Substrate specificity in steroid side-chain degradation by microorganisms

BIOCHIMICA ET BIOPHYSICA ACTA

4II

BBA 25 142

SUBSTRATE SPECIFICITY IN STEROID SIDE-CHAIN DEGRADATION BY MICROORGANISMS O. E L - T A Y E B , S. O. K N I G H T AND C H A R L E S J. S I H "

Department of Flacteviology and School of Pharmacy, Umversily of l|'isco~sin, 3Iadison, IVisc. (U.S.A.) (Received March 24th, ~964)

SUMMARY

Two side-chain cleavage organisms, Penicillium citrinum and Cylindrocarpon radicicola appear to have significant differences in substrate specificity. P. citrinum was incapable of cleaving the side chain of I7e-hydroxy-pregn-4-ene-3,2o-dione (I). Instead, I was transformed into three hydroxyl derivatives; two of which have been characterized as I2fl,I7o~-dihydroxy-pregn-4-ene-3,2o-dione and i5fl,I7e-dihydroxypregn-4-ene-3,2o-dione. On the other hand, C. radicicola cleaved the side chain of pregna-4,I6-diene-3,eo-dione and I with the eventual formation of I7a-oxa-androsta 1,4-diene-3,I7-dione as the end product. I7a-Methyl-pregn-4-ene-3,2o-dione and I7flacetyl-amino-androst-4-ene-3-one were only converted to their I-dehydro analogs.

INTRODUCTION

The microbiological degradation of steroid side chain is of interest because it represents a step in the total oxidation of the steroid molecule x. VISCHER AND WETTSTEIN* first reported that a Fusarium species was capable of cleaving the methyl ketone or the hydroxymethyl ketone but not the dihydroxymethyl ketone side chain of several 5a and 5fl pregnanes. Since then extensive reports on the scope of side-chain cleavage reactions by species of Penicillium, Streptomyces, Aspergillus, Mucor, Fusarium, Gliocladium, and Cylindrocarpon have appeared 3,4. In general, the final products of fermentation by these organisms are usually androst-4-ene-3,I7-dione, x7~hydroxy-androst-4-ene-3-one, I7a-oxa-androst-4-ene-3,I7-dione or their I-dehydro analogs. However, different microorganisms appear to have altered specificity with respect to the steroid side chain; some organisms are unable to attack steroid side chains bearing substituents at C-I6 or C-I 7 whereas other organisms are unaffected by substituents at these positions. PETERSON et al. s, demonstrated that Cylindrocarpon radicicola degrades pregn4-ene-3,2o-dione via the sequence: Pregn-4-ene-3,2o-dione--~ pregna-I,4-diene-3,2odione ~ I7fl-hydroxy-androsta-i,4-diene-3-one --7 androsta-I,4-diene-3,I7-dione --~ I7a-oxa-androsta-I,4-diene-3,I7-dione. More recently, FONKEN et al. 6, isolated I7Bacetoxy-androst-4-ene-3-one after exposure of pregn-4-ene-3,2o-dione to Clado" All requests for r e p r i n t s should be addressed to the last author.

Biochim. Biophys..4eta, 93 (x964) 411-417

412

O. EL-TAYEB, S. G. KNIGHT, C. J. SIH

sporium resinae. PRAIRIE AND TALALAYr succeeded in o b t a i n i n g an e n z y m e p r e p a ration from Penicillium lilacinum, capable of converting androst-4-ene-3,I7-dione into I7a-oxa-androst-4-enc-3,IT-dione. F r o m these results, the current conjecture for the conversion of pregn-4-ene-3,2o-dione into I 7 a - o x a - a n d r o s t - 4 - e n e - 3 , I 7 - d i o n e b y microorganisms m a y be t e n t a t i v e l y represented as follows:

