i s m of I, I I a n d XV were c o m p a r e d (Figs. I -3). The results show t h a t I a n d I I were m e t a b o l i z e d at a b o u t the same rate a n d the ratio of the final p r o d u c t s ( I V : V - 3.4) was also similar. On the o t h e r hand, XV was m e t a b o l i z e d slower and gave a different ratio of final p r o d u c t s ( I V : V = 1.7). F r o m the foregoing d a t a , the following m e c h a n i s m , t h o u g h speculative albeit p e r t i n e n t , could t e n t a t i v e l y 1)e p o s t u l a t e d for the f o r m a t i o n of IV a n d V (Scheme I).
o
~
~
H3
CH 3
I
c=o
HC-OH ... o
o
o
1 :
II
CH3
CH 3
I
I HC-OH
C=O . .- ,:
o
CH 3
VI
I
I
C=O
I
&.o
o ~ I11
'?
vii 1
o/H
O
OH
.OH
0 /
~
~
VIII
~Q"
o
O
Ix
V
L OH O
IV
O x y g e n a t i o n a n d d e h y d r o g e n a t i o n of I would give I7#-acetoxy-I6~,i7cc-oxidoa n d r o s t a - I , 4 - d i e n e - 3 - o n e (VII) which after h y d r o l y s i s b y an esterase would afford I 7 # - h y d r o x y - I 6 c c , I 7 ~ - o x i d o - a n d r o s t a - I , 4 - d i e n e - 3 - o n e ( V I I I ) ; the l a t t e r p r o d u c t could s p o n t a n e o u s l y rearrange 13 to yield IX. R e d u c t i o n of I X via pyridine-nucleotide linked steroid h y d r o x y d e h y d r o g e n a s e would give V whereas o x y g e n a t i o n of I X via a Baeyer--Villiger t y p e would afford IV. 14iochim. Biophys. Acta, 93 (1904) 4°, 4 lo
STEROID EPOXIDE CLEAVAGE BY C. radicicola
409
The proposed mechanism as outlined in Scheme I has ample precedence. The conversion of pregn-4-ene-3,2o-dione into I7/~-hydroxyandrost-4-ene-3-one and acetic acid via the intermediate, I7~-acetoxyandrost-4-ene-3-one has been conclusively established in Cladosporium resinae la. The enzymes responsible for the conversion of I7j~-hydroxyandrost-4-ene-3-one into androst-4-ene-3,I7-dione and further into I7aoxa-androst-4-ene-3,i7-dione had been thoroughly studied in Penicillium lilacinum ~5. The fact that V is resistant to further oxidation is also consistent with the observation of TALALAY .aNn MARCL'S~6 who reported that/~-hydroxy steroid dehydrogenase from Pseudomonas testosteroni was unable to oxidize I7~-hydroxyl steroids bearing oxygen functions at C-I6. However, the reverse reaction obviously can take place since XV was readily reduced to IV b y the organism. t3ecause XV was metabolized slower than either I or II and neither I X or XV was detected in the fermentation broth, their role as key precursors in the formation of IV and V is somewhat attenuated. However, differences in permeability and enzyme inducibility could account for this anomaly. The microbiological reduction of the 2o-carbonyl function to the corresponding 20~-hydroxyl derivative is well documented whereas reduction to the 20~-hydroxyl derivative is less common 17. The role of the 20-dihydro compounds as possible intermediates in side-chain degradation was first raised by SEBEK el al. TM, who reported that P. lilacinum reduced pregn-4-ene-3,2o-dione into its 2off-hydroxyl derivative in the first few hours of fermentation. Similarly, II~-hydroxy-pregn-4-ene-3,20-dione was transformed into its corresponding 20fl-dihydro derivative in the early stages of fermentation. However, no conclusive evidence was available in defining their role in side chain degradation. From our results, the role of the 2o~-hydroxy derivatives is also not clear. However, since II is metabolized at a slightly slower rate than I, it is unlikely that II is an intermediate in the formation of IV and V from I. Since pregn-4-ene-3,2o-dione is metabolized at a faster rate than I with no accumulation of any 2o-hydroxy compound ~9, it appears that the C-2o reductase is probably competing with side-chain degrading enzymes for I. Thus, substrates which are metabolized rapidly by side-chain degrading enzymes would accumulate little if any 2o-hvdroxv coml)ounds. On the other hand, substrates which are slowly metabolized by side-chain degrading enzymes would accumulate 2o-dihydro derivatives. Conelusive answer to this question awaits the study using cell-free systems. ACKNOWI.ED(;EMEN'FS
The authors wish to thank Dr. G. S. FOXKEX of The Upjohn Co. for a sample of I6~-hydroxy-pregn-4-ene-3,2o-dione and Dr. J. FRIED of E.R. Squibb & Sons for a sample of I6~-hydroxy-I7a-oxa-androsta-I,4-diene-3,I7-dione. This work was supported in part by research grants (A-4687) and (AI-oI 2Ol) of The National Institutes of Health. R E F E R Ir.NCES 1 p. '2 B. 3 Z. 4 G. 5 C.
