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Application of thin-layer chromatographic techniques to the separation of Δ16-C19-steroids and related compounds
~zv~Lm1c.41,
20,
BIoCHEXIIF~RY
Application
of to the
7745
Thin-Layer Separation Related
B. P. LISBOA
(1067)
Chromatographic of *lG-C1 &teroids
Techniques and
Compounds AiXD
R. F. PALMER
The Wenner Gren Institute, University of Stockholm; Hormonlaboratoriet, Karolinska sjukhuset, Stockholm 60, Sweden, and Department of Clinical Endocrinology, Our Lady oj Lourdes Hospital, Drogheda, Ireland
Received December 2,
1968
5a-Androst-16-en-3o-ol (~wA~~-~~-oI) has been shown to occur in human urine (1, 2) and to be excreted in increased quantities following stimulation with ACTH (2, 3) ; the characterization of other A16-unsaturated C,, steroids in pooled human urine has also been reported (4). Alfi-Unsaturated C&,-steroids have also been detected or isolated during in vitro studies, e.g., after incubation of testicular tissue with acetate (5)) testosterone (6)) and 3P-hydroxypregn-5-en-20-one (7)) human liver homogenates with testosterone (S), and adrenocortical tissue with dehydroepiandrosterone (9). In spite of these studies on the biosynthesis of the androstenol analogs, the chromatographic separation and characterization of these steroids presents serious difficulty if they occur toget.her with their saturated analogs or with other weakly polar steroids. Therefore, for the isolation of the 16-dehydro-&,-steroids, as well as for their specific determination, it was necessary to develop new analytical methods in order to obtain separation from other weakly polar steroids. By the formation of T-complexes on silver nitrate impregnated silica gel layers, it has been possible to obtain polar derivatives of the A16dehydrosteroids and, therefore, to separate them from their closely related saturated analogs. Also, the resolution of the A16-dehydro-Cssteroids has been achieved with both free steroids and as n-complex derivatives. ESPERIMESTX 1. General Ascending one-dimensional thin-layer chromatography was performed as previously described using single or multiple developments (10). In 77
name
name
Androstenedione Allopregnanedione Androstane-1784 Androstane-3or-ol Etiocholane-3&ol Androstane-384 Etiocholane-3cu-ol Androst-5-enol A%Androstene-3cu-ol A%4ndrostene-3~-ol Ar6-Etiocholene-3cu-ol Androstadienol Pregnane-3ot-ol Allopregnane3~-ol PregnS-enol Dehydroepiandrosterone A”-Dehydroepiandrosterone A4-Dehydroandrosterone
Androstan-3-one Androstan-17-one Pregnan-3-one
Trivial
5Lu-A-3-one 5cu-A-17-one 5j3-P-3-one 5a-A=-7,17-one A4-3,17-one 5a-P-3,20-one 5a-A-178-01 ~CX-A-3a-al 5@A-3j3-01 5cr-A-38-01 5j3-A-3or-01 A6-3j3-ol 5~-A’Q~-ol 5a-A=3j3-01 5j3-A’%~-ol A646-3j3-ol 5j3-P-3a-01 5orP38-01 P%q3-o1 3@-ol-As-17-one 3j3-o1-A’-17ane 3c+ol-A-417-one
Abbreviation
eh h
C
Fi a a a a e f f f f i i g
E, c
a a
EhlY!e
(anisaldehyde-sulfuric
acid)
Yellow-yellow brown Bright violet-blue violet Yellow orange-pink Olive Yellow orange-brown Yellow-red brown Blue Lilac-blue Lilac-blue Lilac-blue Lilac-blue Lilac-blue Bright-violebblue Bright violet-blue Bright violet-blue Bright violet-blue Bright violet-violet blue Bright violet-violet blue Blue Lilac-gray blue Lilac-gray blue Lilac-gray blue
Color
Acid
Sources: (a) Prof. W. Klyne, M. R. C. Steroid Collection, London, (b) Dr. J. Joska, Academy of Sciences, Prague, Czechoslovakia, (c) Prof. J. Ufer, Berlin, Germany, (d) Dipl. Ing. I. Konyves, Halsingborg, Sweden, (e) Prof. E. F. Baulieu, Faculte de Medecine, Paris, (f) Dr. D. B. Gower, Guy’s Hospital, London, (g) Dr. S. Bernstein, Pearl River, N.Y., (h) Dr. B. L.&n, Department of Experimental Biology, Worcester Foundation, Shrewsbury, Mass,, (i) Steraloids Inc., Flushing, N.Y.
