1198
Vol. 7 6
NOTES
terized further. The major activitv resides in a cell-free supernatant of a phosphate buffer extract, pH 7.4, of rat liver ground in a meat grinder. The activity was retained in the supernatant of an acetone powder of the ground rat livers. Dialvsis of this supernatant lowered the conversion of the 3ketone to the Sa-hydroxy group to 30yo,but with the addition of 0.002 M DPN, the conversion increased to 9570. Further studies of this reaction are in progress.
140, 1 ,50-1.51 ether.
Iraq
recrystallized from ether-petroleum
3n-Hvdroxyetiocholan-17-one by Oxidation of 3n,17n,21Trihydroxypregnan-20-one with Sodium Bismuthate .h-Tm
mg. of : h , l i.r,?l -trihydroxypregn~Ln-?O-onr was dissolved in 1 nil. of glricinl acetic acid, followed h v the addition 01 1 ml. of water to a 25-ml. erlenmeyer flaqk covered w i t l i aluminum foil. An excess of 50 mg. of sodium bismuthatv w a y added and the flask shaken for 0.5 hour in a n'arburg a t room temperature. The mixture was diluted with water, filtered and thoroughly extracted with ether. The ether solution was washed once with 1 N sodium hydroxide, fnlloirrd by water. The dried extract consisted of white Experimental6 crystalline material weighing 7 mg. (iOojoo), m.p. 140". Hydrolysis of 2 l-Acetoxy-17a-hydroxypregnane-3,20-di- Recrystaliixation from ether-petroleum ether gave needles one.-Three grams of 21-acetoxy-170-hydroxypregnane-3,- of 3ol-hvdror\-etiocholan-li-one,m . p . 140, l.50-151°. 38-Hydroxvandrostan-17-onefrom 38,17a,2 1-Trihydroxy20-dione was hydrolyzed with KHCOa in methanol a t room temperature for 24 hours to yield 2.74 g. of white allopregnan-?@-oneby Oxidation with Sodium Bismuthate .26.3 mg. of 3?, I7n,?l-trihydroxyallopregnan-20-one was discrystalline material, m.p. 185-195". The material was chromatographed on a column containing 90 g. of silica gel solved in 2 ml. of glacial acetic acid followed by the addition of 2 nil. of wxter in a 25-ml. erlenmeyer flask covered with and eluted with benzene, benzene-ethyl acetate mixtures and methanol. Elution with benzene ethyl acetate ( 2 : 1'1 aluminum foil. An excess of 101) mg. of solid sodium bisyielded 2.3 g. of crystalline material which was recrrstal- muthate was added and the flask shaken for 0.5 hour in :I \Tarburg at room temperature. The extraction with ether lized from acetone and ethanol to give crystals melting a t :IS previously described yielded ?3 mg. (8iajoo', of a white 200-202', [CY]~'D+ 6 2 O (ethanol). Incubation of 17~~,21-Dihydroxypregnane-3,20-dione (Di- crystalline product, in .p. 167-1 70'. Recrystallization from methanol yirlded 3B-h!-dros!.3tldrostan-li-one, m .p. 17% hydro S) with Rat Liver Homogenate.-The livers of adult 176O. male albino rats were quickly removed following decapitaAn attempt to oxidize a 21-desoxy analog, 38,17a-dihvtion and homogenized for 20 sec. in a Waring blender in 0.1 dros~allopregnane-2O-one, m .p. 2-12-549', with uodium bisilf phosphate buffer, pH 7.4. A total of 820 mg. of dihydro S was incubated (10 mg. of steroid per 10 g. equivalent of muthate was unsuccessful and yielded only the starting material. tissue wet weight) by shaking in air at 38' in a Warburg for 1.5 hours. Following acetone precipitation of the proteins, 18) C . J . Brooks and J. IC. Sorymberski, Chem. i- I n d . . 804 (10.52). and a 70'% methanol-petroleum ether partition to remove the lipids, the crude extract (2.77 9.) was chromatographed on ~TORCESTER FOESDATTOS FOR EXPERIMENTAL Rro~ocu a column containing 170 g. of silica gel. SHREV'SBURT, LIASSACHUSETTS Isolation of 3a,17~,21-Trihydroxypregnan-2O-one .