TALALAY,Physiol. l?ev., 37 (x957) 362. CAMERINO, R. MODELLI AND C. SPALLA, Go,z,?'. Chim. ltal., 86 (i956) I219. PROCHAZKA, J. FAJKOS, J. JOSKA AND F. -~OR.M, Coll. Czech. Chem. Commun., 26 (I00i) 2068. E. PETERSEN, R. X,V.THOMA, D. PERLMAN AND J. FRIED, J. Bacteriol., 74 (I957) 684. J. SIH, ,q..~. KUPCHAN, N. I(ATSUI AND (). EL-TAYI.:B, J. Org. Chem., 28 (I9()3) 854.
Biochim. Biophys. Acta, 93 (x964) 4oa-41o
410
O. E L - T A Y E B ,
S. G. KNIGHT,
C. J. .¢,IH
6 A. ZAFFARONI. R. ]~. ]3t:RTON AND E. H. KEUT.',IANN, Science, I11 (I95o) 0. "/ \ ¥ . J. HAINES ANI) N. A. DRAKE, Federation Proc., 9 (195 o) ISO. s C. J. SIH AND l{. E. BENNETT, Biochim. Biophys. ,4cta, 38 ( i 9 6 o ) 378. 9 0 . MANCERA, G. I(OSENKRANZ AND F. SONSHEIMER, f . Chem. Soc., (I953) 2189. 10 G. 1. P o o s , G. E. ARTH, R. E. BEYLER ANt) L. H. SARETT, f . . 4 m . Chem. Soc., 75 (I953) 422. 11 B. CAMERINO AND C. (3. ALI3ERTI, Gaze. Chim. Ital., 85 (I955) 5 I. 12 p. 1,. JULIAN, E. \V. ~ E Y E R , XV. J. [{ARPEL AND ~,¥. COLE, J. Am. Chem. Soc., 73 (1951) 1982. 13 N. S. I,EEOS, D. I'~. FUKUSHIMA AND T. F. GALLAGHER, J. ,4m. Chem. Soc., 76 (1954) 2943. 14 G. S. FONKV:N, H. C. MtJRRAY AND L. M. REINECKE, J. Am. Chem. Sot., 82 (196o) 55o7 . 15 I{. L. PRAIRIE AND l ). TALALAY, Biochemistry, 2 ( i 9 6 3 ) 2o3. le l~. TALALAY AND I >. 1. 3~ARCUS, ./. Biol. Chem., 218 (i95(~) 675. 17 F. CORVAJAL, (). F. X/ITALE, M. S. GENTLES, H. L. HERZOG AND E. I3. HERSHBERG, J. Org. Chem., 24 (1959) 695. 18 O. K. SEBEK, L. M. REINECKE AND I). H. PETERSON, J. Bacteriol., 83 (1962) 1327. 19 O . EL-TAYEB, S. (~. KNIGHT AND C. J. SIH, u n p u b l i s h e d r e s u l t s .
Biochim. Biophys..4cla, 93 ( I 9 0 4 ) 4 ° z - 4 Io
BIOCHIMICA ET BIOPI-[YSICA ACTA
BBA 2 5 1 4 i
STEROID
CLEAVAGE
EPOXIDE
BY C YLINDROCA RPON (). E L - T A Y E B , S. (;. K N I G H T
RA D I C I C O L A *
AND C H A R L E S
J. S i l t ' *
Departmenl of Bacteriology a~,d School of Pharmacy, Umversil), of liqsconsin, Madison, Wise. (U.S.A.) ( R e c e i v e d March 17th, I964)
SUMMAIC.Y
When I6a,I7a-oxido-pregn-4-ene-3,2o-dione (I) was incubated with Cylindrocarpon radicicola four products were obtained. These have been characterized as 2oa-hydroxyI6~,I7~-oxido-pregn-4-ene-3-one (II), 2o~-hydroxy-I6a,I7~-oxido-pregna-I,4-diene3-one (III), I6~-hydroxy-I7a-oxa-androsta-I,4-diene-3,I7-dione (IV) and i6a,i7a-dihydroxy-androsta-I,4-diene-3-one (V). When iI was exposed to this organism it was readily transformed into I I I which in turn was converted into IV and V. V was resistant to further a t t a c k by this organism. Ibe{-hydroxy-pregn-4-ene-3,eo-dione (XVI) was converted into V by C. radicicola but no IV was detected. The relative rates of metabolism of I and lI were found to be similar whereas I 6 a - h y d r o x y - a n d r o s t - 4ene-3,I7-dione (XV) was metabolized at a much slower rate into IV and V. A possible mechanism of epoxide opening by C. radicicola has been proposed.