5cu-Androsta&-one Scu-Androstan-17-one 5j3-Pregnan-3ane 5a-Androst-2-ene-7,17dione Androst&ene-3,17dione 5a-Pregnane-3,20dione 5a-Androstan-1784 5or-Androstan-3a-al 5,9-Androstan-3@ol 5a-Androstan-38-01 5,%Androstan-3a-01 Androstd-en3@ol 5a-Androst-16-en&-ol 5c&ndrost-16-en-3&ol 5fi-Androst-16-en-3a-al Androstad,lGdien-384 5@-Pregnan-30t-ol 5a-Pregnan-38-01 Pregn-5-en-36-01 3fl-HydroxyandrosbS-en-17-one 3,%Hydroxyandrost4en-17-one 3a-Hydroxyandrostn-17-one
Systematic
TABLE 1 Systematic Names, Trivial Names, Abbreviations, Sources, and Colors Developed with the Anisaldehyde-Sulfuric Reaction for the Steroids Used in Thii Investigation
ISOLATION
OF
16-DEHYDRO-Cl
79
,-STEROIDS
these experiments silica gel G (Merck A. G.? Darmstadt, Germany) was used as adsorbent. For the separation of Al”-unsaturated steroids after I;-complex formation, chromatoplates were coated with silica gel G impregnated with 25% silver nitrate (w/w). To obtain this level of impregnation the plates were prepared by thoroughly mixing 40 gm of silica gel with 100 ml of a 10% aqueous solution of silver nitrate; the plates were dried in air, stored away from light, and activated only before use by heating for 20 min to 95-100°C. All the reagents used were of analytical grade and the solvents were distilled before use. Rectangular tanks (21 X 21 X 9 cm, Desaga, Heidelberg) were employed and the chromatoplates were developed singiy under saturated conditions. When multiple developments were used the solvent was allowed to move to the top of the plate, i.e., to a distance of 17.5 cm from the starting line. Between two developments the tank was allowed to re-equilibrate for at least 2 hr before being re-used. The sources of the steroids used in this investigation are summarized in Table 1. 2. Detection
of the Spots
After chromatography the chromatoplates were dried in air and then sprayed with a 1% anisaldehyde solution in sulfuric acid/glacial acetic acid mixture (2/98 v/v), and heated at 95°C for 6-10 min. The colors produced for the different steroids are shown in Table 1. 3. Solvent Systems In this work the following solvent systems were empIoyed for the separation of weakly polar steroids: Ethyl acetate/cyclohexane/absolute Ethyl acetate/cyclohexane Ethyl acetate/n-hexane Ethyl acetate/benzene Benzene/absolute ethanol Benzene/absolute ethanol Chloroform/absolute ethanol
ethanol
45/45/10 50/50 25/75 50/50 95/5 98/2 98/2
(System A) (System C) (System 0)
(SystemQ) (System N) (System P)
For the separation of rr-complexes formed on silver nitrate impregnated plates, two other systems were also employed in addition to systems A, ,O, C, Q, and N: chloroform/ethanol 95/5 (System L) and ethyl acetate/ n-hexane 75/25. RESULTS In Table 2 are shown the Rf values for 22 weakly polar steroids of the C,, and C,, series in seven solvent systems; each Rf value is the average of three determinations.
80
LISBOA
AKD
PALMER
These results show that all the four saturated andostanol isomers as well as the three 16-dehydroandrostanols have been resolved on silica gel G layers; for a complete separation between 5P-androstan-S&o1 and 5@-androstan-3a-01, multiple one-dimensional chromatography with two or three runs was required, as shown using system 0 (Table 4). TABLE 2 RI Values Obtained for Twenty-Two Weakly Polar Steroids in Seven Solvent Systems with Ascending One-Dimensional Thin-Layer Chromatography on Silica Gel G (R, value is the mean of at least three experiments. For further particulars, see “Methods.“) Solvent EtAc Steroid
Sa-A-3-one 5a-A-17-one 5&P-3-one 5or-AZ-7,17-one Ae3,17-one 5a-P-3,20-one 5aA-17@01 501A-3a-al 5@A+-ol 5aA-384~1 5@A-3a-al A6-30-01 5a-A’6-3a-ol 501-A”33,%1 5j3-A’s-3a-01 A6J6-3-38-01 58-P-3a-01 5a-P-3/3-al P5-3p-01 3/3-ol-A6-17-one 3@-ol-A4-17-one 3a-o1-A4-17-one a EtAc = ethyl acetate, ane; Ba = benzene, Chlr
45
ECtYOH 45 10
0.74 0.76 0.76 0.67 0.53 0.64 0.69 0.69 0.65 0.68 0.65 0.68 0.66 0.66 0.66 0.69 0.66 0.66 0.53 0.53 0.53
systems”
EtAc cy
50 50
0.69 0.72 0.73 0.52 0.30 0.52 0.56 0.59 0.57 0.50 0.55 0.49 0.59 0.51 0.56 0.53 0.57 0.51 0.50 0.36 0.35 0.32
Cy = eyclohexane, = chloroform.
EtAo nHx
25 75
0.53 0.57 0.57 0.27 0.08 0.25 0.31 0.31 0.30 0.21 0.25 0.23 0.29 0.20 0.24 0.21 0.27 0.23 0.23 0.08 0.09 0.05 EtOH
EtAc Be
50 50
EtOH Ba
0.67 0.71 0.66
955
Bs EtOH
982
EtOH Chlr
982
0.53 0.56 0.58
G.69 0.71 0.69
0.40 0.55
0.62 0.65 0.65 0.55 0.28 0.48
0.17 0.35
0.52 0.63
0.50 0.50 0.44 0.50 0.45 0.51 0.41 0.48 0.45 0.49 0.45 0.46 0.35 0.38 0.31
0.37 0.35 0.25 0.33 0.28 0.36 0.25 0.30 0.27 0.33 0.27 0.29 0.17 0.1s 0.15
0.30 0.27 0.20 0.25 u.20 0.28 0.18 0.23 0.19 0.25 0.18 0.18 0.14 0.14 0.09
0.48 0.46 0.38 0.43 0 39 0.48 0.36 0.42 0.38 0.45 0.3s 0.39 0.33 0 ,:36 0 44
= absolute
ethanol;
nHx
= n-hex-
The chromatographic mobilities obtained on silica gel G for saturated and 16-dehydroandrostanol pairs differing in the configuration 5~~/3p-o1 and A5/3@-01 show that these steroid pairs are very difficult to separate; however, in the 16-dehydrosteroid series a complete separation of these two compounds was achieved on silica gel plates impregnated with silver nitrate, in all the solvent systems employed (Table 3).
