OXIDATION OF STEROIDS BY MICROORGANISMS. HYDROXYLATION IN POSITION 1 1 AND SYNTHESIS OF CORTISONE FROM REICHSTEIN'S COMPOUND S
( A [ & ~ ] D f407°),4 indicative of the presence of the 11-keto group in the tetraketone derived from 11. Oxidation of Compound S with A . niger afforded two new compounds, A4-pregnene-11a,17a,21-triol,%r : The finding' that the action of the actinomycete 3,20-dione5 (111), 25% yield, m.p. 217-219' ; 242 tnp culture MD-2428 on progesterone could under [a]n +117", (c, 0.46 in alcohol); A&:. proper conditions bring about hydroxylation of the ( E = 15,000); (Anal. Found: C, 69.46; H, [ a I 2 ?+117' ~ latter at carbon atom 16, a position not readily at- 8.39); diacetate: m.p. 206-208'; tacked by chemical oxidants, made it appear hope- (c, 0.84 in CHCIJ and an as yet unidentified isomer [a]% ful that microorganisms could be found capable of of 111 (IV), 15% yield, m.p. 248-250'; introducing oxygen into the important position 11, +97" (c, 0.50 in alcohol); Ai:x, 241 nip ( C = and a successful experiment of this sort has re- 16,000); (Anal. Found: C, 69.45; H, 8.53), cently been reported.2 -4s a result of a systematic separable by fractional crystallization from acetone. study of the metabolic action of a variety of micro- I11 was converted by chromic acid to A4-androorganisms on selected steroidal substrates we have stene-3,11,17-trione (andrenosterone),6 m:p. 22 1independently found that certain members of the 225'; [aIe4D +284' (c, 0.51 in CHCI,) identical genus Aspergillus are capable of effecting the de- in aIl respects (including infrared spectra) with an sired change in practicable yields. In this com- authentic sample of that substance. Acetylation munication we wish to report our experience with of I11 with 1.1 moles of acetic anhydride in pyriAspergillus niger (Wisc. 72-2) using progesterone, dine followed by chromic acid oxidation furnished Reichstein's Compound S, desoxycorticosteroneand in 70% yield cortisone acetate,? m.p. 243-244' (opaque a t 85-95'); Ia]"D +168" (c, 0.63 in I 7a-hydroxyprogesterone as the substrates. All fermentations were carried out in submerged acetone), +204' (G, 0.63 in CHCla), identical in its culture at 25" for periods of 65-70 hours in a physical characteristics with an authentic sample. medium containing corn steep liquor solids, am- This sequence of reactions provides unambiguous monium phosphate, calcium carbonate and soy- proof for the structure of I11 and represents the bean oil. Incubation of A . niger with progesterone final steps of a new and simple synthesis of cortifollowed by extraction of the culture filtrate with sone from readily accessible starting materials. Incubation of A . niger with desoxycorticosterone chloroform yielded a mixture separable by chromatography on alumina into 11a-hydroxyproges- furnished after chromatography on silica a 67% terone2 (I), 3%% yield, m.p. 166-167" : [ a I 2 * ~yield of the hitherto undescribed A4-pregnene-1la,+17S0 (c, 1.22 in CHC18); A$t,a. 241 mp ( e = 17a-diol-3,20-dione (V), m.p. 153-154' ; 168' (c, 0.7 in CHC&),(Anal. Found: C, 72.90; 17,000); A&'$' 2 . 9 1 ~ (OH), 6 . 8 5 ~ (20-keto), i5 . 9 7 ~(A4-3-keto); (Anal. Found: C, 76.12; H, H, 8.76). The structure of V was established by 9.28) and 6i, 11a-dihydroxyprogesterone (11),20% acetylation with 1 mole of acetic anhydride followed by oxidation with chromic acid to the known yield, m.p. 250-253'; [ a I z 3 D +looo (c, 0.28 in 11-dehydrocorticosterone acetate,8 m.p. 178-179", 236 mp ( e = 15,000); 2 . 9 8 ~ [cY]'~D +239" (c, 0.5 in CHCla), identified by comCHC13); (OH), 5 . 8 9 ~(20-keto), 6 . 0 4 ~(AY3-keto); (Anal. parison with an authentic sample. Found: C, 72.66; H, 8.76). In harmony with the The action of A . niger on 17a-hydroxyprogesdata of Peterson and Murray,3 I formed a mono- terone produced two new hydroxylated steroids acetate, m.p. 172-174"; [ a ] " ~$155" ( E , 0.38 separable by chromatography on silica. Elution in CHC13) and on oxidation with chromic acid fur- with 5% acetone in chloroform afforded 17anished the known 11-ketoprogestero~~e,~ m.p. methyl - D - homo - A4 - androstene - l l a , l 7 a - diol170-172", [ Q ] * ~ D +276' (c, 0.29 in CHCb), +229" 3,17-dioneY (VI), 25%, m.p. 261-262'; [a]23~ (c, 0.52 in acetone) ; identical in all respects with +46" (c, 0.74 in CHC13); (Anal. Found: C, an authentic sample prepared according to Reichstein and F ~ c h s . I1 ~ formed a diacetate2m.p. 154- 72.92; H, 8.84), and subsequent elution with 10% 155'; [ a ] " ~+81° (c, 0.92 in CHCl3) and 011 oxida- acetone in chloroform yielded 11a,l7m-dihydroxy[a]23~ tion with chromic acid afforded what appears to progesterone (VII), 15%, m.p. 219-221'; 4-87' (c, 0.57 in CHClr); (Anal. Found: C, be 6,ll-diketoprogesterone,1n.p. 144-146' ; [a]% f143' (c, 0.87 in CHCI,); (AnaE. Found: C , 73.11; H, S.70) ; monoacetate: m.p. 205-208' ; 73.77; H, 7.66) on the basis of its ultraviolet spec- [ a ] " ~+65" (c, 0.61 in CHC13). Oxidation of VI tra in alcohol 249 mp ( E = 11,500)) and meth- and VI1 with chromic acid afforded respectively the anolic KOH (Amax. 255 m,u and 370 mp ( e = 9800 hitherto undescribed 17a-methyl-D-homo-A4-an(4) The contribution for the 11-keto yroup in 11-ketoprogesterone is and 8900 resp.)), identical with those shown by 6ketoprogesterone, and of the difference of its +302' (CHCls). (5) I11 was found to be inactive by Dr. R. W. Bates in the rat liver molecular rotation and that of 6-ketoprogesterone glycogen assay (hl. L. Pabst, R. Sheppard and M. H. Kuizenga, 11.'