I NTROI)UCTION
Although microbial epooxidations of steroids have been well documented 1, only two examples of microbial opening of steroid epoxides have been recorded. CAMr:mNO et al. 2 noted the conversi(m of x6~,IT~-oxido-pregn-4-ene-3,xT-dione into I6c~,2o~-dihydroxy-I7fl-methyl-xS-nor-I7~-pregna-4,I3-die.ne-3-one by yeast via a retropinacoline type rearrangement and Ih~OCHAZKA et al. 3 reported the conversion of 3flhydroxy-5,b-oxido-B-nor-androstan-IT-one into 3fl,5~,6fl-trihydroxy-B-nor-andro stan-x7-one in low yields by Rhizopus nigricans. C3,lindrocarpon radicicola is an organism capable of oxidizing pregn-4-ene-3,zodionc into x7a-oxa-androsta-I,4-diene-3,I7-dione via the following sequence of reactions : pregn-4-ene-3,2o-dione - ---, pregna-I,4-diene-3,eo-dione --> x7fl-hydroxy-androsta-i,4-diene-3-one .- ~ androsta-i,4-diene-3,i7-dione - ~ i7a-oxa-androsta-x,4diene- 3, IT-dione 4. In an a t t e m p t to gain an insight as to the mechanism of side-chain degradation, i6c~,I7a-oxidopregn-4-cne- 3, IT-dione wa.~ incubated with C. radicicola and it was found that its epoxide had becn cleaved. In view of the uniqueness of opoxide cleavage bv microorganisms in general, we wish to record our observations which are " I ' r c s e n t e d a t the 03rd m e e t i n g of the A m e r i c a n Soc i e t y for Microbiology, C l e ve l a nd, Ohio, May 5 0 , ]9()Y * * .Xll r e q u e s t s for r e p r i n t s should be a d d r e s s e d to t h e l a s t a u t h o r .
Biochim. t3iophys..4eta, 93 (1904) 4 o z - 4 n o
STEROID EPOXIDE CLEAVAGEBY C. radiciccda
403
significant in establishing the opening of the oxide of I6a,I7~-oxidopregn-4-ene3,2o-dione by C. radicicola. EXPERIMENTAL
Materials and methods Stock cultures of C. radicicola (ATCC I I O I I ) were maintained on nutrient agar slants supplemented with 1% glucose and 1%, yeast extract. The fermentation medium used throughout this work has been described previously 5. Small-scale fermentations were carried out in 25o-ml erlenmeyer flasks containing 5° ml of medium; large-scale fermentations were carried out in 2-1 erlenmeyer flasks containing 400 ml of medium. The flasks were incubated at 27 ° on a rotary shaker. I6~,I7~-Oxido-pregn-4-ene-3,2odione was a product of Nutritional Biochemicals, Cleveland, Ohio. I6~-Hydroxyandrost-4-ene-3,IT-dione was prepared by incubation of androst-4-ene-3,I7-dione with Streptomyces roseochromogenes (ATCC 1340o ). All melting points were uncorrected and were determined on a T h o m a s - Hoover capillary melting point apparatus. Ultraviolet absorption spectra were determined in 95 % ethanol on a Cary recording spectrophotometer (Model I i MS). Infrared spectra were recorded on a Beckman IR 5 A double beam infrared recording spectrophotometer. Microanalyses were carried out by Mr. J. ALIClXO of Metuchen, N.J. The procedure for quantitative estimation of steroids by paper chromatography has been previously described ~.
