Deuterization of Steroids and Their Use in Isotope Dilution Analysis STANLEY L. JONES', IAN
D. ROBINSONZ,BYRON H. ARISON,
and NELSON R. TRENNER
Research Laboratorks, Merck & Co., Inc., Rahway, N. J.
The combined filtrates were vacuum-distilled to near dryness, then the residue was dissolved in 100 ml. of methanol and permitted to stand overnight to remove labile deuterium. The methanol was finally removed by vacuum distillation and the product was recrystallized from ethyl acetate. The infrared absorption of the product was examined in the 0 - D region a t 3.98 microns to assure the absence of detectable amounts of labile deuterium of this kind. The absence of other kinds of labile deuterium is demonstrated by the data in Table 11. The F and S acetate tracers mere hydrolyzed to the corresponding alcohols for use in the analysis of broth. The hydrolysis was carried out as follows: The acetate was dissolved in purified methanol by warming to 50' C. and then cooling to 30" C. Sodium methoxide, dissolved in purified methanol, was added with stirring under nitrogen. After 9 minutes, dilute acetic acid was added until the solution was just acid to litmus. The steroid alcohol mas isolated by removing most of the methanol by vacuum distillation, adding water, and lctting the crystallization take place in the refrigerator. The product was filtered, dried, and recrystallized from ethyl acetate.
A workable and reliable isotope dilution assay for the steroids, compound S and compound F, i n fcrmentation liquors has been developed. Data on the catalytic introduction of deuterium into several new steroids, some of thein cortical hormones, are presented.
IN
STUDIES of the microbial oxidation of compound S (17-hydroxy-11-desoxycorticosterone) to compound F (17hydroxycorticosterone) in the Merck laboratories, it became necessary to know accurately the amounts of the above steroids in the fermentation liquors. Methods dependent upon quantitative recovery of the steroids from the fermentation broths presented almost insurmountable difficulties because of the complex nature of the medium. An isotope dilution technique, on the other hand, requires only that a representative pure specimen of the substance for which assay is sought be obtained. The necessity for quantitative recovery is avoided. Previous experience led to the selection of deuterium as the tracing isotope. The deuterium was introduced into the steroid molecule by a technique similar to that used by Fulrushima and Gallagher (6) in which platinum catalyst and 700/, deuterioacetic acid was used. Known amounts of deuterio compound S (17-hydroxy-1 1-desoxycorticosterone) and deuterio compound F (17-hydroxycorticosterone) were added directly to the whole fermentation liquor, intimately mixed, then extracted, separated, and purified. From the dilution of the deuterium in the isolated steroids, the original steroid content of the broth can be calculated.
The atom per cent deuterium in the final product is determined by combustion of the steroid and assay of the resulting water by infrared spectrophotometry, essentially along the lines described by Trenner, Arison, and Walker (8). Since the appearance of this article ( 8 ) , a modified procedure of greater precision and convenience has been developed (9). The same batch of catalyst was used in all experiments of Table I except experiments 3 and 4. The 70% deuterioacetic acid mas prepared by stirring for 1 hour 80 ml. of acetic anhydride and 56 ml. of deuterium oxide (99.570) a t room temperature. Quoted yields are crude products before recrystallization from ethyl acetate. COUNTERCURRENT SEPARATION OF COAIPOUNDS F AND S
REAGENTS
Compound F (hydrocortisone or 17-hydroxycorticostcrone); compound S (17-hydroxy-11-desoxycorticosterone); compound E (cortisone or 17-hydroxy-11-dehydrocorticosterone); A'-dehydro17-hydroxycorticosterone; and 4,5-dihydro-17-hydroxy-ll-dehydrocorticosterone were prepared. These compounds were all converted to the 21 acetates for deuterization to improve their stability. The deuterized F and S acetates were subsequently hydrolyzed to the corresponding alcohols for use in analysis. The catalyst, consisting of 13% platinum on acid-washed charcoal, was prepared as described by Trenner (7). Deuterioacetic acid (70% by weight) was prepared by mixing the proper proportions of blerck reagent grade acetic anhydride and 99.5% deuterium oxide obtained from the Stuart Oxygen Co. (by license from the Atomic Energy Commission). PREPARATION O F TRACERS
The steroids listed in Table I were all deuterized by the platinuni catalyst-deuterioacetic acid method previously described (1-3, 6, 6 ) . The reaction mixture consisting of steroid, catalyst, and deutcrioacetic acid, was placed in a large borosilicate glass ampoule equipped with a narrow neck and containing a magnetic stirring bar. The ampoule was immersed in a dry ice-acetone bath, evacuated, and sealed, prior to being placed in the heating bath. The reaction mixture was constantly stirred by the magnetic stirrer and heated by the vapor of a refluxing liquid of tlie appropriate boiling point.. After the proper time, the contents of the ampoule were filtered through a medium porosity sintered-glass funnel and the residue mas washed with four 25-ml. portions of acetone, each wash being allowed to stand with the residue about 30 minutes before filtering. This procedure aids in the desorption of deuterized steroid from the charcoal.
