Microchemical Detection of Characteristic Functional Groups in Steroids Side Chains LEONARD R. AXELROD Department of Radiation Biology, University of Rochester School of Medic
Procedures for the qualitative differential determination of steroid functional groups are useful in the fields of chromatography and organic chemical analysis. Micromethods have been devised to elucidate the side chain structures of adrenal cortical and sex hormones, using GO y or less of each compound. Periodic acid, lead tetraacetate, acetic anhydride, chromium trioxide, lithium aluminum hydride, and aluminum isopropoxide, together with triphengltetrazoliuni chloride and m-dinitrobenzene, have been applied for the identification of nine side chains. These tests will be found an aid to the identification of steroid compounds found in micro quantities.
METHODS
Whatman No. 1 filter paper is cut into 3 X 1 inch strips and lt5y of the steroid is applied a t one end of the paper on the smooth side, by means of a micropipet within a circle of approximately 0.7 cm. A stream of nitrogen is used intermittently to evaporate the solvent. The various organic chemical reactions are carried out by immersion of the filter paper, bearing the steroid, into one of the reagents contained in a porcelain evaporating dish. The procedures used for the reactions are described below with alphabetical correspondence to the previously listed reagents. A. The test strip is dipped into the periodic acid reagent and placed on a glass plate. The glass is then heated on the hot plate until the paper is dry. I n this and all subsequent tests, the strip must have complete contact x-ith the glass plate. An aid in keeping the filter paper flat is to apply glass rods at both ends of the paper (anal- from the spot) diiring the drying period.
CH,OH
I
CHzOH
CH,
CH,OH
CH,OH
I
I
I
I
2.
I.
CH,
CH,
CHOH
C=O
C=O
A.H
6.
I
h.oH
2
5
4.
3.
CH,
I
3IATERIALS A S D REAGENTS
Hot Plate (Lindberg), with 3tepless control. (Unless otherwise stated, the surface temperature is about 80” C.) Glass Plates, approximately 3 X 5 inches. Glass Rods, approximately 4 inches long and ‘/a inch in diameter. Porcelain Evaporating Dishes, 6.0 t o 7 . 5 em. in diameter. Filter Paper, Whatman KO.1. Reagents. -4. PERIODIC ACID, a 3% solution of periodic acid (G Frederick Smith Chemical Co.) in methanol-I+-ater (1 to 1 by volume). This reagent is stable for a t least 3 months in a brown bottle B CHROMIC ACID. a 0 4% solution of C.P chromium trioxide in 90y0 acetic acid. The solution is usable until the reddish brown color begins to show greenish overcasts. C. L E ~ D TETRAACETATE a saturated solution of lead tetraacetate (8) in 19 volumes of glacial acetic acid and 1 volume of acetic anhydride. The reagent is stable for months in a tightlv stoppered h o r n bottle. !LYHYDRIDE, a 0.2% SOlUD. C H R O M I C N TRIOXIDE--~~CETIC tion of C.P. chromium trioxide in acetic anhydride. This reagent is usable onlr for 36 hours. E. ALUMISUMISOPROPOXIDE, a 1% solution of aluminum isopropoxide in toluene-cyclohexanone (1 t o 1 by volume). This
N. Y.
modified Oppenauer reagent is not stable because of the decomposition of the cyclohexanone and must be prepared before use. F. LITHIUM A L m m n f HYDRIDE. Finely divided lithium aluminum hydride is suspended in absolute ether (approximately 1 to 2% “solution”). The suspension is prepared freshly before use. G. TRIPHEKYLTETRAZOLIUM CHLORIDE( T P T Z ) (6),a 0.2% aqueous solution of triphenyltetrazolium chloride mixed just before use with a n equal part of 3.L)- aqueous sodium hydroxide. H. DINITROBESZEXE (S), 2 parts of a 1% methanolic solution of m-dinitrobenzene mixed with 1 part of aqueous 15% potassium hj-droxide just before use.
ULIEROUS color tc
have been devised in the field of paper chronmtogrnphy (1-6, 10-14) 16-19, 22-25) for the detection of steroid compounds. LIost of these are not specific and may give positive reactions with any fiteroid which contains one or more hydroxyl or ketone functional groups. Two of the above tests IT-hich are applicable to steroid side chains, hon-ever, are fairly selective. The dye, triphenyltetrazolium chloride (5), reacts in alkaline solution with 0-keto1 and dihydroxyacetone reducing side chains to form a red complex; also, Zimmermann’s reagent as modified by Kochaltian ( 1 3 ) and .iselrod (3) gives a violet color ivith 17-ketonrs and a blue color a-ith 3-ketones. (By convention, 0-kctol and dihydroxyacetone side chains are designated, respectively, as 20-keto, 2l-hydros>-, and 20-keto, 17,2l-dihydroxy groupings.) This paper dewribes methods of sequentially applying known chemical reactions to micro quantities (15 y ) of steroids on filter paper and thereby identif>-ing from the deiivativcs the known side chains of adrenocortical and sex hormone?. These reactions will he found helpful in the field of paper chromatography for the identifieation of micro quantities of unknown steroids and in steroid chemistry for the rapid identification of side chains in synthetic or analytical procedures.
e and Den1 try, Rochester,
I
hH .