CIH3

CIH3

C=O I

C--O

OH

0

I

~

o

o

P. citrinum was r e p o r t e d to convert pregn-4-ene-3,2o-dione or I 7 a - h y d r o x y pregn-4-ene-3,2o-dione (I) into I7/3-hydroxy-androst-4-ene-3-one ; I I = - h y d r o x y - p r e g n 4 - e n e - 3 , 2 o - d i o n e or p r e g n - 4 - e n e - 3 , I I , 2 o - t r i o n e was c o n v e r t e d into I7/3-hydroxyandrost-4-ene-3, I I - d i o n e a n d i I ~, 17/3-dihydroxy-androst-4-ene-3-one respectively ~. R e c e n t l y , SHIRASAKA AND OZAKI9 r e p o r t e d t h a t P. citrinum c o n v e r t e d pregn-4-ene3,2o-dione, I 7 a - h y d r o x y - p r e g n - 4 - e n e - 3 , 2 o - d i o n e , a n d r o s t - 4 - e n e - 3 , I 7 - d i o n e a n d 21h y d r o x y - p r e g n - 4 - e n e - 3 , 2 o - d i o n e into I 7 a - o x a - a n d r o s t a - i , 4 - d i e n e - 3 , i 7 - d i o n c ; i 7 a , 2 i d i h y d r o x y - p r e g n - 4 - e n e - 3 , 2 o - d i o n e was reduced to its 2ofl-hydroxy d e r i v a t i v e a n d no action was o b s e r v e d on 2 i - h y d r o x y p r e g n - 4 - e n e - 3 , i I , 2 o - t r i o n e , i 7 a , 2 i - d i h y d r o x y pregn-4-ene-3, I 1,2o-trione a n d 11/3,17cq2 I-trihydroxy-pregn-4-ene-3,2o-dione. In an a t t e m p t to e l a b o r a t e on the m e c h a n i s m of side-chain d e g r a d a t i o n , P. citrinum a n d C. radicicola were e x p o s e d to various s u b s t r a t e s with a view of o b t a i n i n g i n t e r m e d i a t e s which m a y shed light on the m e c h a n i s m of side-chain breakdown. In this c o m m u n i c a t i o n , we wish to r e p o r t our o b s e r v a t i o n s on the differences in s u b s t r a t e speciticity of these two organisms. EXPERIMENTAL

Materials and methods S t o c k cultures of d'. radicicola (ATC(7 i i O l i ) a n d P. citrinum (University of Wisconsin, class culture 806) were m a i n t a i n e d on n u t r i e n t agar slants s u p p l e m e n t e d with 1 % glucose a n d 1 % yeast e x t r a c t . The f e r m e n t a t i o n mediuin for P. cilrinum consisted of I °o glucose, 2 % p e p t o n e (Difeo) a n d 0.25 % corn steel.) liquor solids. The ferme.ntation m e d i u m for C. radicicola has been described previously m. Smallscale f e r m e n t a t i o n s were carried out in 25o-ml e r l e n m e y e r flasks containing 5 o m l of m e d i u m ; large-scale f e r m e n t a t i o n s were carried out in 2-1 e r l e n m e y e r ttasks containing 40o nil of m e d i u m . T h e flasks were i n c u b a t e d at 27 ° on a r o t a r y shaker. The procedure for p a p e r c h r o m a t o g r a p h y has also been described p r e v i o u s l y m. All melting p o i n t s were uncorrected a n d were d e t e r m i n e d on a T h o m a s -Hoover capillary melting

lqiochim. Biophy.< Acta, 93 (1964) 41 r-417

SUBSTRATE

S P E C I F I C I T Y IN S T E R O I D

SIDE-CHAIN

DEGRADATION

413

point apparatus. Ultraviolet absorption spectra were determined in 95 o, :o ethanol on a ('ary recording spectrophotometer (Model I I MS). Infrared spectra were recorded on a Beckman I R 5 A double beam infrared recording spectrophotometer. Microanalyses were carried out by Mr. J. ALICINO of Metuchen, N.J. and Dr. S. M. NAGY and associates, Massachusetts Institute of Technology. The nuclear magnetic resonance spectrum was determined on a Varian associates recording spectrometer (A6o) at 6o mcycles in deuterated chloroform. Chemical shifts are reported in v values (ppm).