ISOLATION
OF
81
16-DEHYDRO-‘.&-STEROIDS
On silica gel plates it was impossible to separate 16-dehydroandrostanols from the corresponding saturated compounds; separation was achieved, however, on silver nitrate impregnated plates. The Al”-steroids form a x-complex with silver nitrate which resulted in a large increase in their polarity. This permitted a very great separation from the corresponding saturated compound,, q the latter having the same mobility as when chromatographed on untreated silica gel G. The Rf values obtained under these conditions for the unsaturated steroids are summarized in Tahlc 3.
RI Values
Obtained
for (Each
,\"Sp-01 ~~~Jq3-01 5a-i\‘“-3a-01 5ol-~~‘~-:z~-ol 5@A’“&-01 P-38-01
TABLE 3 Six Unsaturated Steroids in Seven Solvent Nitrate Impregnated Silica Gel G Layers R, value is the mean of t,hree chromatograme.)
0.51 tt.09 0.10 0.14 0.06
a For abbreviations,
see footnote
0.67 0.32 0.37 0.37 0.34
to Table
0.26 0.04 0.06 0.11 0.06 0.26
0.5i 0.25 0.30 0.31 0.15
0.50 0.14 0.21 0.23 0.02;
Systems
0.45 0.12 0.15 0.18 0.07 0.4.’
on
Silver
0.24 0.03 0 .03 0 .04 0.03 0.31
2.
From the results given in Table 3 and in Figure 1 it can be seen that on silica gel-silver nitrate plates the unsaturated steroids can not only be separated from the saturated ones, but can also be resolved from each other. The two isomeric androstanones investigated here (androstan-3-one and androstan-17-one) could be partially separated on silica gel G, using solvent systems ethyl acetate/benzene 50/50 or systems of the benzene/ethanol type. They are completely resolved after two developments in solvent systems P or 0 (Table 4). Androstane-3-one and androsktne17-one also develop different colors with the anisaldehyde-sulfuric acid reagents. For t’he less polar steroids considered in this study, the following sequence of polarity has been found for the three hydroxyl groups: 3454
5 3fl(5/3) < 345p) < 3/3(Aj) < 3/3(5a).
This sequence is slightly different from that found with 17-substituted Clg-steroids and from pregnane steroids.
82
LISBOA
After formation or x-complexes, ing order of polarity:
AND
PALMER
this sequence changed to the follow-
3P(5a) < 3cr(5a) < 3P(A”) < 3a(5P). The three following steroid pairs could not be resolved: androst-5-en3/L01 and pregn-5-en-3/Sol, 5/3-pregnan-301-01 and 5&androstan-L-01, and 5a-pregnan-3P-ol and 5a-androstan-3,&01. Thus, for these steroid groups, the androstane derivative was only slightly more polar than the corresponding pregnane analog (Table 4).
------------------------------es
FIG. 1. Ascending one-dimensional thin-layer chromatography on silver nitratesilica gel G layers of four 16dehydro-C1e-steroids and the corresponding lbdihydro compounds using solvent system C (ethyl acetate/cyclohexane 50/50). Steroids: (a) androst&en-3/3-01, (b) androst-5,16dien-3/3-01, (c) 5a-androstan-3a-01, (d) 5arandrost-16en-3&ol, (e) Fwandrostan-3/3-01, (f) 5/3-androst-M-en&-01, (g) 5~ androst-16-en-k-01.
DISCUSSION
In the present investigation thin-layer chromatography has been employed to separate very weakly polar steroids of the CID and Czl series. Most of these monohydroxylated, monoketonic, and diketonic steroids
ISOLATIOS
OF
x4
16-DEHYDRO-C1,-sTEROIn’
are isomers, or differ from each other by the presence of only one lsolated bond. Their chromatographic mobilities are greater than those of androstenedione and progesterone in the systems of cyclohexane/ 01 n-hexane/ethyl acetate type (e.g., solvent systems 0, C, and -4) (10. 11) ; this permitted a group separation in these systems between the very weakly polar steroids considered here and t’hose of greater polarity such as the monohydroxy-monoketonic steroids. TABLE 4 Mobility of Twenty Weakly Polar Steroids on Silica Gel G Obtained in Three Solvent Systems Using Ascending One-Dimensional Multiple Thin-Layer Chromatography, Following Two Developments ( T T j (For the composition of the solvent systems and further part.ic\dars, see “Methods.“, hkhility Steroid
5a-A-3-one 5a-A-17-one 5@P-3-one A”-3,17-one 58-A-38-01 5a-A-3a-01 5@-A-3or-01 501-A-3,9-01 AK-38-01 5a-A16-3a-01 A5J6-3/3-01 5&A16-301-ol 5c~-A’~-3&01 58-P3ot-01 5o(-P-3P-u1 P63&01 3Lu-o1-A’-17-one 3@ol-A4-17-one 3&ol-AE-17-one 3/3-ol-P”-20-onea a3fi-ITydroxypregn-Fen-2O-one Center, Palo Alto, Calif.
rtt
of solvent
nystems ctt
ott
13.9 14.4 14.4 6.3 8.7 9.6 8.2 6.7 7.2 9.2 7.1 8.1 6.9 8.4 6.9 7.4 3.9 4.9 4.8 5.4 obtained
(in cm)
13.8 14.0 14.1 8.0 12.1 12.2 Il.7 I0.S 11.4 Il.9 10.9 11.5 10.9
13.1
13.9 13.9 2.8 9.3 9 .6
of Dr.
