Gas-chromatographic behavior of trimethylsilyl ethers of steroids

PRELIMINARY NOTES

599

This study was supported in part by Grants A-II32, D 1197 and D I523 of the U.S. Public Health Service. We are indebted to Eli Lilly and Company for a generous gift of parathyroid extract.

Radioisotope Service, P eterans Administration Hospital and DAVID V. COHN Department of Biochemistry, University of Kansas City School BERNARD K. FORSCHER of Dentistry, Kansas City, 3Io. (U.S.A.) 1 D. M. LASKIN AND M. B. ENGEL,A. M. A. Arch. Pathol., 62 (1956) 296. G. R. MARTIN, H. E. FIRSCHEIN, B. J. MULRYAN AND W. F. NEUM&N, J. Am. Chem. Soc., 80 (I958) 62Ol. 3 E. C. LEKAN, D. i . LASKIN AND M. B. ENGEL, Am. J. Physiol., 199 (1961) 856. 4 j . E. VARNER, in S. P. COLOWlCK AND N. O. KAPLAN, Methods in Enzymology, Vol. 3, A c a d e m i c Press, Inc., N e w York, 1957, p. 397. 5 A. B. BORLE, N. NICHOLS AND G. NICHOLS, JR., J. Biol. Chem., 235 (I96O) 12o6.

Received July 24th ,

1961

Biochim. Biophys. Acta, 52 (1961) 596-599

Gas-chromatographic behavior of trimethylsilyl ethers of steroids The separation of closely related hydroxy- and keto-substituted steroids is important in many biological problems. When gas-chromatographic procedures are used, there are three ways in which effective separations may be achieved for these compounds. These are (I) the use of high-resolution columns, in which high plate efficiency is used to resolve compounds with closely related structures, (2) the use of selective phases which will separate epimeric hydroxy compounds from each other and from the

NGS 206" O3 0,..

b t~

E~-TMS; E2~iMS'/ PIREG-TMS;

r

5

i

fO

TIME (MIN) Fig. I. G a s - c h r o m a t o g r a p h i c s e p a r a t i o n of a m i x t u r e of estradiol di-(trimethylsilyl) e t h e r (EzTMSi), estriol tri-(trimethylsilyl) e t h e r (Ea-TMSi), p r e g n e n o l o n e t r i m e t h y l s i l y l e t h e r ( P R E G TMSi) a n d e s t r o n e t r i m e t h y l s i l y l e t h e r (Ez-TMSi). C o l u m n c o n d i t i o n s : 6 ft × 5 m m glass c o l u m n ; 0.75 % n e o p e n t y l glycol s u c c i n a t e p o l y e s t e r (NGS) on lOO-14o m e s h G a s - C h r o m P; 206°; 151 b/in 2.

Biochim. Biophys. Acta, 52 (1961) 599 6o1

600

PRELIMINARY NOTES TABLE 1 RELATIVE RETENTION TIMES FOR TRIMETHYLSILYL ETHERS (RELATIVE TO CHOLESTANE)

Relative rentention time

Compound*

Cholestane Cholesterol Cholestanol Epicholestanol Cholesteryl trimethylsilyl ether Cholestanyl trimethylsilyl ether Epicholestanyl trimethylsilyl ether 5~- Pregnane-3fl, 2 ofl-diol 5a-Pregnane-3fl,2 oa-diol 5~-Pregnane-2 ofl-ol-3-one

SE-.3o**

PhSi***

QF-z ~

NGS§§

i .oo i .95 2.00 i .97 2.56 2.61 2.04 0.67 0.72 0.72

i .oo 3.32 3.16 3.0(7 2.50 2.38 1.68 i .44 1.6o 1.7I

t .oo 3.39 3.7 ° 3. t8 2.28 2.4 ° 1.78 1.94 2.16 3.7 °

i .oo 6.85 6.34 5-79 2.20 2.14 1.45 6.47 7.64 7.64 6..52

5a-Pregnane-3fl-ol-zo-one

0.67

1.55

2.98

5a-Pregnane-3,2 o-dione

0.72

t .79

5.93

7.20

3fl,2ofl-Di-(trimethylsilyloxy)-5~-pregnane

i. t i

0.92

1.13

o.92

3fl,2o~-Di- (trimethylsilyloxy)-5~-pregnane 2ofl-Trimethylsilyloxy-5~-pregnane-3-one 3fl-Trimethylsilyloxy-5~-pregnane-2o-one Estrone Estradiol Estriol E s t r o n e trimethylsilyl ether Estradiol di-(trimethylsilyl) ether Estriol tri-(trimethyl) ether Pregnenolone Pregnenolone trimethylsilyl ether