Xzx.
(1) Preceding communicatiou: D. Perlman. E. Titii? and J . Fried, TRISJOURNAL, 74, 2126 (1952).
(2) D. H. Peterson and H. C. Murray, ibid., 14, 1871 (1952). The microbiological conversion of progcsterone into llor-hydroxyprogesterone and an unidentified dihgdroxyprogcsterone by the fungus RRimfiz~suvrhieus is described. C f , also, the more recent publication by D K. Colingsworth, M. P. Brunner and W. J. Halnes (ibid.. 74, 2381 (19521) reporting tile oxidation of Compound S t o 17a-hydroxycorticosterone in low yield by Stregtomyccs fradinc. ( 3 ; T.Reichstein and H. G.Fuchr, Helu. Chim. Acto, 18, 684 (1940).
Ertdocriwlogy. 41, 56 (1947)) in doses of 800 mcg. per animal. (6) T. Reichstein, Bsiu. Chim. Acta, 19,29 (1936). (7) T.Reichstein, ibid., 80,978 (1837); L.R. Sarett, J. Bid. Chem., 168, 601 (1946). (8)T. Reichstein, Heio. Chim. Acta, 80, 953 (1937). (9) That the formatloo of the D-homosteroid VI wag effected hg the mold and not by one or more of the constituents of the fermentation medium was shown by t h e fact t h a t incubation of 17a-hydroxyproges. terone with the medium in the a b i e n c e ~i i h e orgwnistn caused n o ex.: pavpion of ring I>"
COMMUNICATIONS TO THE EDITOR
Aug. 5 , 1 9 3
3963
An 11-atom ring structure is possible,4consistent with the published X-ray data and with all of Pauling and Corey's postulates regarding bond angles and distances, except that the N-C* bond is not in the C-C'O-NH plane, but makes an angle of about 30' with it. This is not unreasonable, on the basis of their estimate of about equal contributions of structures containing coplanar nitrogen and tetrahedral nitrogen. On the other hand, approxiJOURNAL, 70, 1454 (1948); T. H.Kritchev(10) L.H. Sarett, THIS mate coplanarity has been found in glycylglycines sky, D. .I Garmaise and T. F. Gallagher, ibid., 74, 483 (1952). (11) J. van Euw and T. Reichstein, Helu. Chim. Acta, 24, 879 and acetylglycine6 crystals; this would seem to (1941 1. favor the 13-atom ring structure, which permits JOSEFFRIED such coplanarity. However, since the energy RICHARD W. THOMA THE SQTJIBB INSTITUTE FOR JOHNR. GERKE difference associated with the difference in bond MEDICAL RESEARCH JOSEFE. HERZ orientation is probably small and may be counterNEWBRUNSWICK, NEWJERSEY MILTONN. DONIN acted by environmental differences, this evidence D . PERLMAN is not very strong. RECEIVED JUNE30, 1952 I n neither the 11-atom ring structure nor the 13-atom ring structure is the C==O bond tilted with respect to the axis of the helix more than the POLYPEPTIDE HELICES IN PROTEINS N-H bond, unless the assumptions made are conSir : siderably in error. Hence, the infrared spectrum About fifteen years ago' I discussed the principles differences, tentatively and cautiously attributed underlying protein structure and proposed that by Bamford and co-workers' to such a difference in the polypeptide chains in proteins, when not nearly angle of tilt, should probably be interpreted in some fully extended, have folded or helical structures, other way. with adjacent folds or turns of the helix connected In agreement with Bamford and his colleagues, I by N-H . O hydrogen bonds. Considerable evi- believe that, pending further experimental data, dence has since accumulated in favor of these pro- both of these structures should be considered posposals and they are now generally accepted. sible for the alpha synthetic polypeptides, the alpha -4s the simplest examples illustrating these prin- fibrous proteins and corpuscular proteins. Perciples, I discussed a folded structure containing haps both types are sometimes present together, 7-atom rings in fibrous natural proteins for example. All other -C-NH-CHR-CO-Ntypes of structure seem to be definitely elimi0 . . . . . . . . . . . .. - H nated, at least for the alpha synthetic polypepand helices containing 8-atom rings tides, by the X-ray data.7-9
drostene-17a-ol-3,11,17-trione, m.p. 238-242" ; [ a I z 34-121" ~ (c, 0.48 in CHClg) and the known A4-pregnene-17cr-ol-3,l1,20-trione,1° m.p. 232D (c, 0.33 in CHCb). The con235'; [ L Y ] ~ ~+lSS" version of VI1 into VI under conditions reported1' to effect the expansion of ring D in 17a-hydroxyprogesterone served to establish the structure of VI.
