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changed I (peak A) was identified by recrystallization with carrier from water to constant specific activity. The radioactivity in peak B could be volatilized by treatment with ammonia and acidification with formic acid, identifying it indirectly as II.’ Peak D was shown to be hydantoin-5propionic acid (IV) by recrystallization with carrier to constant specific activity from both ethanol-benzene and water and co-chromatography in six solvents with the synthetic compound.8 Synthetic IV (Found: C, 41.69; H, 4.GO; N, 16.29) prepared from L-glutamic acidg was eluted in the identical position as peak D. The dotted line in Fig. 1 is synthetic radioactive IV. Peaks A-D have 72, 1.1, 1.5 and 8.5% of the urinary radioactivity, respectively. Incubation of I with rat liver slices forms small amounts of IV in the presence or absence of 111; radioactive IV is present in monkey and human urine after intravenous C14 histidine. The biochemical steps from I to IV have not yet been elucidated, Acknowledgment.-The authors are grateful to Dr. Herbert Tabor for his helpful criticism. ( 7 ) U. A. Borek and H. Waelsch, J . Bioi. Chem., 205, 459 (1953). ( 5 ) Synthetic I V sprayed with 0.1 X I AgKOs: 0.1 M NHIOH (1: 1) is white against a brown background. Radioautograph spots matched the outline and position of stained spots exactly. RF values for benzene: 1-butano1:methanol:HzO (1:l:Z:I ) , 2-hutano1:formic acid:HzO (lQ:Z:G),acetic acid:l-butano1:ethyl acetate:H20 ( l : l : l : l )ethanol: , etber:Hs0:7.4 N NHa (4:5:1:0.1), 1-propano1:l N acetic acid (3:1), 2-propanol:NHa:H?O ( 8 : l : l ) were 0.64, 0.07, 0.68, 0.13, O.GS, 0.07, respectively. 19) €1. D. Dakin. Biorhem. J . , 1 3 , 396 (1919). LABORATORY OF CLINICAL SCIENCE NATIONAL INSTITUTE O F MENTAL DONALD D. BROWN
IIEAL~H MARIANW. KIES INSTITUTES O F HEALTH BETHESDA, MARYLAND RECEIVED JULY31, 105s NATIONAL
MICROBIOLOGICAL TRANSFORMATIONS. III.’ THE HYDROXYLATION OF STEROIDS AT C-9 Sir:
when treated with pyridine and acetic anhydride. Fermentation of I with a species of Arthobacter (B 20-17s) that converts 4-androstene-3,l’i-dionc to 1,4-androstadiene-3,17-dionein excellent yield, gave 9,lO-seco-3-hydroxy-1,3,5(10)-androstatriene9,17-dione. The latter compound was purified as its acetate, n1.p. 143.5-146’, which proved to be identical in all respects (m.p., mixed m.p., and 10)infrared) with the 9,10-seco-3-acetoxy-l,3,5( androstatriene-9,17-dione reported previously.’ Thus, the pOSitiOJlS of the three oxygen atonis in 1 were established. The Sa-configuration was assigned to the new hydroxyl group because of its molecular rotatory contribution ( A M D ~ ~ I = - l S ) 4 and because of the recent evidence that microbiologically introduced hydroxyl groups have the same configuration as the hydrogens replaced.5 I n the aromatization-degradation of 4-androstene-3,17-dione i t seems probable that this species of Nocardia6 first hydroxylates a t C-9 then introduces the AI-double bond. This is just the opposite sequence originally found with Psezidonzonns. A paper chromatographic study of the fermentawith tion of 9~u-hydroxy-4-androstene-X,l7-dione Pseiidomonas showed the formation of, a t most, only trace quantities of phenolic material. Tl-itli Pseudomonas the sequence in which the reactions occur seems to be limited. ( 4 ) T h e molecular rotatory contribution o f the Da-hydroxyl groui) A. S. IIallsworth and €1. 1%. in 38-acetoxyergostau-~a-ol was -31. Henbest, J . Chem. SOL., 4604 (19;7). T h e nlolecular rotatory COIItribution o f the new ( 8 or 9) hydroxyl group in the steroids h y J r o x y l ated with Nrlicosljliiwa piv:;ic>vntr, S f x i o r parasilictcs, Xi‘u~ov c!’i.vrflcyaiiiis and A’eurospova S I ’ U S S I ~ (Ref. 2) indicates t h e prohaliility of Sa, rather t h a n 8 p , hydruxylation. See: S. H . Eppstein, P. D. hleister, H. C. Murray and D. H. Peterson, mones,” Vol. S I V , 388 (1956), Academic Press, Inc., iiew T o r k , li,Y. However, t h e specific rotation of the previously described 88 (or 9a)-hydroxy-4-androstene-3,17-dione,la m.p. ?14-217‘o, [a][> 1 G A O (CHCIa), obtained via the hydroxylation of 11-deoxycrirtiwl wit11 11. Pirifoume, does not agree with ours. ( 5 ) (a) M. H a y a n o , &I. G u t , R. I. Dorfman, 0. K. Sebek aud D . H. Peterson, Trixs J O U K N A L . 80, 2376 (1955); (b) E. J . Corey. G. A. Gregoriou and D. €1. Peterson, ibid.,8 0 , 2338 (1928). (0) n’e have isolated another strain of Nucardia (A20 0 ) which apparently follows the alternnt e w l i i e n c e .
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We wish to report the microbiological prepara- G. D. SEARLE ASD COMPANY tion and proof of structure of 9a-hydroxy-4-andro- P. 0. Box ,5110 11. hI. Donsox I
