COMBIUXICATIONS TO THE EDITOR
May 5 , l93S
tifications were established by comparisons against infra-red spectra of known standards. Compounds isolated from the above experiments are listed in Table I together with their specific activities. The results showed a loss of 69% of the total count of pregnanedione-H3 on 11a-hydroxylation, and essentially no loss (2-3%) on llp-hydroxylation. Mild chromic acid oxidation according to the method of P00s5 of the hydroxylated products to their keto analogs (Table 11) showed a loss of 70% of the total count in the case of the l l p hydroxylated structure, while there was essentially no change (lye) in counts in the l l a series.
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THE STEREOCHEMISTRY OF 7a-HYDROXYLATION IN THE BIOSYNTHESIS OF CHOLIC ACID FROM CHOLESTEROL Sir :
Recent evidence on the course of enz,ymatic hydroxylation of steroids a t a saturated carbon atom indicates that such reactions occur by a direct replacement mechanism and not by hydration of olefinic However, the chemical nature of the enzymatic reagent and the type of mechanism involved are still undetermined. The stereochemistry of enzymatic hydroxylation a t C, of the steroid nucleus has now been examined to provide geometrical evidence regarding mechanism. TABLE I1 During the biochemical conversion of cholesterol CHROMIC ACID OXIDATION OF HYDROXYLATED STEROIDS to cholic acid a 7a-hydroxyl group is introduced, quite possibly as the initial step since nuclear 11-Keto-pregnanedione 11or-Hydroxy-pregnanedione hydroxylation of cholesterol proceeds side-chain m.p. 123-125' m.p. 159-161" degradation4 and since 7a-hydroxycholesterol is 8.43 X l o 4 counts/min./ 8.49 X lo4 counts/min./ transformed into cholic acid in the rat.5 The P M P M stereochemical course of this 7a-hydroxylation in Corticosterone-21-acetate 11-Dehydro-corticosterone- rats has been investigated by double-labelling ex21-acetate periments using cholesterol stereospecifically lam.p. 156-157" m.p. 180-182" belled with tritium a t position 76 and cholesterol0 . 9 8 X lo4counts/tnin./
3 . 2 1 X l o 4 counts/min./ ILM
4-'4C.7
P M
TABLE I
A schematic representation of the results is Dresented. 1lor-Hydroxylase
r
I
11P-Hydroxylase 100 €13
/y\
-o\v
I
Chromic acid
30 H3 'Chromic acid
7
Activity ratio T/C14
1 2 3 4 5 6 7 8 9
Cholesterol-4-14C-7a-t Cholic acid rat 4 Cholic acid rat 5 Cholesterol-4-14C-7c-t Cholic acid rat 6 Cholic acid rat 7 Cholester01-4-'~C-7p-t Cholic acid rat 9 Cholic acid rat 10
1.75 0.31 0.12 1.24 0.07 0.09 1.04 0.95 1.03
Per c:ent. retention of tritium in cholic acid
7.4 6.8 5.6 7.2 91.5 100
+
When cholesterol- [4-14C 7a-t] was administered to rats the isolated cholic acid retained only ca. 7% of tritium relative to radiocarbon (Table I).
(1) M. Hayano and R. I. Dorfman, J . Biol. Chem., 211, 227 (1954). (2) B. M. Bloom and G. M. Shull, THISJOURNAL, 71, 5767 (1955). (3) M. Hayano, A. Saito, D. Stone and R. I. Dorfman, Biochem. From the data it can be concluded that enzy- and Biophys. Acta, 21, 380 (1956). (4) See S. Bergstrom and B. Bergstrom, A n n . Rew. Biochem., 2 6 , matic steroid hydroxylations proceed by a mechanism in which there is a simple replacement of the 177( 5 (1956). ) S . Linstedt, Acta Chem. Scand.. 11, 417 (1957). hydrogen in the position to be hydroxylated. This (6) (a) Cholesterol-7a-1 and cholesterol-7a-d were synthesized is the second instance noted in points of similarity stereospecifically by the sequence: 7a-bromo-6-ketocholestanyl acetate (Zn-"HOAc) 7~~H-68in the mechanism of reaction of the steroid hydroxyl- acetate -c 7~~-~H-6-ketorholestanyl 7a-nH-cholesterol (POClshydroxycholestanyl acetate (NaBH4) ases, irrespective of source, the first being their CsHaN, followed by LiAlHI). Cholesterol-7,3-t and cholesterol-78-d ability of the utilization of molecular oxygen di- were prepared by a similar process starting with 5a,78-"Hn-7a-bromorectly in the formation of the hydroxyl function.6.7 6-ketocholestanyl acetate using unlabelled acetic acid in the debroFurther mechanism studies involving this group mination step. The cholesterol-7a-d and -78-d (infrared max 2102, 2127 cm.-1 and 2147, 2160 cm.