-The various fractions of the silica gel chromatogram mere eluted with a system of benzene, benzene-ethyl acetate mixtures, Observations on the Crystalline Forms of Galactose ethyl acetate and methanol. Elution with benzene-ethyl acetate (2: 1) yielded a total of 660 mg. of crystalline prodBY 1 4 . L. ~TOLFROM, MAX S C H L A M O W A NID~ Z ~ uct. Aliquots of the various eluates were spot-checked bv A . THOMPSON~ paper partition chromatography using the toluene-propylRECEIVED OCTOBER30, 1953 ene glycol systems for four hours. The dihydro S and tetrahydro S zones were visualized with the triphenyltetrazolium In an investigation3 concerned with the examinachloride reagent. tion of the hydrolyzate from a polysaccharide, it Recrystallization of the pooled homogeneous fractions yielded 389 mg. of pure 3a,l7a,21-trihydroxypregnan-20- was necessary to recognize and identify the common one. T h e remainder of the crystalline material (271 mg.) crystalline forms of galactose. Five crystalline proved to he a mixture of unchanged substrate and tetrahydro S isomers. 3@,17a,21-Trihydroxypregnan-20-one, phases exist for this sugar. These are the racemate and the a - and /3-pyranose forms for each enantiom.p. 224-226', was isolated from this mixture (2.0% yield). 3a,l7a,21-Trihydroxypregnan-20-one was recrvstallized morph. a-D-Galactopyranose is the stable form from acetone and finally ethanol t o give small platelets, m.p. obtainable under the usual laboratory conditions. 200-204'. Various melting points were obtained x i t h different crystallization procedures, the highest melting crvs- KO record of the melting point of /3-D-galactopyrantals, obtained from ethanol, melting a t 214-2163", [ a l Z 7 D ose could be found. X sample of this material +60° (ethanol). The diacetate melted at 201-206°, was prepared according to the directions of Hudson [ a I n D f77' (ethanol). A mixture with 3a,21-diacetoxvand Yanovsky.4 I t exhibited the mutarotatory 17a-hydroxypregnan-20-one (m.p. 195202') melted a t 19.5properties reported by these authors and its X-ray 204O.7 3a-Hydroxyetiocholan-17-oneby Oxidation of 3a,21-Di- powder diffraction diagram was that recorded by acetoxy-17cu-hydroxypregnan-20-onewith Chromic Acid.%?ernerj except that we found no evidence of con40.5 mg. of 3a,21-diacetoxy-17a-hydr~~ypregnan-20-one,tamination with the a-D-anomer. This preparam.p. 201-206O, was dissolved in 4 ml. of glacial acetic acid. tion melted a t 11:3-11.5°6 when heated raFir!ly to To this was added 20 mg. of CrOs in 0.4 ml. of water and the near its melting point. On maintaining the temsolution was allowed t o stand a t room temperature for 3 hours. Excess CrOa was reduced, the mixture diluted with perature a t 160°, the substance resolidified comwater t o 50 ml., and extracted with ether exhaustively to I n some pletely and then remelted a t 164-167'. give a crude crystalline product (30.3 mg.) melting a t 80preparations an initial melting point a t 16'7-1 50' 90'. Following sodium hydroxide hydrolysis a t 50' for 1 hour, 11.2 mg. of 3a-hydroxyetiocholan-17-one, m . p . was found and under continued slow heating,
resolidification was noted a t 15'7-15,7°
with re-
(6) All melting points were taken on a Fisher-John apparatus and are uncorrected. Infrared analyses were carried out with a Perkin( I ) Postdoctoral Research Fellow of the 3-ational Institutes of Elmer model 12-C infrared spectrometer. The analyses and interHealth, United States Public Health Service. pretations were carried out by Dr. H. Rosenkrantz and hlr. P. Skog(2) Research Associate of the Corn Industries Research Foundation. strom in our laboratories. (3) SI. L. Wolfrom, G. Sutherland and SI. Schlamowitz, THIS 74, 4883 (1952). (7) B. A. Koechlin, T . H . Kritchevsky and T. F. Gallagher, THIS JOURNAL, JOURNAL, 73, 189 (1951), reported for 3a,21-diacetoxy-17a-hydroxy(4) C.S. Hudson and E. Yanovsky, i b i d . , 39, 1013 (1017). pregnan-2O-one, m.p. 205-206°, [ a ]f 8~8 O (ethanol). A sample was (5) I. Werner, Microchsmie per. Micvochim. A d a , 39, 133 (1952). generously supplied for comparison and proved to be identical as (6) All melting points are corrected and were observed in Pyrex shown by infrared analysis. glass.