1.2o 0.93 0.80 o.46 0.48 0.99 0.52 6.68 1.38 0.62 o.77

1.oo i .38 i. i i 1.98 1.83 4.77 1.21 o.82 i .54 1.57 1.17

t.27 3.04 2.o 4 2.63 t.63 4.30 J.83 0.87 1.58 2.68 1.81

I.Ol 2.8 i 2.04 I6.1 I8. 3 2.67 o.97 i .4 i 7.17 2.08

* Trimethylsilyl ethers were p r e p a r e d in t e t r a h y d r o f u r a n solution with hexamethyldisilazane (trimethylchlorosilane was used as a catalyst). Overnight s t a n d i n g at r o o m t e m p e r a t u r e resulted in complete reaction. Derivatives of cholesterol, cholestancl, epicholestanol, estrone, estradio] and estriol were isolated and h a d satisfactory elementary analyses. Chole~tanyl trimethylsilyl ether was collected after c h r o m a t o g r a p h y and found to be unchanged (infrared). Other derivatives were p r e p a r e d b y a s h o r t procedure as follows: The reaction m i x t u r e was centrifuged and the supern a t a n t liquid was removed. The solvents were removed (nitrogen), and the residue was t r i t u r a t e d with hexane. This m i x t u r e was centrifuged and the hexane solution removed. The solvent was e v a p o r a t e d (nitrogen), and the p r o d u c t dissolved in t e t r a h y d r o f u r a n for c h r o m a t o g r a p h y . Keto g r o u p s were not affected b y the silylation procedure. ** Column conditions: 0.75% silicone SE-3 o (General Electric Company) on lOO-14o mesh Gas-Chrom P; 6 I t × 4 m m ; 205°; 14 lb/in2; cholestane time lO.6 rain, *** Column conditions: i % phenylsilicone p o l y m e r 191-43 (Dow Corning Corp.) on lOO-14o mesh Gas-Chrom P; 6 ft × 4 m m ; 207°; 16 lb/in~; cholestane time 5.8 min. § Column conditions: i % fluoralkyl silicone p o l y m e r QF-I-OO65 (IOOOO CS.) (Dow-Corning Corp.) on lOO-14o m e s h Gas-Chrom P; 6 ft × 5 ram; 195°; 12 lb/inZ; cholestane time 4.2 rain. §§ Column conditions: o.75 % neopentyl glycol succinate polyester (gift from Dr. J. CORSE) on lOO-14o m e s h Gas-Chrom P; 6 f t x 5 m m ; 2 I o ° ; 15 lb/in2; cholestane time 3.8 rain.

corresponding ketones, and (3) the use of derivatives which will emphasize structural differences and which will permit a better resolution of the compounds under study. Examples of these methods are the use of a high-resolution column packed with silicone SE-3o to separate urinary I7-keto steroids 1, the use of phase fluoralkyl silicone polymer QF-I-OO65 to separate epimeric sterols and the corresponding ketones 2, and the use of trifluoracetyl derivatives of polyhydroxy steroids a. The present work describes the properties of trimethylsilyl ethers of steroids; these derivatives of Biochim. Biophys, Acta, 52 (196I) 599-6Ol

601

PRELIMINARY NOTES

phenolic and hydroxyl-substituted steroids are particularly useful in work with epimers and with the estrogens*. Table I contains the relative (to cholestane) retention times for a number of steroids and their trimethylsilyl ether derivatives. With the non-selective phase SE-3o the retention times for the ethers were somewhat greater than those for the parent compound. This is not unexpected, since separations with an SE-3o phase follow approximately the order of molecular weight. With selective phases, the retention times of the ethers are significantly less than those of the parent compound, and the effect is particularly great for polyhydroxy compounds. For example, with a neopentyl glycol succinate polyester column the di-(trimethylsilyl) ether of estradiol is eluted about 2o times faster than estradiol, and the tri-(trimethylsilyl) ether of estriol is eluted in about 5 rain under conditions where estriol itself would require m a n y hours for elution. These dramatic differences are due to the fact that the trimethylsilyl ether group behaves very much like a non-polar functional group which is sufficiently bulky to affect the molecular size and shape, but which does not show selective functional-group retention effects. Several useful properties have been observed for these derivatives. Epimer separation factors m a y be materially increased; for example, the cholesterol/ epicholestanol separation factor is 1.o3 on a phenylsilicone phase, but the value for the trimethylsilyl ethers under the same conditions is 1.42. The separation of estrone from estradiol is difficult when a non-selective phase is used; the separation is good with selective phases but estriol is difficult to chromatograph without loss with a selective phase, and the separation of all three estrogens in a mixture is difficult for this reason**. The relatively rapid elution and excellent resolution with all four phases of the three phenolic steroids as trimethylsilyl ethers suggests that these derivatives m a y be particularly useful in work with estrogens. T . L U U K K A I N E N * **

Laboratory o/Chemistry o/ Natural Products, National Heart Institute, Bethesda, Md. (U.S.A.)

W . J . A . VANDENHEUVEL E . O . A . HAAHTI§ E. C. HORNING

1 E. O. A. HA&HTI, W. J. A. VANDENHEUVEL AND E. C. HORNING, Anal. Biochem., 2 (196I) 182. 2 w . J. A. VANDENHEUVEL, E. O. A. I-~AAHTI AND E. C. HORNING, J. Am. Chem. Soc., 83 (1961) 1513. 3 W. J. A. VANDENHEUVEL, J. SJOVALL AND E. C. HORNING, Biochim. Biophys. Acta, 48 (1961) 596. 4 E. V. HEDGLEY AND W. G. OVEREND, Chem. and Ind.(London), (196o) 378. H. LANGER, P. PANTAGES AND I. WENDLER, Chem. and Ind. (London), (1958) 1664. 6 H. M. WOTIZ AND H. F. MARTIN, J. Biol. Chem., 236 (1961) 1312.

Received July 4th, 1961 * G a s - c h r o m a t o g r a p h i c p r o p e r t i e s of t r i m e t h y l s i l y l e t h e r s of c a r b o h y d r a t e s a a n d p h e n o l s 5 h a v e been r e p o r t e d . ** WOTIZ AND MARTIN 6 r e c e n t l y d e s c r i b e d t h e s e p a r a t i o n of t h e a c e t a t e s of estrone, e s t r a d i o l a n d estriol w i t h silicone grease c o l u m n s in t i m e s v a r y i n g from a few m i n u t e s a t 280 ° t o s e v e r a l hours a t 235 ° . *** U n i v e r s i t y of H e l s i n k i (Finland). § U n i v e r s i t y of T u r k u (Finland).

Biochim. Biophys. Acta, 52 (1961) 599-6Ol