Reaction of z7o~-hydroxy-pregn-4-ene-3,2o-dione (I) with P. citrinmn P. citrim~m was grown in 4.8 1 of the fermentation medium (12 2-1 erlenmeyer flasks). After 24 h of incubation, 2. 4 g of I, dissolved in 19 ml of dimethylformamide was distributed equally to the flasks. After 96 h, the culture broth was filtered and the filtrate was extracted three times with 3-1 portions of chloroform. The' combined chloroform extract was dried over Na2SO 4 and taken down to dryness. The residue was taken up in chloroform, streaked across 24 sheets of Whatman No. I paper (8 in ~, 18.5 in), and developed for 36 h in the toluene-propylene glycol system 1°. Three distinct bands appeared as viewed under the ultraviolet scanner. The bands were cut, eluted with methanol.--chloroform ( I : I , v/v) and concentrated to dryness. The excess propylene glycol was removed by dissolving the residue in chloroform and washed with water three times. The least polar band (Fraction I) weighed 720 mg after evaporation of the solvent. Crystallization from acetone afforded 640 mg (27 %) of I2fl,I7c~-dihydroxy-pregn-4-ene-3,2o-dione (II), m.p. 215-217 °. An analytical sample was obtained by recrvstallization from acetone-light petroleum (b.p. 6o-8o°), m.p. 218-219°; !cc!~6 -t-64 ° in chloroform (c, o.5); nuclear magnetic resonance 9.16, 8.78, 7-57, 6.1o, and 4.25 z; light-absorption max. in ethanol 24o m/, (e I5OOO), and in nulol 2.9o, 5.9 o, 6.12 and 6.20 ~. Calc. for (',,1H:30()4: C, 72.79; H, 8.73. Found: C, 72.42; H, 8.70 Oo. The middle band (Fraction II) afforded 412 mg of residue. Crystallization from acetone gave 318 mg (13 %) of IS/~,I7:c-dihydroxy-pregn-4-ene-3,2o-dione (III), m.p. 252 -255 °. Two recrystallizations from acetone--light petroleum (b.p. 6o .8o °) yMded an analytical sample, m.p. 254-256'~; [~ifis i-57 ° in chloroform (c, I.O); light-absorption max. in ethanol 240 m/~ (e I5OOo), and in nujol 2.92, 5.90, 6.02 and 6.22 ~. Calc. for C,~tH3004: C, 72.79; H, 8.73. Found: C, 72.73; H, 8.73%. The most polar band (Fraction III) gave 224 mg of residue. After two crystallizations from acetone, 186 mg (8 °o) of crystals w-,re obtained. Two recrvstallizations from acetone gave an anal\,tical sample of IX", m.p. 194-196°. ,~i~.~r, -i93 ° in chloroform (c, I.O); light-absorption max. in ethanol 24o mp, (~" 16ooo), and in nujol 2.92, 5.88, 6.o2 and 6.22 t*. Calc. for C.a~lta00~: C, 72-79; ti, 8.73. Found: C, 72.o7 ; H, 8.90 %.

I25-Acetoxy-r7~-h:vdrox~,-pregn-4-ene-3,2o-dione (V) 5o mg of II were dissolved in 1.5 ml of pyridine and 1.5 ml of acetic anhydride. The reaction mixture was left standing at room temperature for 16 h. After the solvents were removed in vacuo, the residue was crystallized from acetone to yield 42 mg of crystals. Recrystallization from ethyl acetate gave 36 mg of V, m.p. 187-188°; ~c~i~ -! 980 in chloroform (c, I.O); light-absorption max. in ethanol 24o m j, (e 18ooo), and in nujol 2.9o, 5.8(7, 5.9 o, 6.o5 and 6.22/,. Calc. for C2aH:~205: C, 71.11; H, 8.31. Found: C, 71.54; H, 7.83 %. Hiochiln. Bioph.rs..-tcta, 93 (19~,4) 41 L-417