1:. Kincl,
I1.Q
11.0 11.6 7.4 s.5 s.4 9.4 Synt,ex
Research
In the systems specially developed for the steroids investigated here, separation can normally be achieved after a single development but, the use of one-dimensional multiple chromatography permitted a better separation of some steroid pairs; for instance, 5a-androstan-3a-ol and 5/3-androstan-3b-ol were completely resolved after two runs in sp&em P on silica gel G. The separation of A5-3&hydroxysteroid and its related 3@hydroxy$acompound (e.g., dehydroepiandrosterone and epiandrosterone) has been
84
LISBOA
AND
PALMER
reported to be difficult on thin-layer chromatography (12) even when multiple chromatography was employed (13, 14). Indeed both 5a-androstan-3p-ol/androst-5-en-3fi-o1 and &-pregnan-3/3-ol/pregn-5-en-3@-ol steroid pairs have been completely resolved by multiple one-dimensional chromatography in systems P or C. Using the conventional techniques of thin-layer chromatography on silica gel G, Gower (15) could not separate androstanedienol and A16androstene-3&ol as free steroids in any one of the systems employed, and even after four runs their separation was incomplete. Neither was it possible to separate these compounds on silicic acid impregnated paper either as free or as acetylated compounds. Also with gas-liquid chromatography after trimethyl silyl ether formation using T S-1265 as stationary phase (4), only a small degree of separation could be achieved. The separation of these two steroids, partially achieved on silica gel G in solvent system Q, was complete when silver nitrate-silica gel G layers were used. Thus the data presented in this paper on the chromatographic mobilities of very weakly polar steroids can be used in conjunction with data in previously published papers (10, 11, 13, 16) to allow separation of complex mixtures of weakly polar steroids and those of greater polarity. SUMMARY
The behavior of weakly polar steroids and their corresponding A’“compounds on thin-layer chromatography was studied. A method is presented for the separation of the four androstanol isomers as well as a complete separation of these from AIs -compounds using a-complex formation on AgN03 treated chromatoplates. This technique also permitted complete separation of the four A1%ompounds from each other. The results are compared to those obtained by the use of untreated plates and to both reverse-phase chromatography on paper and gas-liquid chromatography. ACKNOWLEDGMENTS For a generous supply of the steroids used in this work, the authors are greatly indebted to Prof. E. E. Baulieu, Faculte de MBdecine, Paris, France; Dr. S. Bernstein, American Cyanamid Co., Pearl River, N. Y.; Drs. D. B. Gower and B. W. L. Brooksbank, Guy’s Hospital, London, England; Dr. J. Joska, Academy of Sciences, Prague, Czechoslovakia; Prof. W. Klyne, M. R. C. Steroid Collection, London, England; Dipl. Ing. I. Konyves, Leo AB, Hiilsingborg, Sweden; Dr. J. Ufer, Schering AG, Berlin, Germany; Dr. F. Kincl, Syntex Research Center, Palo Alto, Calif. For a personal grant in Reproductive Endocrinology, one of us (B.P.L.) is pleased to express his gratitude to Schering AG, Berlin, Germany. Part of the expenses of this investigation were defrayed by Hoffmann-La Roche, Basle, in a grant to one of us (R.F.P.).
ISOLATIOA-
OF
16-DEHYDRO-C,
x.-l
,-STEROIDS
REFERE&CES 1. BROOKSBANK,
B.
W.
L.,
.IND
HASLE~OOD,
2. BROOKSBANK, B. W. L., J. Endocrinol. 3. PALMER, R. F., Ph.D. Thesis, University 4.
5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
15. 16.
G.
24,
A.
D.,
LJZOCILCMI.
1. 80,
488
(1961~
435 (1962). of Birmingham, 1963. BRM)KSBANK, B. W. L., AND GOTVER, D. B., Steroids 4, 787 (1964). GOWEH, D. B., AND HASLEWOOD, G. A. D., J. Endocrird. 23, 253 (1961). STYLIANOG, M., FORCHIELLI, E., AND DORFM~W, R. 1.: J. Viol. Ckenr 236, 131X (1961). AHMAD, N., AND GOWER, D. B., Biochem. J. 98, 25 (1966). STYLUNOU, M., FORCHIELLI, E., TUMILLO, M.. .~sI) DORFXIS, 8.. I., 1. Blol. Chem. 236, 692 (1961). GOWER, D. B., J. Endocrinol. 96, 173 (1963). LISBOA, B. P., Steroids 6, 605 (1965. LISBOA, B. P.. J. Chromatog. 19, 81 (1965). CORNY, V., .Jos~a, J. AND L.~BLER, L., Collection Czech. Chern. Comw~tcn. 26. 1658 (1961). LISBOA, B. I’., J. Chromatog. 19, 333 (1965). GALLETII, F., Research on Steroids, Vol. 2, p. 189. 11 Ppnsiero Hcient,ifico, Rome,. 1965. GOWER, D. U.: J. Chromatog. 14, 424 (1964). LISBOA, B. P., (‘Zin. Chim. Acta 13, 179 (19661.