Fermentation of I6o~,z7o~-oxido-pregn-4-ene-3,2o-dione (I) C. radicicola was grown in 9.6 1 of the fermentation medium (24 2-1 erlenmeyer flasks). After 24 h of incubation, 4.8 g of I, dissolved in 38 ml of dimethylformamide was added equally to the flasks. After 17 h, the culture broth was filtered and the filtrate was extracted three times with 3-1 portions of chloroform. An aliquot of the chloroform extract was spotted on Whatman No. i paper and developed in a toluene.propylcne glycol system n for 3 h. Four products were observed as viewed under an ultraviolet scanneff ; the RF values were 0.47, 0.34, o.15 and o.oo.The combined chh)roform extract was dried over NaoSO 4 and taken down to dryness to give 5.12 g of residue. 2.56 g of this residue was chromatographed over a cellulose-powder column ~ (3.4 cm .. 54 cm), using propylene glycol as the stationary phase. The mobile phase eonsistt.d of toluene--cvclohexane (75: 25, v/v), saturated with propylene glycoh IO ml fractions were collected. After Fraction I2o, the mobile phase was changed to toluene, saturated with propylene glycol. After analysis of the fractions by paper chromatography, homogeneous fractions were pooled, evaporated down to dryness and taken tip in chloroform. The chloroform solution was washed three times with water, dried over Na~S04 and again taken down to dryness. Fractions 62-74 gave 1.21 g of solids. Recrvstallization from acetone afforded 0.65 g of the starting material, 1, m.p. 206-208 ° , identical in all respects to an authentic sample. Fractions 127-131 afforded 132 nag of residue. Two reerystallizations from acetone yielded lO6 mg of I I, m.p. 246-247 ~' '~cdJ~ 6 ! - I O 2 ° in chloroform (c, 0.5) ; light-absorption max. in ethanol 24o m/~ (e 1570o) and in chloroform 2.92, 6.06 and 6.20/~. Calc. for C2~H3oO~: C, 76.32; H, 9.15. Found: C, 76.15; H, 9.13°.3 . Fractions 139-148 yielded 320 mg of solids. Two recrystallizations from acetone, followed by one recrystallization from acetone-light petroleum (b.p. 60-.80 °) afforded Biochim. Bioph.vs..4cla, 93 (I{}64) 4°2 4 to
404
O. EL-TAYEI:~, S. G. KNIGHT,
C. J. SIH
205 mg of I I I , m.p. 2 2 7 - 2 2 8 " [~7~)6 -,-420 in chloroform (c, I.O)' light-absorption max. in ethanol 244 m/x (,: 153o0 ), and in chloroform 2.92, 6.05, 6.20 and 6.28/x. Calc. for C2xlI.,~O3: C, 7~).79; H, 8.59. F o u n d : C, 76.86 • H, 8.6!)"o. The dr\" weight obtained from Fractions I6O-I8~ was 670 nag. Two recrvstallizations from a c e t o n e - l i g h t petroleum (h.p. (;0--80") gave 48o nag of IX,.',m.p. 206-208°; [ c ~ ---(~6° in chloroform (c, I.O)" light-absorption max. in ethanol 240 mr* (~: I~)7oo), and in nujol 2.94, 5.60, 6.o6, 6.2o and 6.25/~. Calc. for C19H24() ~" (', 72.17; H, 7.65, F o u n d : C, 71.96; I-I, 7.72 %. After 2oo fractions, the mobile phase was changed to claloroform-methanol (I "I, v/v). When Fractions I99-2IO were pooled, I24 mg of residue was obtained. After two recrvstallizations from acetone, followed bv one recrystallization in a c e t o n e - l i g h t r -.2s 4- 2 = in chloropetroleum (b.p. 60-80':) 88 mg of V was obtained, m.p. 2oo--2o1"; '_~i~ form (c, i.o); light-ahsorption m a x i m u m in ethanol 241 m/x (e I57OO), and ill nujol 2.92, 6.06, 6.20 and 6.26 ~. Cale. for C~9H260 ~" C, 75.46; H, 8.67. F o u n d : C, 75.31;
H, 8.61%. 2oe¢-Hydroxv-r6~,z7~-oxido-pregn-4-ene-3-one pregn-4-ene-3-one (XI)
(11) and 2ofL~,droxy-z6~,z7,-oxido-
173 mg of NaBH4 in 5 ml of absolute methanol was added dropwise with stirring to a solution containing 500 nag of I, in 5 ml of absolute methanol. The mixture was stirred overnight at room temperature. 20 ml of water was then added, followed hv a few drops of o.I N HC1. The mixture was extracted with chloroform; the combined chloroform extract was washed successively with 5 % NaHCO:3 and water, dried over Na2S() 4 and the volume of chloroform was brought down to IOO ml. 5 g of Mn() 2 (ref. 9) was added to the chloroform solution and the mixture was stirred for 24 h. Upon filtration and evaporation of the solvent, 44 ° mg of a white powder was obtained. Repeated fractional crystallizations from hot acetone afforded two fractions: 267 m g o f X I , m.p. 199-2o2 ° and 75 mg of II, m.p. 242-246° . Repeated crystallizations from acetone afforded 46 mg of X I , m.p. 2o2-2o3 °" .~aD :26 ,_ 94 ° in chloroform (c, o.5); light-absorption max. in ethanol 240 m/~ (e 19000 ), and in chloroform 2.92 , 6.06 and 6.20 /x. Calc. for C2~Ha0()a: C, 76.32; H, 9.15. F o u n d : (', 75.94; H, 9.15 %. Three recrystallizations of II from acetone gave pure II, m.p. 246-247 ', identical in all respects (mixed melting point, infrared spectrum and mixed paper chromatography) to I I ohtained by fermentation.