414

o. EL-TAYEB, S. G. KNIGHT, C. J. SIH

t 7~-Hydroxy-pregn-4-ene-3,I e ,eo-trione (V I) 50 mg of I I was treated with CrzO s in acetone at room temperature for 6 h. After destroying the excess chromic acid with ethanol, the mixture was diluted with water and extracted with chloroform. After evaporation of solvent and crystallization from acetone-light petroleum (b.p. 6o-8o°), 42 mg of VI were obtained, m.p. 227-228 ° ; [~]~)6 + 145 ° in chloroform (c, I.O); light-absorption max. in ethanol 240 m/, (e I6OOO), and in nujol 2.87, 5.9 o, 6.05 and 6.20 t~. Calc. for (;21H2sO4: C, 73.22; H, 8.19. F o u n d : C, 73.69; H, 8.24 %.

z 5fl-A cetoxv-i 7~-h3,dro.Lv-pregn-4-ene- 3,2o-dione (VII) 5o mg of I I I were dissolved in 1.5 ml of pyridine and 1.5 ml of acetic anhydride and the mixture was left standing for 16 h at room temperature. The residue was c h r o m a t o g r a p h e d over silicic acid 1°. Elution with m e t h a n o l - c h l o r o f o r m (2:98, v/v) afforded 32 mg of crude crystals. Two recrvstallizations from a c e t o n e - l i g h t petroleum (b.p. 60 .80 °) gave 24 mg of VII, m.p. 215-217°; )cJ~)4 - - 2 0 '0 in chloroform (c, I.O); light-absorption max. in ethanol 240 m~ (e I8OOO), and in nujol 2.92, 5.76, 5.9 o, 6.05 and 6.20/~. ('ale. for Cz3H3z()~: C, 71.11; H, 8.31. F o u n d : C, 70.56; H, 8.64°'°.

I7~-Hydroxv-pregn-4~ene-3,15,2o-trione

(VIII)

IOO mg of I I I were treated with Cr~Oz in acetone. After the usual work up and crystallization from acetone, 80 mg of crystals, m.p. 265 -267 °, were obtained. Recrystallization from a c e t o n e - l i g h t petroleum (b.p. 6o-8o ") gave an analytical sample, m.p. 2t)7-268:" 1~]~ - - 9 8 ° in chloroform (c, I.O); light-absorption max. in ethanol 240 n'lbt. (6 I5OOO), and in nujol 2.92, 5-74, 5 .88, 6.02 and 6.20/z. Calc. for C2~H2804: C, 73.22; H, 8.19 . F o u n d : (', 73.11; H, 8.o9%.

Reaction of 2Tc~-hydroxv-pre~n-4-cne-3,2o-dione (I) with C. radicicola C. radicicola was grown in 1.2 l of the medium (three 2-1 erlenmeyer flasks). After 24 h of incubation, o. 7 g of I, dissolved in 0 ml of dimethvlformamide was distributed equally to the flasks and the fermentation was continued for 96 h. The culture broth was then filtered and the filtrate was extracted three times with three 3oo-ml portions of chloroform. The combined chloroform extract was dried over NazSO 4 and taken down to dryness to v M d 725 mg of a pale yellow residue. Crystallization from actone gave 51o nag (73 %) of crystals, m.p. 214-217L Recrvstallization from acetone gave a sample, m.p. 218-22o -~, identical with an authentic sample of I7a-oxa-androsta- r,4-diene-3, I7-dione (IX).

Reaction of pregna-4,r6-diene-3,2o-dione (X) with (?. radicicola Under similar conditions, 65o ing of X afforded 47 ° mg (72 %) of IX, na.p. 218-22o : after exposure to C. radicicola for 96 h.

