Androgen receptor affinity chromatography: Synthesis and properties of 17α-epoxypropyl- dihydrotestosterone sepharose

J. steroid Biochem. Vol. 24, No. 6, pp. Printed in Great Britain.

1111-1115,1986

0022-4731/86 $3.00 + 0.00 Pergamon Journals Ltd

ANDROGEN RECEPTOR AFFINITY CHROMATOGRAPHY: SYNTHESIS AND PROPERTIES OF 17a-EPOXYPROPYLDIHYDROTESTOSTERONE SEPHAROSE” SUPRABHAT RAY?& MOHAMMADSALMAN~,ANDREWA. RuIzt, PHILIP L. STOTTER~ and GARY C. CHAMNESS~~ TDepartment of Medicine/Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78284 and SDivision of Earth and Physical Sciences, University of Texas at San Antonio, San Antonio, TX 78285, USA (Received 2 December

1985)

Summary-We have prepared a new affinity chromatography reagent, 17a-epoxypropyl-dihydrotestosterone linked to Thiopropyl-Sepharose, with potential for use in purification of androgen receptor and other specific androgen binding proteins. The linkage is stable, and the ligand has reasonably high affinity for the receptor. Starting with Su-androstane-3/?-ol-17-one, we synthesized in two steps 17a-allyl-dihydrotestosterone, which was then oxidized to 17u-epoxypropyl-DHT yielding 2 diastereomers in about a 4:1 ratio. The 17cr-allyl-DHT had about 50% of DHT’s affinity for rat uterine androgen receptor, while the affinity of the major epoxide isomer was 9% and that of the minor isomer was 4%. Reaction of the epoxides with Thiopropyl-Sepharose-6B gave about 7 pmol of covalently bound DHT per ml of beads. These beads took up 83% of the androgen receptor from a rat uterine cytosol in a preliminary study, which more than equalled the performance of identically prepared estradiol beads successfully used for estrogen receptor purification. The use of the new DHT beads in purifications of the androgen receptor and other binding proteins is now being explored by other laboratories.

INTRODUCTION Study of the function and properties of the androgen receptor has been hampered by the absence of a convenient purification method. Affinity chromatography seems by far the most promising approach to effective purification, but requires synthesis of an androgen derivative which can be linked with high stability to an insoluble matrix without losing affinity for the receptor. Such a ligand for estrogen receptor has been successfully prepared and used by Greene and Jensen[l]. We here report the synthesis and properties of a similar ligand for androgen receptor, 17a-(2’,3’-epoxypropyl)-S~-androstan-17B-ol-3-one, or 17a -epoxypropyl-DHT. The epoxy group provides a site for extremely stable covalent linkage to *This work was presented in preliminary form at the 65rh Annual Meeting of the Endocrine Society, San Antonio, Texas, June 1983 (Abstract no. 653). §Present address: Central Drug Research Institute, ChattarManzil Palace, Lucknow 226-001, India. ~TO whom correspondence should be addressed. Abbreviations and &ivinl names : 17~ -Epoxypropyl-dihydrotestosterone or 1711 -eooxvproovl-DHT. 17~-(2’.3’17~laliylepoxypropyl)-5a:-androscan-i?fi-oi-3-one; DHT, 17a-allyl-5a-androstan-l7/3-ol-3-one; DHT or 5u-androstan-17/?-ol-3-one; E, dihydrotestosterone, or estradiol, estra-1,3,5(10)-triene-3,17p-diol; R5020 promegestone, 17u,21-dimethyl-19-nor-pregna:,6(10)-d’ tene-3,20-dione; TMS, tetramethylsilane; THF, tetrahydrofuran; DMF, N,N-dimethyl formamide; EDTA, ethylenediaminetratraacetic acid; TLC, thinlayer chromatography; AR, androgen receptor; ER, estrogen receptor; PgR, progesterone receptor.