20,
BIoCHEXIIF~RY
Application
of to the
7745
Thin-Layer Separation Related
B. P. LISBOA
(1067)
Chromatographic of *lG-C1 &teroids
Techniques and
Compounds AiXD
R. F. PALMER
The Wenner Gren Institute, University of Stockholm; Hormonlaboratoriet, Karolinska sjukhuset, Stockholm 60, Sweden, and Department of Clinical Endocrinology, Our Lady oj Lourdes Hospital, Drogheda, Ireland
Received December 2,
1968
5a-Androst-16-en-3o-ol (~wA~~-~~-oI) has been shown to occur in human urine (1, 2) and to be excreted in increased quantities following stimulation with ACTH (2, 3) ; the characterization of other A16-unsaturated C,, steroids in pooled human urine has also been reported (4). Alfi-Unsaturated C&,-steroids have also been detected or isolated during in vitro studies, e.g., after incubation of testicular tissue with acetate (5)) testosterone (6)) and 3P-hydroxypregn-5-en-20-one (7)) human liver homogenates with testosterone (S), and adrenocortical tissue with dehydroepiandrosterone (9). In spite of these studies on the biosynthesis of the androstenol analogs, the chromatographic separation and characterization of these steroids presents serious difficulty if they occur toget.her with their saturated analogs or with other weakly polar steroids. Therefore, for the isolation of the 16-dehydro-&,-steroids, as well as for their specific determination, it was necessary to develop new analytical methods in order to obtain separation from other weakly polar steroids. By the formation of T-complexes on silver nitrate impregnated silica gel layers, it has been possible to obtain polar derivatives of the A16dehydrosteroids and, therefore, to separate them from their closely related saturated analogs. Also, the resolution of the A16-dehydro-Cssteroids has been achieved with both free steroids and as n-complex derivatives. ESPERIMESTX 1. General Ascending one-dimensional thin-layer chromatography was performed as previously described using single or multiple developments (10). In 77
name
name
Androstenedione Allopregnanedione Androstane-1784 Androstane-3or-ol Etiocholane-3&ol Androstane-384 Etiocholane-3cu-ol Androst-5-enol A%Androstene-3cu-ol A%4ndrostene-3~-ol Ar6-Etiocholene-3cu-ol Androstadienol Pregnane-3ot-ol Allopregnane3~-ol PregnS-enol Dehydroepiandrosterone A”-Dehydroepiandrosterone A4-Dehydroandrosterone
Androstan-3-one Androstan-17-one Pregnan-3-one
Trivial
5Lu-A-3-one 5cu-A-17-one 5j3-P-3-one 5a-A=-7,17-one A4-3,17-one 5a-P-3,20-one 5a-A-178-01 ~CX-A-3a-al 5@A-3j3-01 5cr-A-38-01 5j3-A-3or-01 A6-3j3-ol 5~-A’Q~-ol 5a-A=3j3-01 5j3-A’%~-ol A646-3j3-ol 5j3-P-3a-01 5orP38-01 P%q3-o1 3@-ol-As-17-one 3j3-o1-A’-17ane 3c+ol-A-417-one
Abbreviation
eh h
C
Fi a a a a e f f f f i i g
E, c
a a
EhlY!e
(anisaldehyde-sulfuric
acid)
Yellow-yellow brown Bright violet-blue violet Yellow orange-pink Olive Yellow orange-brown Yellow-red brown Blue Lilac-blue Lilac-blue Lilac-blue Lilac-blue Lilac-blue Bright-violebblue Bright violet-blue Bright violet-blue Bright violet-blue Bright violet-violet blue Bright violet-violet blue Blue Lilac-gray blue Lilac-gray blue Lilac-gray blue
Color
Acid
Sources: (a) Prof. W. Klyne, M. R. C. Steroid Collection, London, (b) Dr. J. Joska, Academy of Sciences, Prague, Czechoslovakia, (c) Prof. J. Ufer, Berlin, Germany, (d) Dipl. Ing. I. Konyves, Halsingborg, Sweden, (e) Prof. E. F. Baulieu, Faculte de Medecine, Paris, (f) Dr. D. B. Gower, Guy’s Hospital, London, (g) Dr. S. Bernstein, Pearl River, N.Y., (h) Dr. B. L.&n, Department of Experimental Biology, Worcester Foundation, Shrewsbury, Mass,, (i) Steraloids Inc., Flushing, N.Y.