Transformation of rTfl-acetvl-amino-androst-4-ene-3-one (XIII) to z7f3-ace~.'l-aminoandrosta-z,4-diene-3-one (XIV) b3, C. radicicola 6oo mg of X I I I n were exposed to C. radicicola for 12o h. After the usual work up, 580 mg of a pale yellow residue was obtained. Paper c h r o m a t o g r a p h y showed two spots with 1@ values of o.3o and o.14 in the toluene--propylene glycol system. The mixture was separated by chromatograt)hy over silica gel. Elution with chloroform--

Biochim. l~iophys..4eta, 93 (1964) 41 I...t f 7

S U B S T R A T E S P E C I F I C I T Y IN S T E R O I D S I D E - C H A I N D E G R A D A T I O N

415

methanol (98:2, v/v) afforded 32o mg of crude crystals. Recrystallizations from hot acetone gave 25o mg of XIV, m.p. 286-288 ° ; [~z]L7 + 13° in chloroform (c, I.O)" lightabsorption max. in ethanol 243 mv~ (e i6ooo), and in chloroform 2.99, 6.02, 6.20 and 6.24 V,. Calc. for C21H~902N: C, 77.o2; H, 8.93; N, 4.28. Found: C, 77.13; H, 9.09; N, 4.04%.

Transformation of r7o~-methyl-pregn-4-ene-3,2o-dione diene-3,2o-dione (XVI) by C. radicicola

(XV) to ~7~-methyl-pregna-x,4 -

250 mg of XV were exposed to C. radicicola for 96 h. After the usual work up 235 mg of a pale yellow residue was obtained. Paper chromatography showed two spots in the isooctane-2-methoxyethanol system with RF values of 0.37 and o.23. The mixture was separated on a cellulose powder column using 2-methoxyethanol as the stationary phase and isooctane, saturated with 2-methoxyethanol as the mobile phase. The first fraction contained 165 mg of the starting material, XV. The second fraction gave 46 mg of crude crystals which upon crystallization from ethyl a c e t a t e light petroleum (b.p. 60°-80 °) afforded 32 mg of XVI, m.p. I6O°-I62~; [cc]~ --51° in chloroform (c, 0.4); light-absorption max. in ethanol 242 m~ (e I55OO), and in chloroform 5.9 o, 6.02, 6.2o and 6.24/~. RESULTS

When i7ce-hydroxy-pregn-4-ene-3,20-dione (I) was exposed to P. citrinum, three products were formed. The first product, II, was assigned the structure i2fl,i7oedihydroxy-pregn-4-ene-3,2o-dione on the basis of the following data. C and H analysis was in good agreement with C21H3004; its infrared spectrum showed bands at 2.90 (OH), 5.9 °/~ (2o-carbonyl), 6.1o and 6.20 ~ (A4-3-ketone). Acetylation of II afforded a monoacetate (V). Oxidation of I I with chromic acid in acetone gave VI, identical in all respects (mixed melting point and infrared spectrum) to an attthentic sample of I7~-hydroxy-pregn-4-ene-3,I2,2o-trione. The configuration of the hydroxyl at (;-12 was established by its nuclear magnetic resonance spectrum, which showed a broad band at 6.1 r (I H, J at least 14 cycles/sec) which indicates that the methine proton on C-I2 exhibits an axial-axial and axial-equatorial spin couplings ~2. Hence, the methine proton is axial and the hydroxyl on (;-12 is equatorial. The second product, 11I, was assigned the structure I5fl,i7ce-dihydroxy-pregn-4-ene-3,2o-dione. C and H analysis was consistent with the empirical formula C21H3oO4. Oxidation of I I I with chromic acid in acetone afforded a compound which showed a band at 5.74 ~ in its infrared spectrum which is characteristic for the presence of a 5-membered ring ketone; hence the hydroxyl in I I I must be situated either at positions 15 or 16. The molecular rotatory contribution of the. hydroxyl group in I I I was (MD(III) - - MD(I)) --- --12o ~. This is in close agreement with the MD values reported for I5fl-hydroxyl groupsla: AMr~ for I5fl-hydroxyl = --114°; for I5ce-hydroxyl= --87°; for I6flhydroxyl = -~-38°; and for I6ce-hydroxyl . . . . . 64 °. Since the physical properties of I I I were not identical to I6oqI7ce-dihydroxypregn-4-ene-3,2o-dionO4O s, the hydroxyl group in I I I was assigned the I5/3-configuration. Furthermore, it was observed that I I I was difficult to acetylate under the conventional procedure using acetic anhydride-pyridine at room temperature. This is consistent with the observation that I5/3-hydroxy steroids are difficult to acetvlate