-1, respectively) were analyzed b y of enzymes are now in progress. infrared absorption [E. J. Corey, M. G. Howell, A. Boston, R. L. WORCESTER FOUNDATION FOR MIKAHAYANO Young and R. A. Sneen, THISJOURNAL, 78, 5036 (1956)I and the isoEXPERIMENTAL BIOLOGY MARCELGUT tope orientation was found to be stereospecific within the analytical SNREWSBURY, MASS.,A N D R. I. DORFMAN sensitivity of &4%. It follows t h a t the tritium labelling is 96 =k 4 % THEUPJOHNCOMPANY 0. K. SEBEK stereospecific; (b) see E. J. Corep and G. A. Gregoriou, Abstracts, D. H. PETERSON 131st A.C.S. meeting, p. 15-0. KALAMAZOO, MICH. (7) Ca. 2 mg. of a mixture of cholesterol-4-*'C and cholesterolRECEIVED JANUARY 28, 1958 7a-# or cholesterol-78-1 (one microcurie of each isotope) was injected (5) G. I. Poos, G. E. Arth, R. E. Beyler and L. H. Sarett, THIS intraperitoneally into rats with bile fistula. The bile was collected JOURNAL, 76, 422 (1953). and cholic acid was isolated as described earlier [S.Bergstrom and A. (6) M. Hayano, M. C. Lindberg, R. I. Dorfman, J. E. H. Hancock, Norman, Proc. Sot. E x p f l . Biol. Med., 83, 71 (1953)l. further purified and W. v. E. Doering, Arch. Biochem. Biophys., 69, 529 (1955). by dilution with 100 mg. of unlabelled cholic acid and recrystallization, (7) M. Hayano, A. Saito, D. Stone and R. I. Dorfman. Biochim. and analyzed for isotopes according t o R. Glascock, Iaolopic Gas Biophys. Acta, 21, 380 (1956). Analysis for Biochemisls, Academic Press, N. Y..1954.
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COMRITJNICATIONS TO TIIE EDITOR
2338
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When cho1esterol-[4-l4C 7/3-t] was used practically all the tritium remained in the cholic acid molecule. Mild oxidation of the isolated cholic acid to its 7-keto derivative resulted in complete loss of the tritium label. Consequently, 7ahydroxylation involves displacement of the 7ahydrogen with at least 9370 and possibly complete specificity. The same stereochemical course has been observed for the hydroxylation of steroids a t C ~ Ii.e., , displacement with retention of configuration.sfY These data are reminiscent of the observation that hydroxylation of cis- and trans-decalin by ozone proceeds with retention of configuration to cis- and trans-9 hydroxydecalin, respectively, lo and are in agreement with Bloom's evidence.2 In addition, i t seems relevant that in chemical systems electrophilic displacement a t a saturated carbon atom has been found to occur preferentially with retention of configuration.6a,11 This work was supported by the National Institutes of Health (Grant H2843) and the Alfred P. Sloan Foundation. ( 8 ) M. Hayano, M. G u t and D. H . Peterson, private communication. (9) E . J. Corey. G. A . Gregoriou and D. H . Peterson 1'111s JOURNAL, 80, 2338 (1958). (10) J. R. Durland and H. Adkins, ibid., 61, 429 (1930). (11) S. Winstein, T. G . Traylor and C. S. Garner, i b i d . , 11, 3711 (1955): S. Winstein and T. G . Traylor, ibid., 11, 3747 (1955), 1 8 , 2,597 (195fi).
Vol. 80
niques previously described3 and yielded 1lahydroxypregnane-3,20-dione-llp-d containing 0.98 + 0.02 deuterium/molecule. Similar oxidation of pregnane-3,2O-dione-11a-d having additional deuterium a t CS and C l aand a total of 2.50 deuterium! molecule4 resulted in complete loss of lla-deuterium since the 11a-hydroxypregnane-3,20-dione which was produced possessed 1.77 deuterium/ molecule. Enzymatic hydroxylation of steroids a t the llP-5 and 7a-positions6 also has been found to proceed with retention of configuration, a course which, though under the control of specific enzymatic interactions as usual, may also be favored by the electrophilic nature of the displacing reagent .6 All the data accumulated thus far5r6indicate a lack of hydrogen isotope effect on the rate of oxidation and permit an additional conclusion: either C-H bond rupture occurs after the rate determining step of the reaction or else chemical reaction is preceded by a t least one slow physical step, e.g., adsorption, which is insensitive to H isotope. We take pleasure in thanking Mr. Josef Nemeth for the deuterium analyses, Dr. Robert Levin for gifts of steroids, and Mr. 0. I