Thiopropyl-Sepharose-6B, and the bound compound retains very high selective affinity for the androgen receptor. EXPERIMENTAL Chemistry

Melting points were determined in open capillary tubes either immersed in a heated sulfuric acid bath or with an Electrothermal& melting point apparatus, and are uncorrected. i.r. Spectra were recorded on a Perkin-Elmer 283B spectrophotometer and values are expressed in cm-‘. [‘H]NMR spectra were taken in deuterated chloroform unless stated other-wise, on either a Varian EM-90 or a Jeol FX 90Q spectrometer using TMS as internal standard; only salient resonances are reported, with chemical shift values expressed in ppm (6) relative to the standard, and coupling constants (J) in Hz. Variable chemical shifts of exchangeable protons are not reported. Mass spectra were obtained on a Hewlett-Packard Model 5982 quadrupole mass spectrometer with a HewlettPackard Model 5933 data system. The relative intensity of the salient fragment ions to the base peak (100) is given in parentheses. Homogeneity of all the compounds was routinely checked by TLC using silica gel (Type G, Sigma, St Louis, MO.). Preparative TLC was also performed on silica gel (Type G, Sigma), prepared as a slurry in water. The desired compounds were isolated from silica gel bands by 5a-Androstanextraction with ethyl acetate. 3/3-o]- 17-one was purchased from Steraloids, Wilton

1111

1112

SUPKABHAT

RAY

ef ul.

3 Fig. I. Synthesis of 17a-epoxypropyl-DHT.

N.H., and Thiopropyl-Sepharose-6B was purchased from Pharmacia P-L Biochemicals, Piscataway N.J. EDTA, Tris-HCI, and dithiothreitol were purchased from Sigma. All other reagents and solvents were purchased from Aldrich, Milwaukee, Wis. Anhydrous tetrahydrofuran containing less than 1% water was further purified by distillation from butyl lithium. The synthetic scheme is outlined in Fig. 1. The detailed reaction conditions as described below are not necessarily optimized. 17a-Ally/-_%-androstan-3/?,178-diol

(2)

A mixture of 5a-androstan-3/J-ol-17-one (l), (1 g, 3.45 mmol) and ally1 bromide (4.19 g, 34.6 mmol) in dry THF (40ml) was gradually added to the Grignard reagent prepared from excess magnesium (1 g, 41.66 mmol) and ally1 bromide (1.4 g, 11.57 mmol) at 1&12”C with stirring. The reaction mixture was brought to room temperature and stirred for 3 h. Saturated aqueous ammonium chloride was then added and the resulting mixture was filtered through celite. The residue was washed with THF (40 ml) and the filtrate was dried over anhydrous sodium sulfate. Removal of solvent in vacua afforded a white solid which was recrystallized from benzene-hexane to afford 2: yield 1.12 g (98%); m.p. 172-173°C; I:$ 3450, 3240, 2940 and 1645; 6 (CDCI, + DMSO-d,): 0.78 (s, 3H, 18 CH3), 0.82 (s, 3H, 19 CH,), 2.0-2.27 (m, 2H, C&CH=), 3.19-3.64 (m, lH, 3aH,), 4.84-5.16 (m, 2H, CH==CH,) and 5.63-6.11 (m, lH, CH=CH,); M+ m/e 332 (6.7), fragment ions at m/e 291 (42) and m/e 273 (100). 17a-Allyl-Scl-androstan-l7~-ol-3-one

(3)

Jones reagent [2] (0.3 ml) was added rapidly to a solution of 2 (300 mg, 0.9 mmol) in anhydrous acetone (50 ml) under nitrogen at lO_12°C. The reaction mixture was stirred for 3 min, treated with methanol (0.1 ml), and poured over water (250 ml). The precipitate thus obtained was collected by filtration, washed with water (100 ml), and air-dried. Recrystallization

from benzene-hexane afforded 3: yield 160mg (53%); m.p. 120-121°C; Ii2 3460, 2940, 1710 and 1650; 6: 0.91 (s, 3H, 18 Cc,), 1.03 (s, 3H, 19 CH& 4.92-5.28 (m, 2H, CH=CH,) and 5.7-6.17 (m, lH, CI+CH,); M+ m/e 330 (9.5), fragment ions at m/e 289 (42), m/e 271 (83) and m/e 55 (100).