OH A H
8.
9.
STEROID SIDE CHAINS
Figure 1. Side chains of adrenocortical and sex steroids at carbon-17 detected by appropriately applied tests Only in side chain 9 a r e isomeric forms noted
B. -4portion of the chromic acid solution is boiled, at which time the test strip is momentarily immersed in the boiling liquid and then dried on a glass plate preheated and still on the hot plate. C. After a test strip has been dipped into the lead tetraacetate reagent, i t is dried on the hot plate on a preheated glass plate until the paper turns light brown. The browning is due to the reduction of the excess lead tetraacetate by the moisture in the air. Usually the immediate area containing the unknown spot remains free of coloration.
1308
V O L U M E 2 7 , N O . 8, A U G U S T 1 9 5 5
1309
1-9
on its underside with filter paper, and returned to the hot plate until i t is dry. G. T h e strip is dipped into the reagent,, held for a few seconds, blotted on one side with filter paper, and allowed to develop on another piece of filter paper. H. This reagent is used exactly as previously described (3). R e a c t i o n rvith e i t h e r Reagents G or II is alTYays the last step in any series of reactions on a test strip.
I
Strip One
1
A
S t r i i Two
1
G
1
G Red( 1,2) I
S o color(5-9) I
RESULTS AND DISCUSSION
The above reagent.; used in proper seyuenre n-iih 15-y por1 tioiis of an unfinon-n steroid F o n p a p e r ri-ill Ppecifically i. h .I identify all of the k n o ~ nside 1 chairis of adrenocortical nnd H sex hormones. The reactions of these reagents with each of the groups representing the Violet(2) steroid side chains are known ( 7 , 9. 16, 20, 2f) and the end products represent derivatives. I n general, the schemes of analysis depend upon the following organic reactions. Of all the side chains in Figure 1, only 3 and 4 are s o color(6,8,9) Red(5) oxidized t o 1i-ketones b y I periodic acid (Reagent A ) . Strip Five Chromic acid (Reagent B) 1 C will oxidize side chains 2, 3, 4, 1 and 9 to 1T-ketones, and side G chain G to a nictliyl ketone (Scheme I, Strip 6). The lead tetraacetate then oxidizes the Red(8) S o c&x(6.0) methyl ketone t o an a-keto1 I acetate which yields a red Strip Six color with Reagent G. Al1 B though i t is known t h a t 1 chromic acid will oxidize side C chain i to a 17-ketone, under I the conditions of the test and using the reagent a8 described above, side chain T is not appreciably oxidized to a 17Red( 6 ) S o color(9) ketone in usc,ful quantities in Sc eme I. Scheme of ana-;& to determine the identity of side chains 1 to 9. these micro quantities for this Letters refer to reagents described in text. Arabic numbers refer to side test series. chains shown in Figure 1 Oxidation by lead tetraacetate (l!eagent C ) will produce a D. Chromium trioxide-acetic anhydride oxidation is carried dihydrox3--acetoneacetate and an a-keto1 acetate from side chains out by dipping the test paper into the reagent, placing i t on a 7 and 8, respectively. All others are diesimilarly oxidized (if a t glass plate, and allon-ing most of the acetic anhydride to evaporate. The plate is then transferred to the hot plate and the paper Chromium triosideacetic anhydride (Reagent D ) oxidizes side thoroughly dried. E. Oppenaiier oxidation is effected hy immersing the test chains 4 and 5 to dihydroxyacetone acetate and a-keto1 acetate strip in the rengellt,, placing i t on the hot plate 011 a Preheated side chains, respectivelJ-. Side chains 3 and G to 9 do not yield glass plate, and covering i t n-ith another glass plate. After 2 to these required structures upon oxidation and suhscquently do 3 minutes, the top ,,late is removed and the paper thoroughly dried. not react, with Reagent G. Side chains 1 and 2 are acetylated F. The lithium alumininn hydride reagent is covered and by thip reagent 1)ut are otherxipe unaltered. allowed to boil, and the strip is then momentarily immersed oppenauer oxidation (Reagent E ) produces a li-ketone only until the vigorous bubbling ceases (about 7 to 10 seconds). It is from 9. then immediate1)- placed between two glass plates and heated for Lithium aluminum hydride (Reagent F)reduces the 20-ketone 2 minutes on the hot ~h~ top is removed and the in side chains 1, 2 , 7, and 8 to hydroxyl groups. The reagpnt strip dried. The strip is then dipped into t a p water until bubbling ceases (destruction of the lithium aluminum hydride), blotted also reduces the 3-, 11-, and li-ketones of steroids. Red( 4 )