5cu-Androsta&-one Scu-Androstan-17-one 5j3-Pregnan-3ane 5a-Androst-2-ene-7,17dione Androst&ene-3,17dione 5a-Pregnane-3,20dione 5a-Androstan-1784 5or-Androstan-3a-al 5,9-Androstan-3@ol 5a-Androstan-38-01 5,%Androstan-3a-01 Androstd-en3@ol 5a-Androst-16-en&-ol 5c&ndrost-16-en-3&ol 5fi-Androst-16-en-3a-al Androstad,lGdien-384 5@-Pregnan-30t-ol 5a-Pregnan-38-01 Pregn-5-en-36-01 3fl-HydroxyandrosbS-en-17-one 3,%Hydroxyandrost4en-17-one 3a-Hydroxyandrostn-17-one
Systematic
TABLE 1 Systematic Names, Trivial Names, Abbreviations, Sources, and Colors Developed with the Anisaldehyde-Sulfuric Reaction for the Steroids Used in Thii Investigation
ISOLATION
OF
16-DEHYDRO-Cl
79
,-STEROIDS
these experiments silica gel G (Merck A. G.? Darmstadt, Germany) was used as adsorbent. For the separation of Al”-unsaturated steroids after I;-complex formation, chromatoplates were coated with silica gel G impregnated with 25% silver nitrate (w/w). To obtain this level of impregnation the plates were prepared by thoroughly mixing 40 gm of silica gel with 100 ml of a 10% aqueous solution of silver nitrate; the plates were dried in air, stored away from light, and activated only before use by heating for 20 min to 95-100°C. All the reagents used were of analytical grade and the solvents were distilled before use. Rectangular tanks (21 X 21 X 9 cm, Desaga, Heidelberg) were employed and the chromatoplates were developed singiy under saturated conditions. When multiple developments were used the solvent was allowed to move to the top of the plate, i.e., to a distance of 17.5 cm from the starting line. Between two developments the tank was allowed to re-equilibrate for at least 2 hr before being re-used. The sources of the steroids used in this investigation are summarized in Table 1. 2. Detection
of the Spots
After chromatography the chromatoplates were dried in air and then sprayed with a 1% anisaldehyde solution in sulfuric acid/glacial acetic acid mixture (2/98 v/v), and heated at 95°C for 6-10 min. The colors produced for the different steroids are shown in Table 1. 3. Solvent Systems In this work the following solvent systems were empIoyed for the separation of weakly polar steroids: Ethyl acetate/cyclohexane/absolute Ethyl acetate/cyclohexane Ethyl acetate/n-hexane Ethyl acetate/benzene Benzene/absolute ethanol Benzene/absolute ethanol Chloroform/absolute ethanol
ethanol
45/45/10 50/50 25/75 50/50 95/5 98/2 98/2
(System A) (System C) (System 0)
(SystemQ) (System N) (System P)
For the separation of rr-complexes formed on silver nitrate impregnated plates, two other systems were also employed in addition to systems A, ,O, C, Q, and N: chloroform/ethanol 95/5 (System L) and ethyl acetate/ n-hexane 75/25. RESULTS In Table 2 are shown the Rf values for 22 weakly polar steroids of the C,, and C,, series in seven solvent systems; each Rf value is the average of three determinations.
80
LISBOA
AKD
PALMER
These results show that all the four saturated andostanol isomers as well as the three 16-dehydroandrostanols have been resolved on silica gel G layers; for a complete separation between 5P-androstan-S&o1 and 5@-androstan-3a-01, multiple one-dimensional chromatography with two or three runs was required, as shown using system 0 (Table 4). TABLE 2 RI Values Obtained for Twenty-Two Weakly Polar Steroids in Seven Solvent Systems with Ascending One-Dimensional Thin-Layer Chromatography on Silica Gel G (R, value is the mean of at least three experiments. For further particulars, see “Methods.“) Solvent EtAc Steroid
Sa-A-3-one 5a-A-17-one 5&P-3-one 5or-AZ-7,17-one Ae3,17-one 5a-P-3,20-one 5aA-17@01 501A-3a-al 5@A+-ol 5aA-384~1 5@A-3a-al A6-30-01 5a-A’6-3a-ol 501-A”33,%1 5j3-A’s-3a-01 A6J6-3-38-01 58-P-3a-01 5a-P-3/3-al P5-3p-01 3/3-ol-A6-17-one 3@-ol-A4-17-one 3a-o1-A4-17-one a EtAc = ethyl acetate, ane; Ba = benzene, Chlr
45
ECtYOH 45 10
0.74 0.76 0.76 0.67 0.53 0.64 0.69 0.69 0.65 0.68 0.65 0.68 0.66 0.66 0.66 0.69 0.66 0.66 0.53 0.53 0.53
systems”
EtAc cy
50 50
0.69 0.72 0.73 0.52 0.30 0.52 0.56 0.59 0.57 0.50 0.55 0.49 0.59 0.51 0.56 0.53 0.57 0.51 0.50 0.36 0.35 0.32
Cy = eyclohexane, = chloroform.
EtAo nHx
25 75
0.53 0.57 0.57 0.27 0.08 0.25 0.31 0.31 0.30 0.21 0.25 0.23 0.29 0.20 0.24 0.21 0.27 0.23 0.23 0.08 0.09 0.05 EtOH
EtAc Be
50 50
EtOH Ba
0.67 0.71 0.66
955
Bs EtOH
982
EtOH Chlr
982
0.53 0.56 0.58
G.69 0.71 0.69
0.40 0.55
0.62 0.65 0.65 0.55 0.28 0.48
0.17 0.35
0.52 0.63
0.50 0.50 0.44 0.50 0.45 0.51 0.41 0.48 0.45 0.49 0.45 0.46 0.35 0.38 0.31
0.37 0.35 0.25 0.33 0.28 0.36 0.25 0.30 0.27 0.33 0.27 0.29 0.17 0.1s 0.15
0.30 0.27 0.20 0.25 u.20 0.28 0.18 0.23 0.19 0.25 0.18 0.18 0.14 0.14 0.09
0.48 0.46 0.38 0.43 0 39 0.48 0.36 0.42 0.38 0.45 0.3s 0.39 0.33 0 ,:36 0 44
= absolute
ethanol;
nHx
= n-hex-
The chromatographic mobilities obtained on silica gel G for saturated and 16-dehydroandrostanol pairs differing in the configuration 5~~/3p-o1 and A5/3@-01 show that these steroid pairs are very difficult to separate; however, in the 16-dehydrosteroid series a complete separation of these two compounds was achieved on silica gel plates impregnated with silver nitrate, in all the solvent systems employed (Table 3).