In order to separate compounds F and S alcohols, a countercurrent system using butyl alcohol-iso-octane as the upper phaee, and water-butyl alcohol as the lower phase was developed. The system is prepared by equilibrating equal volumes of 20% butyl alcohol in iso-octane (by volume) and water. At equilibrium about one third of the butyl alcohol is in the lower, aqueous phase. In this system compound S free alcohol has a distribution ratio of about 7.3 (defined as concentration in upper phase over concentration in lower phase), n-hile compound F free alcohol has a distribution ratio of about 0.57. Thus, in a five-transfer (six-tube, numbered 0, 1, 2, 3, 4, 5 ) system, moving the lower (aqueous) phase, the above steroids distribute in such a way that tube zero, it the end of the distribution, contains approximately 53% of the :ompound S alcohol and less than 1% of the compound F alcohol in the total system. Tubes 3 and 4, on the other hand, contain a i,otal of about 63% of the compoupd F alcohol, and less than 1% of the compound S alcohol (4). PROCEDURE
An amount of whole fermentation broth estimated to contain 100 mg. of compound F alcohol is chosen as the sample and is placed in a high speed (Waring) blender. One hundred milligrams each of deuterio compound F and deuterio compound S in methanol solution are added to the broth sample and the whole is ~ g i t a t e din the blender for about 20 minutes. The mixture ia then transferred t o a round-bottomed flask and reduced by Tmuum distillation t o about one fifth of its original volume. 'Thus with an original sample of 250 ml., 50 ml. or less will remain a t this point, The concentrate is now placed in the blender again and blended about 10 minutes with 500 ml. of ethylene dichloride. The slurry is separated in a separatory funnel and the ethylene di-
1 Present address, Materials and Processes Laboratory, General Electric Co., Scheneotady 5 , N. Y . * Present address, Sterling Laboratory, Yale University, New Haven, Conn.
482
V O L U M E 28, NO. 4, A P R I L 1 9 5 6
483
Table I.
used to calculate the amount of steroid originally present in the unknown sample.
Preparation of Deuterized Steroids
Weight Expt. No. 1 2 3
4 5 6
7
Steroid Added (Acetates) CompoundF Compound F Compound S CompoundS AI-Deliydro F Compound E 4,5-Dihydro E
Weight
of
Steroid Added, Grams 2.0 2.0
Catalyst, Gram 0.4
1.0 2.0 2.0 2.0 2.0
0.2 0.37 0.4 0.4
of
os
0.4
% ’ D in Deuterio- Volume D acetic Acetic Acid T2mp., Acid Added, M1. C. 70 30 82 70 30 82 GO 23 76 70 25 64 70 30 82 70 30 82 70 30 82
%D Found 8.7 8.2
Time, Hours 67 70 72 168 69 69 70
6.1 7.5 1.7 13.3 9.0
Crude Yield,
’%
E’”
;;(
=
-
1)
43 58
59 87 90
TI’,
= weight steroid origi-
nally present TPt = weight tracer added Ct = atom yo deuterium in ~
Table 11. Lability of Deuterium during Fermentation and Isolation Expt. No. 1 2 3
S Alcohol Tracer .4dded, 7% D
S Blcohol Recovered, % D
9.2 9.1
...