Diastereomeric 17a-(2’,3’-epoxypropyl)-5u-androstan17fl-ol-3-ones (4a and 4b) To a solution of 3 (660 mg, 2 mmol) in methylene chloride (35 ml) was added vanadyl acetylacetonate (53 mg, 0.2 mmol), and to the resuiting green solution, t-butyl hydroperoxide (286.2 mg, 2.7 mmol) in methylene chloride (5 ml) was added slowly. The reaction mixture changed to dark red and was then stirred at room temperature until the red color had faded to light brown (1.5 h), at which time it was diluted with methylene chloride (40 ml). Aqueous sodium sulfite (3%, pH 9.0, 25 ml) was then added and the mixture was stirred for 30min at room temperature. The organic phase was washed with 3% aqueous sodium sulfite (pH 9.0, 3 x 40 ml) and finally with water to neutrality, dried, and concentrated in vacua. Crystallization with benzene-hexane afforded a 4: 1 mixture of the 2 diastereomeric epoxides 4a and 4b: yield 540mg (78%); m.p. 166168°C (previous softening at 159°C). Purification by preparative TLC (40% ethyl acetate-benzene) yielded the title compounds, arbitrarily designated 4a (less polar, Rf 0.45) and 4b (more polar, Rf 0.35). Final purification, melting points and other physical data for each of the two diastereomers are listed below: Diastereomer 4a. Recrystallization of chromatographed solid from methylene chloride-hexane afforded 4a: m.p. 118-120°C (solidifies and remelts at 165-l 66°C); I~~;C’2 3420,3060,2990 and 17 15; 6 0.9 1 (s, 3H, 18 CH,), 1.03 (s, 3H, 19 CH,), 2.44-2.62 (m, lH, 3’-proton), 2.75-2.93 (m, lH, 3’-proton) and 3.11-3.33 (m, lH, 2’-proton); M+ m/e 346 (26) fragment ions at m/e 328 (50) and m/e 289 (100).

Androgen receptor affinity chromatography Diastereomer 4b. Recrystallization of chromatographed solid from methylene chloride-hexane afforded 4b; m.p. 98-100°C; I~~clz 3420, 3060, 2995 and 1710; 6 0.89 (s, 3H, 18 CH,), 1.02 (s, 3H, 19 CH,), 2.4-2.58 (m, lH, 3’-proton), 2.692.89 (m, lH, 3’-proton) and 3.1 l-3.34 (m, lH, 2’-proton); M+ m/e 346 (28), fragment ions at m/e 328 (52) m/e 289 (96.8) and m/e 113 (100). Receptor afinity determinations

Androgen receptor (AR) for these experiments was prepared from rat uteri [3,4]. Mature 2-3-month old female Harlan-Sprague-Dawley rats were ovariectomized under ether anesthesia. Two days later uteri were excised, quickly trimmed, frozen in liquid nitrogen, and stored at -70°C until use. To prepare cytosol AR, about 50 mg of this tissue per ml of TEDGM buffer (10 mM Tris-HCl, pH 7.4 at 0°C 1.5 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol, 20 mM sodium molybdate) were homogenized in a Dual1 glass-glass homogenizer with a motor driven pestle; this and all subsequent procedures were carried out at 04°C. The homogenate was centrifuged at 100,000 g for 20 min and the clear supernataht (cytosol) was collected. In each volume of cytosol, the pellet from a half volume of dextrancoated charcoal suspension (10 mM Tri-HCl, pH 8.0 at 0°C 0.25% Norit A, 0.0025% dextran) was suspended for 10 min and then centrifuged 10 min at 2000 g. For relative binding affinity (RBA) determinations, triplicate sets of tubes received 200 ~1 cytosol, 25 ~1 lo-’ M [3H]DHT (172 Ci/mmol, New England Nuclear), and 25 ~1 unlabelled test compound at five or more different concentrations. After 18 h at 0°C all tubes received 500 ~1 dextran-coated charcoal suspension and were shaken every 5 min for 15 min before being centrifuged 10 min at 2000g. Supernatants (500 ~1) were added to 5 ml counting solution (42 ml Amersham-Searle Spectrafluor in 11 of toluene) and counted in a Beckman LS-7000 liquid scintillation counter. After subtraction of nonspecific [3H]DHT binding (determined in the presence of 3 x 10m6M DHT), RBA’s were calculated as the ratio of the concentration of unlabelled DHT giving 50% inhibition of [3H]DHT binding to the concentration of unlabelled test compound giving the same inhibition. Afinity bead preparation