Chromic acid oxidation of 2oa-hydroxy-I6a,z7a-oxido-pregn-4-ene-3-one (II) and 2o~hydro.~v-i6~,I7c~-oxido-pregn-4-ene- 3-one (X I) To 46 mg of X I in 1 ml of pyridine was added 46 mg of Cr203 in I ml of pyridinO °, and the mixture was left standing overnight at room temperature for 16 h. 20 ml of water was then added and the mixture was extracted with three 2o-ml portions of ether. The ethereal laver was washed three times with water, dried over Na2SO , and taken down to dryness to give 45 mg of a white powder. Two recrystallizations from acetone gave 37 mg of I, m.p. 2o~-2o8L identical in all respects (mixed melting point and infrared spectrum) to an authentic sample of I. The procedure was repeated with II, l was obtained in approximately the same yield. Biochim. t¢mph)'s...1 cla, 93 (t 904) 4o2 -4 m
STEROID EPOXIDE CLEAVAGE
2oc~-Acetoxy- I6cc,r7~-oxido-pregn-4-ene-3-one
BY C. radicicola
405
(X I I)
IOO mg of II was dissolved in 3 ml of an acetic anhydride-pyridine mixture ( I : I , v/v) and was left standing at room temperature for 16 h. After removing the solvents the residue was crystallized from acetone. Two recrvstallizations from acetone gave 67 mg of X I I , m.p. 228-229 °. r~-25. :r~ ~-63 ° in chloroform (c, I.O); light-absorption max. in ethanol 240 m/z (e I78OO), and in chloroform 5.72, 6.02, 6.2o and 8.04 V~Calc. for C~3H~2Oa: C, 74.16; H, 8.66. Found: C, 73.99; H, 8.41 o.o.
2o~-A ceto.Lv-z6~,17~-oxido-pregn- 4-ene-3-one (X I I I) 60 mg of XI was dissolved in 2 ml of an acetic anhydride-pyridine mixture (1 : i, v/v) and it was left standing at room temperature for 16 h. After removing the solv,?nts, the residue was crystallized from acetone to yield 56 mg of crystals, m.p. 18o -192:'. Repeated crystallization from hot acetone gave pure X I I I , m.p. 193 -194 ° ; Zc~22s :- 1560 in chloroform (c, 0.8); light-absorption max. in ethanol 240 roll (,: I64OO), and in chloroform 5.74, 6.02, 6.20 and 8.06 ~. Reported ~t m.p. I9O-I93°; !c~o + 165 °.
2oe-Acetoxy-x6~,r7cc-oxido-pregna-z,4-diene-3-one (XIV) 45 mg of I I I was dissolved in 1.5 ml of an acetic anhydride-pyridine (I : I, v/v) and was left standing at room temperature for 16 h. After removing the solvents the residue was crystallized from acetone to yield 38 mg of crystals, m.p. 222-225 °. Recrystallization from acetone gave a sample, m.p. 226-227 °; ~ c ~ - - 5 ° in chloroform (c, 1.21) ; light-absorption max. 241 rap, (e I46OO), and in chloroform 5.78, 6.02, 6.20, 6.24 and 8.05/z. Calc. for C2sHs00,: C, 74.56; H, 8.16. Found: C, 74.17; H, 8.41%.
Fermentation of z6~-hydroxv-androst-4-ene-3,r7-dione (XV) C. radicicola was grown in 400 ml of the fermentation medium (one 2-1 erlenmeyer flask). After 24 tx of incubation 200 mg of XV, dissolved in 16 ml of dimethylformamide was added and the fermentation was continued for 72 h. After removing the mveelia by filtration, the filtrate was extracted with three 2oo-ml portions of chloroform. The combined chloroform extract was dried over Na2SO 4 and evaporated down to dryness to give 18o nag of a crude powder. The residue was chromatographed over 15 g of silicic acid containing IO % celite. The mobile phase consisted of 2 % me.thanol in chloroform and io-ml fractions were collected. Fractions 73-89 were pooled and afforded 65 mg of residue after evaporation of the solvents. Two crystallizations from acetone-light petroleum (b.p. 60-80 °) gave 53 mg of IV, m.p. 206-208 °, identical to an authentic sample of I6~-hydroxy-I7a-oxa-androsta-I,4-diene-3,I7-dione. Fractions 189-215 afforded 45 mg of residue after remoxdng the solvent. Two crystallizations from acetone gave 32 mg of V, m.p. 199.5--2oo.5 °.