9.0
9.1
F Alcohol Recovered, % ’ D 8.6 8.9 8.8
Compound S alcohol, free from compound F alcohol, is recovered from the number zero separatory funnel, while compound F alcohol, substantially free from compound S alcohol, is recovered from combined tubes 3 and 4. The solvents are removed by vacuum distillation or steam bath evaporation under a stream of nitrogen. The dried steroid residues are now dissolved in a small quantity of hot ethyl acetate, treated with small portions of activated charcoal and acid-washed alumina, centrifuged, and crystallized by evaporative concentration and refrigeration.
Table 111. Effect of Blending Time on Assay Expt. No. 1 2 3 4
Time in Blender, Minutes 5 20 30 5
F Alcohd Found, Mg.
S Alcohol Found, M g .
After a second crystallization from ethyl acetate, and to effect further purification of these isolates, the steroid alcohols are converted to the acetates by the following procedure. Two tenths milliliter of dry pyridine and 0.04 ml. of acetic anhydride are added to the dry alcohol (approximately 100 mg.), and the container is placed in a heating bath maintained a t 56’ C. for 75 minutes. The mixture is then cooled, and 2.0 ml. of petroleum ether are added to precipitate the steroid acetate. The precipitated acetate is washed with petroleum ether, dried, and recrystallized twice from ethyl acetate. The final isolate is dried 2 hours a t 100” C., under vacuum. The usual yield of crystalline acetate is about 15 mg., an amount more than sufficient for combustion and infrared determination of the deuterium present in the water formed. The melting point of the final product should be consistent with that of pure material. The atom per cent deuterium, determined from the infrared absorption of the vater of combustion a t 3.98 microns (8),is corrected for the natural abundance mhere necessary, and then is
LIf
59
.~ ...
ohloridc phase is filtered on a Buchner funnel. The clear, usually yellow ethylene dichloride filtrate is washed with two 250-ml. portions of saturated sodium bicarbonate solution and finally evaporated to dryness on the steam bath under a streain of nitrogen. The dried steroid residue is now washed with two 100-ml. portions of petroleum ether, dried, and then dissolved in the number zero tube containing the 20% butyl alcohol-iso-octane and water system, being warmed if necessary to achieve solution of the solids. il five-transfer system using 100-ml. phases in 300-ml. separatory funnels has been found satisfactory for the routine separation of the F and S alcohols. The lower, aqueous phase is the moving phase, and five transfers, involving six separatory funnels, are carried out.
x W’ x Ti
85
~
C , = atom % ’ deuterium found in isolate fila = molecular weight of sample steroid M t = molecular weight of tracer steroid. Since the tracer steroids are added t o the sample as the alcohols, but isolated as the acetates, the value taken for C, must be the atom per cent deuterium calculated for the acetates, not the alcohols. EXPERIMENTAL RESULTS
The blending time for the sample and tracer was varied to determine whether or not mixing was complete. The results, all carried out on equal portions of the same sample, are shown in Table 111. They show no significant difference, indicating that even 5 minutes are sufficient for satisfactory physical equilibration. I n experiment 4,the mixtuie was heated to 100” C. after blending, and held a t that temperature for 1 hour.
Table TV. Analysis of a Iimown Broth Compound F S
Steroid Added, Rlg. 87.5 68.2
Steroid Found, blg. 90 64
Compound S alcohol containing deuterium was added to a fermentation broth a t the beginning of the fermentation, to determine whether appreciable loss of isotope took place during the actual fermentation. The results of three such experiments, shown in Table 11, indicate losses of only a few tenths atom per cent of deuterium, and are almost within experimental error. These results point up the fact that the tracers used contain no significant amounts of deuterium hbile under any of the given experimental conditions. A blank fermentation broth, fermented in the absence of added steroid, was used as a control for the over-all assay. To 250 ml. of such a blank broth known amounts of compound S and compound F alcohols were added, then the assay was carried out by the addition of tracer in the usual manner. The results of such a run, giving a good check on the reliability of the method, are shown in Table IV. LITERATURE CITED
(1) -.inlier, 11. S., Bloch, K., J . A m . Cheni. Soc. 66, 1752 (1944). (2) Bell, J., Thomson, 8. J., J. Chem. SOC.1952, 572. (3) Bloch, K., Rittenberg, D., J . Bbl. Chem. 149,505 (1943). (4) Craig, L. C., Craig, David, in Weissberger, “Technique of Organic Chemistry,” Vol. 111, p. 171, Interscience Publishers, New
York, 1950.
(5) Fukushima, D. I