Feb. 20, 1954
COMMUNICATIONS TO THE EDITOR
melting a t 164-167’. It is believed that these preparations were less pure. The resolidified phase exhibited the X-ray powder diffraction diagram recorded by Werners for a-D-galactopyranose. It is probable that an equilibrium between the two anomers was established in the melt. I n this equilibrium melt the a-D-pyranose anomer was capable of crystallizing, and as it crystallized the equilibrium in the molten part generated more of it, so that the whole melt solidified as a-D-galactopyranose, which then melted a t the higher temperature of 164-1G7’, slightly below that of the pure a-D-form, 168-170’. Equal parts of a-D-galactopyranose, m.p. 168170°, and a-L-galactopyranose, m.p. 168-170°,
1199
were crystallized by dissolving in water, concentrating to a sirup and dissolving the sirup in methanol. The resultant optically inactive, crystalline phase melted a t 145-14s’. Previously reported values are: 163’,’ 140-142°,8 143-144°.9 The X-ray powder diffraction diagram of this preparation was identical with that of a-D-galactopyranose. This racemic phase is therefore a mechanical mixture of the a-Dand a-Lanomers. (7) E. 0. von Lippmann, Bcr., 55, 3038 (1922). (8) E.Fischer and J. Hertz, ibid., 25, 1247 (1892). (9) C. Neuberg and J . Wohlgemuth, 2 . p h y s i o l . Ch ?n (1902).
36, 239
DEPARTMENT OF CHEMISTRY THEOHIO STATE UNIVERSITY 10, OHIO COLUMBUS
COMMUNICATIONS T O T H E EDITOR DISCONTINUITIES JN T H E ADSORPTION ISOTHERM OF %-HEPTANE ON MOLYBDENUM DISULFIDE
Sir :
were observed in the same pressure range. Numerous experiments with benzene and water adsorption on the same surface showed no vertical rises in the adsorption or desorption curves, but in experiments with toluene adsorbate a vertical rise a t p / p 0 = 0.003 was obtained when allowing five to ten minutes between doses. The amaratus used was similar to that described by Harkins and Jura.3
In the course of a recent investigation a t Walker Laboratory of Rensselaer Polytechnic Institute, certain interesting experimental results were found regarding the appearance of discontinuities in the adsorption isotherm of n-heptane on molybdenum disulfide. These results, heretofore referred t o in a discussion of observed transitions by Young, Beebe and Bienes,’ were not included in the original publication on water and benzene adsorption on molybdenum disulfide. Reproducible discontinuities in the adsorption b0. 0.4 M isotherm of n-heptane on molybdenum disulfide a t 26.0’ were observed a t a relative pressure near 0.01 / > o when five to ten minutes was allowed for equilibrium between doses. However, when more time was allowed after the initial and succeeding doses, the discontinuity disappeared, and a smooth isotherm, concave to the pressure axis, resulted. If the sample was reactivated and the shorter time be-0 /O i tween admission of doses resumed, the discontinuity again appeared. This was observed when the sample was previously degassed a t room temI perature as well as when “activated” a t 200’. At higher pressures (above p l p o = 0.05) the time after each dose could be shortened considerably 1 I I without effect. 0.01 0.02 0.03 0.04 The low pressure portion of an n-heptane adRelative pressure, PIP”. sorption isotherm showing the discontinuity as Fig. 1.-The absorption of n-heptane on molybdenum well as the low pressure portion of a smooth isotherm, in which several hours elapsed between the disulfide activated adsorbent 1: 0 , five to ten minutes beinitial and second dose, is shown in Fig. 1. The tween doses; 0, up to eight hours between doses. adsorbent is “activated adsorbent 1” of molybAlthough the appearance of a discontinuity in an denum disulfide, as previously described.2 The desorption isotherms showed the same appearance isotherm may be a non-equilibrium condition which of discontinuities, except that several small steps disappears in sufficient time, this does not eliminate the reality of the observation. As physical ad(1) D. M. Young, R. A. Beebe and H. Bienes, Trans. Faraday Soc., sorption is a rapid process, an adsorbate which 49, 1086 (1953).
c
do I
(2) E. V. Ballou and S. Ross, J . Phys. Chcm., 57, 653 (1953).
(3)
W.D.Harkins and G. Jura,THIS JOURNAL, 68, 1356 (1944).