Fermentation of I6c~-hydroxy-pr egn-4-ene-3,2o-dione (X V I) C. radicicola was grown in 1.2 1 of the fermentation medium (three 2-1 erlenmever flasks). After 24 h of growth, 500 mg of XVI in 4.8 ml of dimethylformamide was added and the fermentation was continued for 72 h. After filtration, the filtrate was extracted with three 4oo-ml portions of chloroform, dried over Na,,SO 4 and taken down to dryness to give 385 mg of residue. This residue was chromatographed over 15 g of silicic acid with IO % Celite. The mobile phase consisted of 2 o~ /o methanol in chloroform and Io-ml fractions were collected.
Biochim. Biophys. Acta, 93 (I9~')4) 4o'-4 lo
406
O. E L - T A Y E B ,
S. (;. K N I G H T ,
C. J . S I H
Fractions 54---69 afforded 36 nag of crude material. Recrystallizations from acetone-light petroleum (b.p. 6o--80 °) gave 12 mg of a compound, m.p. 162-163°; lightabsorption max. in chloroform 2.92, 5.72, 6.Ol, 6.I 5 and 6.22/,. Calc. for C2~H2804: C, 73.22; H, 8.19 . Found: C, 73.66; H, 8.47%. Fractions 79-86 gave I I nag of crude material. Many attempts to crystallize this fiaction have not met with success. Fractions 123-152 yielded 285 mg of crystals. Two recrystallizations from acetone afforded 242 nag of V, m.p. 2oo-2ol ~, identical in all respects (mixed melting point and infrared spectruna) to an authentic sample of V.
RESULTS
AND
DISCUSSION
When I was incubated with C. radicicola four products were obtained. The first product II was initially recognized as a 2o-hydroxy derivative of I by virtue of the absence of a band around 5.85 t* in its infrared spectrum and C and H analysis was in good agreement with C2~Ha00 a. The following series of reactions was used to establish its structure: When I was reduced with NaBH 4 and oxidized with MnO.,, a mixture of two compounds was obtained, one of which, m.p. 246--247 °, was identical with I I, obtained from fermentation; the other (XI) melted at 2o2-2o3 °. However, both II and X1 afforded I after chromic acid oxidation, indicating that they are 2o-dihvdro epimers of I. To establish the configuration of the 2o-hydroxyl group in II and XI, the monoacetates of these epimers were prepared.
2 o - h y d r o x v (I1, X l ) _,o-acetates (XlI, XIII)
.MI~
(Chloroform)
Jr- 3 3 7 " + 235 ~
+ 3 tl~ --. 5 8 i 2
From tile molecular rotation (MD) of the monoacetates, II was assigned tile structure 2ox-hydroxy-x6~,I7~-oxido-pregn-4-ene-3-one and XI as 2o/3-hydroxyi6c
407
STEROID EPOXIDE CLEAVAGE BY C. radicicola
structure V is further confirmed by the fact that both XV and XVI were rapidly converted into V after incubation with C. radicicola. These results suggest that I is metabolized by this organism into IV and V with the possible participation of the 2o=-hydroxyl derivatives (II and III) as intermediates. When II was incubated with this organism, I I I was formed; I I I was in turn transformed into IV and V. The latter compounds IV and V were resistant to further attack by this organism, even after prolonged period of incubation.
•
Ioc
IOO
8(2
x
80
60
::L 4o
40
20
"~..~.
o
20
0 0
4
8
12
16
20
4
Z4
8
Time in Hours
x
16
20
24
Time in Hours
Fig. 2. The rate of metabolism of -o~x-hydroxy16~, 17~-oxido-pregn- 4-ene-3-one (I I). The procedure of assay is the same as those in Fig. x. I I, 2ox-hydroxy- i6(x, 17~-oxido-pregn-4-ene-3one; l I I , 2o~-hydroxy-l(~X, I7~-oxitlo-pregna1,4-diene-3-one; IV, i 6 a - h y d r o x y - I 7a-oxa-an drosta-l,4-diene-3,17-dione ; V, i (xz, z 7~-dihyd r o x y a n d r o s t a - 1,4-diene-3-one.