ISOLATION
OF
81
16-DEHYDRO-‘.&-STEROIDS
On silica gel plates it was impossible to separate 16-dehydroandrostanols from the corresponding saturated compounds; separation was achieved, however, on silver nitrate impregnated plates. The Al”-steroids form a x-complex with silver nitrate which resulted in a large increase in their polarity. This permitted a very great separation from the corresponding saturated compound,, q the latter having the same mobility as when chromatographed on untreated silica gel G. The Rf values obtained under these conditions for the unsaturated steroids are summarized in Tahlc 3.
RI Values
Obtained
for (Each
,\"Sp-01 ~~~Jq3-01 5a-i\‘“-3a-01 5ol-~~‘~-:z~-ol 5@A’“&-01 P-38-01
TABLE 3 Six Unsaturated Steroids in Seven Solvent Nitrate Impregnated Silica Gel G Layers R, value is the mean of t,hree chromatograme.)
0.51 tt.09 0.10 0.14 0.06
a For abbreviations,
see footnote
0.67 0.32 0.37 0.37 0.34
to Table
0.26 0.04 0.06 0.11 0.06 0.26
0.5i 0.25 0.30 0.31 0.15
0.50 0.14 0.21 0.23 0.02;
Systems
0.45 0.12 0.15 0.18 0.07 0.4.’
on
Silver
0.24 0.03 0 .03 0 .04 0.03 0.31
2.
From the results given in Table 3 and in Figure 1 it can be seen that on silica gel-silver nitrate plates the unsaturated steroids can not only be separated from the saturated ones, but can also be resolved from each other. The two isomeric androstanones investigated here (androstan-3-one and androstan-17-one) could be partially separated on silica gel G, using solvent systems ethyl acetate/benzene 50/50 or systems of the benzene/ethanol type. They are completely resolved after two developments in solvent systems P or 0 (Table 4). Androstane-3-one and androsktne17-one also develop different colors with the anisaldehyde-sulfuric acid reagents. For t’he less polar steroids considered in this study, the following sequence of polarity has been found for the three hydroxyl groups: 3454
5 3fl(5/3) < 345p) < 3/3(Aj) < 3/3(5a).
This sequence is slightly different from that found with 17-substituted Clg-steroids and from pregnane steroids.
82
LISBOA
After formation or x-complexes, ing order of polarity:
AND
PALMER
this sequence changed to the follow-
3P(5a) < 3cr(5a) < 3P(A”) < 3a(5P). The three following steroid pairs could not be resolved: androst-5-en3/L01 and pregn-5-en-3/Sol, 5/3-pregnan-301-01 and 5&androstan-L-01, and 5a-pregnan-3P-ol and 5a-androstan-3,&01. Thus, for these steroid groups, the androstane derivative was only slightly more polar than the corresponding pregnane analog (Table 4).
------------------------------es
FIG. 1. Ascending one-dimensional thin-layer chromatography on silver nitratesilica gel G layers of four 16dehydro-C1e-steroids and the corresponding lbdihydro compounds using solvent system C (ethyl acetate/cyclohexane 50/50). Steroids: (a) androst&en-3/3-01, (b) androst-5,16dien-3/3-01, (c) 5a-androstan-3a-01, (d) 5arandrost-16en-3&ol, (e) Fwandrostan-3/3-01, (f) 5/3-androst-M-en&-01, (g) 5~ androst-16-en-k-01.
DISCUSSION
In the present investigation thin-layer chromatography has been employed to separate very weakly polar steroids of the CID and Czl series. Most of these monohydroxylated, monoketonic, and diketonic steroids
ISOLATIOS
OF
x4
16-DEHYDRO-C1,-sTEROIn’
are isomers, or differ from each other by the presence of only one lsolated bond. Their chromatographic mobilities are greater than those of androstenedione and progesterone in the systems of cyclohexane/ 01 n-hexane/ethyl acetate type (e.g., solvent systems 0, C, and -4) (10. 11) ; this permitted a group separation in these systems between the very weakly polar steroids considered here and t’hose of greater polarity such as the monohydroxy-monoketonic steroids. TABLE 4 Mobility of Twenty Weakly Polar Steroids on Silica Gel G Obtained in Three Solvent Systems Using Ascending One-Dimensional Multiple Thin-Layer Chromatography, Following Two Developments ( T T j (For the composition of the solvent systems and further part.ic\dars, see “Methods.“, hkhility Steroid
5a-A-3-one 5a-A-17-one 5@P-3-one A”-3,17-one 58-A-38-01 5a-A-3a-01 5@-A-3or-01 501-A-3,9-01 AK-38-01 5a-A16-3a-01 A5J6-3/3-01 5&A16-301-ol 5c~-A’~-3&01 58-P3ot-01 5o(-P-3P-u1 P63&01 3Lu-o1-A’-17-one 3@ol-A4-17-one 3&ol-AE-17-one 3/3-ol-P”-20-onea a3fi-ITydroxypregn-Fen-2O-one Center, Palo Alto, Calif.
rtt
of solvent
nystems ctt
ott
13.9 14.4 14.4 6.3 8.7 9.6 8.2 6.7 7.2 9.2 7.1 8.1 6.9 8.4 6.9 7.4 3.9 4.9 4.8 5.4 obtained
(in cm)
13.8 14.0 14.1 8.0 12.1 12.2 Il.7 I0.S 11.4 Il.9 10.9 11.5 10.9
13.1
13.9 13.9 2.8 9.3 9 .6
of Dr.