The entire procedure was carried out under an atmosphere of nitrogen. All the solutions and buffers were bubbled with nitrogen for approx 1 h prior to use. Freeze-dried Thiopropyl-Sepharose-6B (15 g) was suspended in buffer A [lo0 ml, pH 8.41 (buffer A = 0.3 M sodium bicarbonate and 1.1 mM EDTA) containing dithiothreitol (6.5 mmol). The flask was charged with nitrogen and shaken for 1 h at 25°C. The activated beads were then filtered, washed with

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15 1 of water containing acetic acid (90 ml), sodium chloride (438.75 g), and EDTA (5.54 g, 16.48 mmol), and finally washed with buffer A (1.5 1, pH 9.2). A bead sample was taken for assay of total free thiol groups (see below). The caked beads were then transferred from the filter to a flask containing diastereomeric epoxides 4a and 4b (155.7 mg, 0.45 mmol) in DMF (45 ml), and buffer A (70 ml, pH 8.4) was added. Under nitrogen the flask was shaken for 20 h at 25°C. The beads were filtered and washed with buffer A (1.5 1, pH 8.4). Another bead sample was removed at this stage for determination of remaining free thiol groups. Iodoacetamide (275 mg, 1.4 mmol) was dissolved in buffer A (90 ml, pH 8.4) and the caked beads were added to this solution. In the dark, the flask was shaken for 1 h at 25°C. The beads were then filtered under nitrogen and washed in sequence with buffer A (4 1, pH 8.4) containing dithiothreitol (48.7 mmol); buffer A (4 1, pH 8.4); Tris-HCl (2 1, 50 mM, pH 7.4); water (2 1) and 21 each of 25, 50 and 70% methanol-water. The beads (approximate vol 60 ml) were stored in 70% methanol-water under nitrogen at 4°C. The degree of substitution was estimated by determining the difference in free thiol groups on aliquots of beads before and after reaction with the epoxides. To 400 ~1 of 1: 1 bead suspensions in Buffer A were added 500 ~1 of Buffer A containing [‘4C]iodoacetamide (23 mCi/mmol, New England Nuclear; 50,000 cpm) and 23 pmol unlabelled iodoacetamide. After 1 h at 25°C the beads were washed 3 times each with 3 ml Buffer A containing 1% dithiothreitol, Tris-HCl (3 ml, 50 mM, pH 7.4) and 3 ml 70% methanol in water. Aliquots of a 1: 1 bead suspension in 70% methanol were counted in 5 ml of the toluene-Spectrafluor scintillation fluid. By this method, it was determined that about 7pmol of ligand were bound per ml of beads. (The same substitution was obtained for beads identically prepared from the analogous 17a-epoxypropylestradiol, where the substitution could be determined by the drop in 280 nm absorbance of the free ligand HPLC peak after the reaction as well as by the labelled iodoacetamide method). Receptor binding to beads

Cytosols were prepared from rat uteri as above for AR and estrogen receptor (ER), and from rat uteri taken 7 days after depo-estradiol injection for progesterone receptor (PgR). Ten volumes of buffer were used for homogenization, but cytosols were further diluted 1:4 before 500 ~1 aliquots were placed in 1.5 ml Eppendorf tubes. Suspensions of DHT beads as prepared above or E, beads prepared identically from 17~-epoxypropyl-estradiol were made 1: 1 in buffer, and 50~1 aliquots were added to the tubes. For some experiments, DHT and E, beads were diluted 1: 10 with non-derivatized Sepharose-6B (Pharmacia), and a 1: 1 suspension of Sepharose-6B

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SUPRABHA~ RAY et

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alone was added to control tubes. All tubes were closed and incubated at 4°C for 3 h, with occasional mixing or with slow continuous rotation. They were then centrifuged briefly to pellet the beads and 200 ~1 aliquots of the supernatants were assayed for receptors remaining unbound (2 x 10e8 M [3H]estradiol for ER, 5 x lo-“M [3H]DHT for AR, 5 x 10m8 M [3H]R5020 for PgR, f IOO-fold excesses of the corresponding unlabelled ligands to determine nonspecific binding, 18 h at 4”C, followed by dextran-coated charcoal treatment and counting as above). RESULTS