Biochim. Biophys. Acta, 93 (1964) 4 x1-417

416

O. E L - T A Y E B ,

S. (;. K N I G H T ,

C. J . S I H

due to the pseudoaxial orientation which exhibits a non-bonded 1,3-interaction with the Ct8 methyl group le. The third product I I I , was also a monohydroxyl derivative of I but unfortunately the quantity was too small to permit its structural elucidation. When I and X were incubated with C. radicicola, I7a-oxa-androsta-I,4-diene3,I7-dione was obtained in good yield. The latter product was identified by comparing with an authentic sample. I7ct-Methyl-pregn-4-ene-3,2o-dione was selected for incubation with C. radicicola with a view of obtaining I7~-methyl-I7/3-acetoxy-androsta1,4-diene-3-one since tertiary acetoxvl grout)s were found to be unsuscet)tible to microbial esterasO 7. However, both i7,~-methyl-pregn-4-ene-3,2o-dione and I7/3acetyl-amino-androst-4-ene-3-one were converted to their I-dehydro analogs. Their structure was established by virtue of the presence of bands in their infrared spectra, characteristic for A~,4-3-ketones. CH3

CH3

I C=O

CH3

I C=O

OH

" 3H

I C=O

"'OH

""OH

Pciteinurn~ ~ 0

+~

0

H

+

IV

0-~ "-,..~ ~,./

[[

I

O III

C.Padlctcola

CH3 I

C=O

~C.Padtcicolo

O.~L~/~j.~ 3

o

[X

X F i g . 2a.

3 CH C=O

CH 3 I C=O I NH

'

NH

0

0

XI[I

XIV

Fig. 2b.

CH3 I C=0 ~ 0

c

H

CH3 I C=O

3

~ ' ~

CH3

C.radicicOlao~ XVI

XV

F i g . 2c.

Hiochim. Biophys..4 eta, 03 ( l 9i,4) 41 [ -4 r 7

SUBSTRATE

S P E C I F I C I T Y IN S T E R O I D

SIDE-CHAIN

DEGRADATION

417

DISCUSSION

There appears to be a significant difference between P. citrinum and C. radicicola in their metabolism of derivatives of pregn-4-ene-3,2o-dione. C. radicicola is capable of transforming pregn-4-ene-3,2o-dione, pregna-4,I6-diene-3,2o-dione and I7~-hydroxy-pregn-4-ene-3,2o-dione into I7a-oxa-androsta-I,4-diene-3,I7-dione as the end product of metabolism. Although P. citrinum is also capable of transforming pregn4-ene-3,2o-dione into ITa-oxa-androst-4-ene-3,I7-dione, it was unable to cleave the side chain of I7~-hydroxy-pregn-4-ene-3,zo-dione; instead it introduced hydroxyl groups into the latter substrate at positions 12 and 15. Normally, P. citrinum is not known to hydroxylatc pregn-4-ene-3,2o-dione. This difference of substrate specificity between these two organisms may be attributed to the different stereo-specificity of the two enzyme systems or alternately, these organisms cleave the side chain via entirely different mechanisms. C. radicicola has always been able to introduce a 1,2 double bond into a variety of steroids, including that which carries a nitrogen atom at C-I7. It is also capable of removing the methyl ketone side chain, regardless whether it is substituted by a hydroxyl, double bond or an epoxide at C-I 7. However, it was surprising to find that a methyl group at C-I 7 appeared to block side-chain degradation completely, indicating that electronic effects may also play an important role in the side-chain cleavage reaction. ACKNOWLEI)GEMENTS