No color(3)
Strip 'Three
Strip 'Three
fi
A
c:
ANALYTICAL CHEMISTRY
1310
1-9 Triphenyltetrazolium chloride Reagent G is ieduced by Strid One the a-keto1 or dihydroxyace1 tone side chain Csed on a C strip after Reagent C, a posi1 G tive reaction has occurred if the spot turns pink-ied after 6 to 8 m i n u t e s , u s u a l l y Red( 7 , s ) No color( 1-6,9) against a very light pink I b a c k g r o u n d on the paper. Strip Two S t r i p Tn.0 The alkali in this reazent must 1 1 first hydiolyze t h e a c e t a t e E F formed a i t h Reagent C and 1 1 ii H then Reagent G reacts, and this accounts for the time lapse. Reaction M ith Reagent D on a test spot folloned by Reagent G peldq a reddish Violet( 7) KOcolor(8) Strip Three pink spot in 2 to 4 minutes 1 which is alrTays, in a positive B test, more intense 111th side 1 C chain 5 than 4 The splitting of the acetate may also account for the time lag. X o background color on the paper is found after this time. ImNo colpr( 1-5) Red(6) mediate deep coloration occurs Strip'Four n i t h side chains 1 and 2 as 1 described by Burton and coD workers ( 5 ) . 1 Although side chains 1 and G 2 may be differentiated by a previous test ( I ) , the method as described in Schemes I and I1 is very reliable in these Strip' Five microquantities and u t i l i z e s 1 already available reagents for G these tests. Dinitrobenzene (Reagent H) reacts with all the li-ketone Red( 1,2) S o color(4,5) d e r i v a t i v e s ( p r o d u c t s of Strid six Strid six Reageite A and B ) a t about 1 1 the +:me rate and t o the Follow d 8a-e extent. One exreption Scheme I 1 H is t h e r e a c t i o n b e t w e e n Reagent H and the product of Oppenauer oxidation. A4 Violet( 4) s o color(5) positive reaction occurs and a brilliant violet ensues with Scheme 11. Scheme of analysis to determine the identity of side chains 1 to 9. Letonly the slightest warming ters refer to reagents described in text. Arabic numbers refer to side chains shown in Figure 1 of the stiip and in less than a minute. Some -light disc o l o r a t i o n of t h e p a p e r , following Scheme I, the analysis of very polar side chains coloring it in parts violet-purple, may also occur at this time. is performed with no more than 45 y (three strips) whereas JIany schemes of analysis are possible in which the above 60 to 90 y (4 to 6 strips) are needpd to analyze the least polar organic reagents are compatible for successive reactions on a side chains. The reverse is true if Scheme I1 is followed, and single test strip. Schemes I and 11, however, represent two of the least polar side chains ale identified with no more than 45 the tiied and successful combinations. (three strips). For paper chromatography, it is assumed that narrow test If the relative polarity of the compound is not known, such as stiips from a chromatogram have been tested with the modified nhen the side chain resulting from a emthetic reaction is to be Zimniermann dinitrobenzene reagent to rule out immediately determined, Schemes I or I1 or modifications of these may apthe presence of a 17-ketone, before eluting the unknown compropriately be applied for its analysis. T o more than 60 y pound for differential chemical testing. (four strips) are required for the determination of any of the Schemes I and I1 are very useful for determining the identity nine side chains in these schemes. Although test strips containof the side chain of compounds after paper chromatography, since ing 15 */ each have been used uniformly throughout this investiliv chromatography the relative polarity of a compound (and gation, many of the chemical reactions can be successively and thcrefoie nn indication of its side chain polarity) is knoR-n. By
-,
V O L U M E 2 7 , NO. 8, A U G U S T 1 9 5 5 mccessfullj- exeru(ed with test strips which contain only 5 t o 6 y of starting conipound in a spot 0.7 em. in diameter. T o conserve further the compound, t h e strip may be cut in half, thus halving the spot, and each half used is a different organic chemical reaction. ACKNOWLEDGMENT
T h e author a.ishes to express his appreciation t o Carmela Santomieri and AIadeline Goldstein for their valuable technical assistnncae. This study n-as made possible by a grant from the Jane Coffin Childs llemorial Fund for Medical Research. LITERATURE CITED
(1) .kxelrod. L. 11.. J . Am. Chem. Soc.. 75, 4074 (1953). (2) Axelrod, L. I
