fied product with tlic system isobutyric acid-water-coriccntrated animonia ( 6 6 : 3 3 :1 ) 3 3 shotved only one spot, with IZi 0.50, w. Rf's of 0.32 for 5'-tliyniid;lic acid, 0.79 for the thymidine isostere I, and 0.69 for thymidine. 1-(cis-4-Carboxy-trans-3-hydroxycyclopentyl)-thymine (XIX).--X solution (pH 9 j of 0.361 g. ( 1 . 5 mmoles) of 1(tvuns-3-hydroxy-cis-4-h~droxymetliyl)-thymine (I), 0.318 g. (3 mmoles) of sodium carbonate and 0.758 g. ( 9 mmoles) of sodium bicarbonate in 15 ml. of water was sealed with platinum catalyst (pre-reduced, from 0.038 g. of platinum oxide) and oxygen at 35 p.s.i. into a 183-ml., stainless steel bomb. This was rocked and heated at 85-90' for 24 hours, cooled, opened, charged Lvith oxygen as before, sealed and rocked at 88-92' for another 4T hours. T h e cooled mixture was acidified and de-ionized by the careful, portionwise addition of 4.0 g. of Dowex-50 polysulfonic acid resin, a n d then filtered. T h e dark filtrate and washes were evaporated to a volunie of ca. 5 ml., adjusted to pH 7 with concentrated aqueous ammonia, allowed to stand for 2 hours, tlien filtered from 0.048 g. of a solid which had separated, i11.p. 317-319". This material gave an infrared spectrum identical with t h a t of th>-mine. T h e filtrate was adjusted to pH 8.5-9.0 with more animonia, then subjected to chro1natography~0oii a 1.6 X 27-cni. columii of Dowex-1 formate. Eluate peaks were located qualitatively by evaporating a droplet from each cut (10-12 nil.) oil filter paper; ( 3 0 ) R. ZetterstrGm and 31. Ljunggren, A c l n Chsin. Scaizd., 5 , 291 ~l !l ,?ll. fi0) I n a n earlier run this ion exchange chromatoyraphy iva5 d m e hy a gradient elution method patterned after a procedure of IT. Buscb, R . B. Hurlbert and 1 '. I a t crmcentratioiis clo\vti t o about 0 Mniolejml. were detected as dark spots when viewed und a n ultraviolet lamp with L: filter giving maximum transmission at 254 mp. Quantitative assessments of peak liomogeiieities were made after appropriate dilutions, using a Reckman DU ultraviolet spectrophotometer, then plotting cut numbers LIS. optical densities 011 graph paper. Water (cuts 1-15) eluted a peak centered a t cut 7 with a shoulder a t c u t 10. Evaporation of cuts 6-8 left a glass which t. It was then washed with ethanol, recovery) of crystals, 1n.p. 210-612". ed spectrum identical with t h a t of the starting diol I . Dilute formic acid (0.05 LV,cuts 16-29) gave a peak centered a t cut 24. Evaporation of cuts 22-26 left 0.021 g. of a solid, m.p. 315-318" dec., whicl~ gave a n infrared spectrum iclentical with t h a t of thymine. Stronger formic acid (0.55 IV,cuts 30-46) gave a, peak centered a t cut 40. Cuts 39-45 were evaporated and residual formic acid wds removed by cvaporation t o dryness with 3 X 1 ml. of water. T h e residue was heated with 6 drops of v-ater, allowed to stand and then collected by filtration; yield 0.018 g. ( 5 ( , i ) , m.p. 250-254': ::A: 273 inp ( e 9,OOC,),niiri. 238 nip ( e 1,680); ::A: 2.90 (-OH), 3.19 SII ( - S H J , 3.9, 8.20axid 8.30 (-COOH). U'IICI~thc pvtassiurn krotnide disk was ground with the calculated :mount of 0.01 S inetiianolic potassium hydroxide, then dried and reprrsw!, it g:t\-t it spectrum in which the 3.0, 2 0 and 8.20 bands ere gone and a new band a t G . 3 S p -COOK) w:ts present.
In a siniilar exlierinient with a heating period of 24 hours, the yield of hydroxy acid XIX was 1.25&,and 6774, of the starting diol I \vas recovered.
T H E MECHANISM OF ESTROGEN BIOSYNTHESIS
is outlined in the precediiig publicaticm. These two compounds were then incubated with a plaOne of the more iniportant biochemical problems cental aromatiziiig system. related to steroid nietabolisni concerns the hioExperimentai.- ---Two sets of iiiculx~tioiiswere synthetic sequence and mechanism involved i i i the carried out mith liuiiiaii placciititl iiiicrosoiiics s u p transformation of androgens to estrogens. I t has ~)leriicr~tctlwith ;L reduced trii)liospliol)~-riditlebeen shown t h a t l0-hydroxyandrost-4-e11e-Y,lir- riucleoticie (TPNII) regeneratiiig system as d e dione and lY-oxoandrost-4-ene-~,l7-dione arc scribed earlier. I n the first experiiiiciit, the excellent suhstratcs for conversion to estrogens iiicubatioiis, run with a total of lii.7 r11g. of 1, using hunirin pla.centa1 microsonic preparations were extractetl with iiielhy-lene chloride. 'rhc and arc coiisitlercd to be 011 the pathway going coiiibiiied crudc extracts were partitioiled betwxii iroiii aiitlrost-'l-eiic-:-:,1i-tlione t o cstrcigen.l I t 3 j ? , aqueous iiieth:~.nol (mobile u p l m pli:.tse) :tiit1 ;tlso Itas been I'ouiitl t h a t the lO-hy(lroxy coiii- carboil tc~tr:icliIoritlc (lo\n;c\r p l i ~ s e ) i n ;t CriiiK c o u i i t ~ ' r i ' ~ti rlistril~uticiii rr~~~ ~qiar;itus(!)!I t raiisfcrs) l m i i i i ( 1 Eoriiis both foriiialdcti~-dcaiid fiiritiic ;Icitl i l l its cciiivc~rsiiiiitci cstrogcii wliilc tlic I9-ciso struc- a i i t l csiroiic V,YI'; ~ J b t a i l l C t l from ihc : t l ) j ) i - o l r i : t l c , IIIattctii1)ting f r i ~ a l ~ c ~ sI I. I malyziiig for radioactivity, saiiii)lc. turt. yields iiiostly forrliic tit thesc facts into :i. iucchaiiistii for cstrcgrii Fori~ix- \wights werc tltti~riniiicdby direct wcigliiiig o f tioil, inforiliation coiiceriiing the stereoclicniistry crystals (iiietlicitl Aij arid by sl)ectroplioto~iietric o f the hydrogen eliniiiiatcd a t C-1 was iieccssary. analyses (method 13) using the absorption I m k at 'This conirnunication is concerned with this point. 242 ~ i i p for androst-kne-3,17-dione and thc T w o samples of androst-4-ene-3, li-dione specifi- Kober reactiori chromogen5 for estrone. In excally labeled with tritium at c-1were prepared. periment '7. the incubations containing a total of The distribution of tritium in compound I was 25% 200 p g . of I1 were extracted with methylene chlol a and 75yc 1p and in compound I1 937* 1 a and ( 3 ) H. J . Brodie, 31. Hayano a n d hf. Gut, J . A m . Chenz. SOL., 84, 746 1,B. The proof for the distribution or' tritium :3766 (1962).
Sir:
(1) T. hlorato, M. Hayano, R. I. Dorfman and I.. K. Axelrod, Oiucheriz. Biogizys. Research Cummiin., 6, 334 (19Gl). ( 2 ) I