F i g z. The kinetics of 16~,17x-oxido-pregn-4ene-3,2o-dione oxidation. At the indicated time intervals, an aliquot of the f e r m e n t a t i o n b r o t h was removed and analyzed by the q u a n t i t a t i v e p a p e r c h r o m a t o g r a p h i c m e t h o d described u n d e r METHODS. I, 16~, I 7c(-oxido-pregn-4-ene-3,zodione; if, 2o~-hydroxy-16~,x7~(-oxido-pregn-4ene-3-one; llI, 2o~-hydroxy- I ~x, [ 7x-oxidopregna- 1,4-cliene-3-one ; IV, 16a-hydroxy- x7aoxa-androsta-l,4-diene-3,17-dione; V, 16~,17~d i h y d r o x y - a n d r o s t a - 1,4-diene-3-one.
oJ
12
ot \
xv
6O
20
,
0 0
16
32
;~ 48
A 64
80
96
Time m Hours
1;ig. 3. The rate of metabolism of i6~-hydroxyandrost-4-ene-3, x7-dione. The conditions and proced u r e for q u a n t i t a t i o n are the same as those in Fig. I. IV, I 6 ~ - h y d r o x y - I T a - o x a - a n d r o s t a - I , 4 diene-3, [ 7-dione ; V, 162, [ 7~-dihydroxy-androsta- 1,4-diene-3-one.
Biochim. Biophys. Acta, 93 (I964) 4 o 2 - 4 I o
4o8
o. EL-TAYEB, S. G. KNIGHT, (. J. SIH
To gain further information as to the reaction sequence of these compounds, the relative rates of m e t a b o l i s m of I, I I a n d XV were c o m p a r e d (Figs. I -3). The results show t h a t I a n d I I were m e t a b o l i z e d at a b o u t the same rate a n d the ratio of the final p r o d u c t s ( I V : V - 3.4) was also similar. On the o t h e r hand, XV was m e t a b o l i z e d slower and gave a different ratio of final p r o d u c t s ( I V : V = 1.7). F r o m the foregoing d a t a , the following m e c h a n i s m , t h o u g h speculative albeit p e r t i n e n t , could t e n t a t i v e l y 1)e p o s t u l a t e d for the f o r m a t i o n of IV a n d V (Scheme I).
o
~
~
H3
CH 3
I
c=o
HC-OH ... o
o
o
1 :
II
CH3
CH 3
I
I HC-OH
C=O . .- ,:
o
CH 3
VI
I
I
C=O
I
&.o
o ~ I11
'?
vii 1
o/H
O
OH
.OH
0 /
~
~
VIII
~Q"
o
O
Ix
V
L OH O
IV
O x y g e n a t i o n a n d d e h y d r o g e n a t i o n of I would give I7#-acetoxy-I6~,i7cc-oxidoa n d r o s t a - I , 4 - d i e n e - 3 - o n e (VII) which after h y d r o l y s i s b y an esterase would afford I 7 # - h y d r o x y - I 6 c c , I 7 ~ - o x i d o - a n d r o s t a - I , 4 - d i e n e - 3 - o n e ( V I I I ) ; the l a t t e r p r o d u c t could s p o n t a n e o u s l y rearrange 13 to yield IX. R e d u c t i o n of I X via pyridine-nucleotide linked steroid h y d r o x y d e h y d r o g e n a s e would give V whereas o x y g e n a t i o n of I X via a Baeyer--Villiger t y p e would afford IV. 14iochim. Biophys. Acta, 93 (1904) 4°, 4 lo
STEROID EPOXIDE CLEAVAGE BY C. radicicola
409
The proposed mechanism as outlined in Scheme I has ample precedence. The conversion of pregn-4-ene-3,2o-dione into I7/~-hydroxyandrost-4-ene-3-one and acetic acid via the intermediate, I7~-acetoxyandrost-4-ene-3-one has been conclusively established in Cladosporium resinae la. The enzymes responsible for the conversion of I7j~-hydroxyandrost-4-ene-3-one into androst-4-ene-3,I7-dione and further into I7aoxa-androst-4-ene-3,i7-dione had been thoroughly studied in Penicillium lilacinum ~5. The fact that V is resistant to further oxidation is also consistent with the observation of TALALAY .aNn MARCL'S~6 who reported that/~-hydroxy steroid dehydrogenase from Pseudomonas testosteroni was unable to oxidize I7~-hydroxyl steroids bearing oxygen functions at C-I6. However, the reverse reaction obviously can take place since XV was readily reduced to IV b y the organism. t3ecause XV was metabolized slower than either I or II and neither I X or XV was detected in the fermentation broth, their role as key precursors in the formation of IV and V is somewhat