a
-NCHR-CO-NH-CHR-CH . . . . . . . . . . . . . . . . . . . .O
and 10-atom rings -C-( NH-CHR-CO)z-N0 . . . . . . . . . . . . . . . .. H
Bragg, Kendrew and Perutz2 have recently considered similar 11-atom ring -N-( CHR-CO-NH)2-CHR-CH . . . . . . . . . . . . . . . . . . . . . . .O
and 13-atom ring -C-( NH-CHR-CO)rN0. . . . . . . . . . . . . . . . .H
helices, assuming in both exactly four amino-acid residues per turn, and Pauling, Corey and Branson3 have advocated the 13-atom ring helix with about 3.7 residues per turn. They pointed out, as I had done in the case of the 10-atom ring structure, that it is not necessary that this number be integral. (At the recent Chemical Conclave I mistakenly believed and stated that their model was merely a refinement of my 10-atom ring structure.) (1) M . L.Huggins, Abstracts, Rochester Meeting, American Chemical Society, B10 (1937); see also Abstracts, Memphis Meeting, A.C.S., P4 (1942); Annual Reuiew of Biochemistry, 11, 27 (1942); Chem. Revs., 34, 195 (1943). (2) W.L. Bragg, J. C. Kendrew and M. F. Perutz, PYOC. Roy. SOC. (London), A¶W, 321 (1960). (3) L. Pauling and R. B. Corcy. TEZSJ O V ~ N A L ,79, 5349 (1950); PYOC.Nal. Acod. Sci., 57, 286, Ml, 256,261,282 (1951); L. Pading, R. B. Corcf and H. P.B-n, iW., 1,206 (1951).
(4) M. L. Huggins, THISJOURNAL, 74, 3963 (1952). (5) E.W.Hughes and W. J. Moore, ibid., 71, 2618 (1949). (6) G.B. Carpenter and J. Donohue, ibid., 72, 2315 (1950). (7) C. H. Bamford, L. Brown, A. Elliott, 6'.E. Hanby and I. F. Trotter, Nature, 169, 357 (1952). (8) M. F. Perutz, ibid., 167, 1053 (1951); 166, 653 (1951); H.E. Huxley and M. F. Perutz, ibid., 167, 1054 (1961). (9) W.Cochran and F. H. C. Crick, i b i d . , 169, 234 (1952).
RESEARCH LABORATORIES EASTMAN KODAKCOMPANY MAURICE L. HUGGINS ROCHESTER 4, NEWYORK RECEIVED JUNE23, 1952 COORDINATES OF THE 11-ATOM RING POLYPEPTIDE HELIX
Sir: In order to facilitate comparison of the 11-atom ring helical polypeptide structure112 with other structures and with experimental data, I have calculated atomic coordinates, on the following assumptions : (1) the translational and rotational shifts per amino-acid residue are 1.47A. and looo as o b ~ e r v e d ~in - ~ poly-(methyl glutamate) ; (2; the bond distances and bond angles are those that some assumed by Pauling and C ~ r e yexcept ,~ (1) M.L. Huggins, THIS JOURNAL, 74, 3963 (1952). (2) C. H. Bamford, L. Brown, A. Elliott, W. E. Hanby and I. F. Trotter, N a u r e , 169,357 (1952). (3) L. Pouling and R. B. Corey, PYOC. N a f . Acad. Sci., 57, 241 (1951); Notwid, 169,494(1952). (4) M. F. Perutz, ibid., 167, 1058 (1951). (6) L. Pauling and R . B. Corey, Proc. Nul. Acod. Sci., 87, 235 (1961).