1:. Kincl,
I1.Q
11.0 11.6 7.4 s.5 s.4 9.4 Synt,ex
Research
In the systems specially developed for the steroids investigated here, separation can normally be achieved after a single development but, the use of one-dimensional multiple chromatography permitted a better separation of some steroid pairs; for instance, 5a-androstan-3a-ol and 5/3-androstan-3b-ol were completely resolved after two runs in sp&em P on silica gel G. The separation of A5-3&hydroxysteroid and its related 3@hydroxy$acompound (e.g., dehydroepiandrosterone and epiandrosterone) has been
84
LISBOA
AND
PALMER
reported to be difficult on thin-layer chromatography (12) even when multiple chromatography was employed (13, 14). Indeed both 5a-androstan-3p-ol/androst-5-en-3fi-o1 and &-pregnan-3/3-ol/pregn-5-en-3@-ol steroid pairs have been completely resolved by multiple one-dimensional chromatography in systems P or C. Using the conventional techniques of thin-layer chromatography on silica gel G, Gower (15) could not separate androstanedienol and A16androstene-3&ol as free steroids in any one of the systems employed, and even after four runs their separation was incomplete. Neither was it possible to separate these compounds on silicic acid impregnated paper either as free or as acetylated compounds. Also with gas-liquid chromatography after trimethyl silyl ether formation using T S-1265 as stationary phase (4), only a small degree of separation could be achieved. The separation of these two steroids, partially achieved on silica gel G in solvent system Q, was complete when silver nitrate-silica gel G layers were used. Thus the data presented in this paper on the chromatographic mobilities of very weakly polar steroids can be used in conjunction with data in previously published papers (10, 11, 13, 16) to allow separation of complex mixtures of weakly polar steroids and those of greater polarity. SUMMARY
The behavior of weakly polar steroids and their corresponding A’“compounds on thin-layer chromatography was studied. A method is presented for the separation of the four androstanol isomers as well as a complete separation of these from AIs -compounds using a-complex formation on AgN03 treated chromatoplates. This technique also permitted complete separation of the four A1%ompounds from each other. The results are compared to those obtained by the use of untreated plates and to both reverse-phase chromatography on paper and gas-liquid chromatography. ACKNOWLEDGMENTS For a generous supply of the steroids used in this work, the authors are greatly indebted to Prof. E. E. Baulieu, Faculte de MBdecine, Paris, France; Dr. S. Bernstein, American Cyanamid Co., Pearl River, N. Y.; Drs. D. B. Gower and B. W. L. Brooksbank, Guy’s Hospital, London, England; Dr. J. Joska, Academy of Sciences, Prague, Czechoslovakia; Prof. W. Klyne, M. R. C. Steroid Collection, London, England; Dipl. Ing. I. Konyves, Leo AB, Hiilsingborg, Sweden; Dr. J. Ufer, Schering AG, Berlin, Germany; Dr. F. Kincl, Syntex Research Center, Palo Alto, Calif. For a personal grant in Reproductive Endocrinology, one of us (B.P.L.) is pleased to express his gratitude to Schering AG, Berlin, Germany. Part of the expenses of this investigation were defrayed by Hoffmann-La Roche, Basle, in a grant to one of us (R.F.P.).
ISOLATIOA-
OF
16-DEHYDRO-C,
x.-l
,-STEROIDS
REFERE&CES 1. BROOKSBANK,
B.
W.
L.,
.IND
HASLE~OOD,
2. BROOKSBANK, B. W. L., J. Endocrinol. 3. PALMER, R. F., Ph.D. Thesis, University 4.
5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
15. 16.
G.
24,
A.
D.,
LJZOCILCMI.
1. 80,
488
(1961~
435 (1962). of Birmingham, 1963. BRM)KSBANK, B. W. L., AND GOTVER, D. B., Steroids 4, 787 (1964). GOWEH, D. B., AND HASLEWOOD, G. A. D., J. Endocrird. 23, 253 (1961). STYLIANOG, M., FORCHIELLI, E., AND DORFM~W, R. 1.: J. Viol. Ckenr 236, 131X (1961). AHMAD, N., AND GOWER, D. B., Biochem. J. 98, 25 (1966). STYLUNOU, M., FORCHIELLI, E., TUMILLO, M.. .~sI) DORFXIS, 8.. I., 1. Blol. Chem. 236, 692 (1961). GOWER, D. B., J. Endocrinol. 96, 173 (1963). LISBOA, B. P., Steroids 6, 605 (1965. LISBOA, B. P.. J. Chromatog. 19, 81 (1965). CORNY, V., .Jos~a, J. AND L.~BLER, L., Collection Czech. Chern. Comw~tcn. 26. 1658 (1961). LISBOA, B. I’., J. Chromatog. 19, 333 (1965). GALLETII, F., Research on Steroids, Vol. 2, p. 189. 11 Ppnsiero Hcient,ifico, Rome,. 1965. GOWER, D. U.: J. Chromatog. 14, 424 (1964). LISBOA, B. P., (‘Zin. Chim. Acta 13, 179 (19661.