attenuated. However, differences in permeability and enzyme inducibility could account for this anomaly. The microbiological reduction of the 2o-carbonyl function to the corresponding 20~-hydroxyl derivative is well documented whereas reduction to the 20~-hydroxyl derivative is less common 17. The role of the 20-dihydro compounds as possible intermediates in side-chain degradation was first raised by SEBEK el al. TM, who reported that P. lilacinum reduced pregn-4-ene-3,2o-dione into its 2off-hydroxyl derivative in the first few hours of fermentation. Similarly, II~-hydroxy-pregn-4-ene-3,20-dione was transformed into its corresponding 20fl-dihydro derivative in the early stages of fermentation. However, no conclusive evidence was available in defining their role in side chain degradation. From our results, the role of the 2o~-hydroxy derivatives is also not clear. However, since II is metabolized at a slightly slower rate than I, it is unlikely that II is an intermediate in the formation of IV and V from I. Since pregn-4-ene-3,2o-dione is metabolized at a faster rate than I with no accumulation of any 2o-hydroxy compound ~9, it appears that the C-2o reductase is probably competing with side-chain degrading enzymes for I. Thus, substrates which are metabolized rapidly by side-chain degrading enzymes would accumulate little if any 2o-hvdroxv coml)ounds. On the other hand, substrates which are slowly metabolized by side-chain degrading enzymes would accumulate 2o-dihydro derivatives. Conelusive answer to this question awaits the study using cell-free systems. ACKNOWI.ED(;EMEN'FS
The authors wish to thank Dr. G. S. FOXKEX of The Upjohn Co. for a sample of I6~-hydroxy-pregn-4-ene-3,2o-dione and Dr. J. FRIED of E.R. Squibb & Sons for a sample of I6~-hydroxy-I7a-oxa-androsta-I,4-diene-3,I7-dione. This work was supported in part by research grants (A-4687) and (AI-oI 2Ol) of The National Institutes of Health. R E F E R Ir.NCES 1 p. '2 B. 3 Z. 4 G. 5 C.
TALALAY,Physiol. l?ev., 37 (x957) 362. CAMERINO, R. MODELLI AND C. SPALLA, Go,z,?'. Chim. ltal., 86 (i956) I219. PROCHAZKA, J. FAJKOS, J. JOSKA AND F. -~OR.M, Coll. Czech. Chem. Commun., 26 (I00i) 2068. E. PETERSEN, R. X,V.THOMA, D. PERLMAN AND J. FRIED, J. Bacteriol., 74 (I957) 684. J. SIH, ,q..~. KUPCHAN, N. I(ATSUI AND (). EL-TAYI.:B, J. Org. Chem., 28 (I9()3) 854.
Biochim. Biophys. Acta, 93 (x964) 4oa-41o
410
O. E L - T A Y E B ,
S. G. KNIGHT,
C. J. .¢,IH
6 A. ZAFFARONI. R. ]~. ]3t:RTON AND E. H. KEUT.',IANN, Science, I11 (I95o) 0. "/ \ ¥ . J. HAINES ANI) N. A. DRAKE, Federation Proc., 9 (195 o) ISO. s C. J. SIH AND l{. E. BENNETT, Biochim. Biophys. ,4cta, 38 ( i 9 6 o ) 378. 9 0 . MANCERA, G. I(OSENKRANZ AND F. SONSHEIMER, f . Chem. Soc., (I953) 2189. 10 G. 1. P o o s , G. E. ARTH, R. E. BEYLER ANt) L. H. SARETT, f . . 4 m . Chem. Soc., 75 (I953) 422. 11 B. CAMERINO AND C. (3. ALI3ERTI, Gaze. Chim. Ital., 85 (I955) 5 I. 12 p. 1,. JULIAN, E. \V. ~ E Y E R , XV. J. [{ARPEL AND ~,¥. COLE, J. Am. Chem. Soc., 73 (1951) 1982. 13 N. S. I,EEOS, D. I'~. FUKUSHIMA AND T. F. GALLAGHER, J. ,4m. Chem. Soc., 76 (1954) 2943. 14 G. S. FONKV:N, H. C. MtJRRAY AND L. M. REINECKE, J. Am. Chem. Sot., 82 (196o) 55o7 . 15 I{. L. PRAIRIE AND l ). TALALAY, Biochemistry, 2 ( i 9 6 3 ) 2o3. le l~. TALALAY AND I >. 1. 3~ARCUS, ./. Biol. Chem., 218 (i95(~) 675. 17 F. CORVAJAL, (). F. X/ITALE, M. S. GENTLES, H. L. HERZOG AND E. I3. HERSHBERG, J. Org. Chem., 24 (1959) 695. 18 O. K. SEBEK, L. M. REINECKE AND I). H. PETERSON, J. Bacteriol., 83 (1962) 1327. 19 O . EL-TAYEB, S. (~. KNIGHT AND C. J. SIH, u n p u b l i s h e d r e s u l t s .
Biochim. Biophys..4cla, 93 ( I 9 0 4 ) 4 ° z - 4 Io