The authors wish to express their thanks to the following: Professor K. RAPER, Department of Bacteriology, University of Wisconsin for the culture of P. citrinum; Dr. E. S. ROTH.~IAN of U.S.D.A. for a sample of I7~-hydroxy-pregn-4-ene-3,i2,2otrione; Dr. R. DEGHRNGHI of Averst, McKenna and Harrison Ltd. for a sample of I7~-methyl-pregn-4-ene-3,2o-dione and Dr. N. BHACCA of Varian Associates for the nuclear magnetic resonance analysis and interpretation. This work was supported in part by research grants (A-4687 and AI-oi 2Ol) of The National Institutes of Health. REFERENCES (-. j. SIH, Hiochim. 14iophys. /tcta, 62 (I962) 541. E. VISCHER AND A. WETTS'rEIX, Experientia, 9 0 9 5 3 ) 37 r. j . FRIED, R. W . TItOMA AND A. KLINGSBERG, J . . 4 m . Chem. Soc., 75 (I953) 5764 • I). H. PETERSON, S. H. EPPSTEIN, P. ]). ]~EISTER, H. C..'~{URRAY, H. M. I.EIGI], A. ~,VEINSTRAUB AND l.. M. REINECKE, J. Am. Chem. Noc., 75 (1953) 5708. 5 G. E. PETERSON, R. W. TItOMA, D. PERL.~IAN AND J. FRI~.'D, J. Bacteriol., 74 (I957) 684. 6 G. S. FONKEN, H. C. MURRAY AND L. M. REINECKE, .[..4m. ('hem. Sot., 82 (I96o) 55o7 . 7 R. L. PRAIRIE AND P. T.'~LALAY, Biochemistry, z (1903) -'03. s O. HA.~C,A. CAPEK, M. T^DRA, K. MACEK AND A. SIMEK, Arzneimittel-Forsch., 7 (I957) I75. 9 ]~'I. SHIRASAKA AND M. (.)ZAKI, J . Agri. Chem. Soc. Japan, 35 ( I 9 6 I ) 206. lo O. EL-TAYEB, S. G. KN1GnT AND C. J. SIH, t3iochim. Biophys...lcta, 93 (I964) 4 o2. it N. J. [)OORENBOOS AND H. SIN(;, f . Pharm. 5ci., 51 (1962) 418. lz l.. M. JACKMAN, Applications of Nuclear ~$Iagnetic Re.~onance .S'pectroscopy in Organic Chemistry, P e r g a m o n , L o n d o n , I959. p. 116. 13 H. I.. HERZOG, l'kI. J. GENTLES, ~V. CHARNEY, I). Su'r'rER, E. TOWNLEY, M. YUDIS, 1), ]XABASAKALIAN AND E. B. HERSHBERG, J. Org. Chem., 24 (1959) 691. 14 G. COOLEY, ]3. ELLIS, F. HARTLFY AND V. PETROW, .]. Chem. Noc., (r955) 4373. 15 G. R. ALLFN AND I~'1. J. WEISS, J. Am. Chem. Hoe., 8 i (I959) 4968. 16 S. I{ERNSTE1N, .X,'l. HELLE'R, L. I. FELDMAN, XV. S. ALLEN, ]{. I1. BLANK AN1) C. E. I.INDER, J. Am. Chem. Soc., 8"- (196o) 3685 . 17 C. J. SIH, J. LAVAL AND A..'~I. RAIIIM, J. Biol. Chem., 238 (I963) 026. t 2 3 4

Biochim. Biophys. Acta, 93 (I964) 41 I - 4 1 7