DHT Eporide A DHT Epoxide S

0.094 0.042

AND DISCUSSION

Chemistry 5cr-Androstan-3P-ol17-one (1) was used as staring material for the synthesis of the desired epoxide. Treatment of 1 with ally1 magnesium bromide and subsequent aqueous work-up afforded 17cr-allyl5a-androstan-3/?,178-diol (2) in excellent yield. The 178 orientation of the OH group in this compound was assigned by analogy with the established stereochemistry of 17u-allylestradiol[5] and on the basis of the known stereochemistry of addition of organometallic compounds to 17-oxosteroids [6]. Jones[2] oxidation of 2 gave 17~ -allyl-5cc -androstan- 178ol-3-one (3). Epoxidation of 3 with t-butyl hydroperoxide in the presence of catalytic amounts of vanadyl acetylacetonate afforded the diastereomeric 17c(-(2’,3’epoxypropyl)-5cc-androstan-l7~-ol-3-ones (4a and 4b in the ratio 4: 1) in good yield. The two diastereomers were separated by preparative TLC. They show nearly identical mass fragmentation patterns, and the [‘H]NMR chemical shifts for the 2’-diastereomeric proton are not sufficiently different for unambiguous configurational assignments. Nor are the differences in the infrared spectra of these compounds helpful in assigning absolute stereochemistry at the 2’-carbon. However, their polarities are quite different; the major product 4a is substantially less polar on TLC than its diastereomer 4b. We have previously epoxidation of diastereoselective reported [7] 17cc-allylestradiol; and, in that report, we tentatively assigned the 2’-S configuration for major, less polar product, based on conformational preferences, steric interactions, and hydrogen bonding arguments; by analogy, 4a might be expected to have the corresponding 2’-S configuration. More detailed investigation of the diastereoselectivity of these and other epoxidations of 17cc-ally1 steroids is continuing in our laboratories and will be reported elsewhere. Receptor

Relative Binding Affinity for AR

aJinity

After ally1 DHT (3) was prepared, we examined its receptor specificity. In particular, we were concerned that the 17a-substitution had increased its affinity for PgR, as might have been predicted from the data of Ojasso and Raynaud[8]. Fortunately, we found the RBA of 3 for rat PgR to be only 0.3% (vs R5020),

Fig. 2. Competition for [‘HIDHT binding to rat uterine androgen receptor by unlabelled DHT and several derivatives. while its RBA for AR was about 50% (vs DHT). No affinity for ER was observed under conditions which would have detected
afinity

beads

The diastereomeric mixture of 4a and 4b was used as such for the preparation of affinity beads. The 2-thiopyridyl protecting group in ThiopropylSepharose-6B was removed by reductive cleavage of the disulfide bond using dithiothreitol in sodium bicarbonate buffer containing EDTA. The resulting unprotected thiol groups reacted readily with diastereomeric epoxides 4a and 4b at pH 8.4 in sodium bicarbonate buffer. Excess thiol groups were finally masked by alkylation using a large excess of iodo-

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Androgen receptor affinity chromatography

E-C,,

Fig. 3. Expected

structure

of DHT

beads

synthesized from Sepharose-6B.

Table I. Percent

uptake of androgen, estrogen and progesterone receptors from rat uterine cytosol by DHT and E2 beads (see

Experimental) AR

ER

PgR

Concentrated I:10

83 39

54 26

33 5

Estradiol Beads Concentrated I:10

4 0

64 18

DHT Beads

acetamide in sodium bicarbonate buffer containing EDTA. The same reaction but with “C-labelled iodoacetamide provided a measure of free thiol groups on the beads before and after reaction with the epoxides. About 7 pmol of ligand per ml of beads were bound according to this measure, which agrees well with this and other measures of an identical bead preparation from 17a-epoxypropyl-estradiol. Aliquots of these DHT beads, whose expected structure is shown in Fig. 3, were rocked gently with 20 vol of rat uterine cytosol to determine their uptake of AR and also ER and PgR. We compared these with E, beads prepared identically from the analogous epoxide of estradiol. (Following Greene and Jensen[l], we routinely use the latter beads for estrogen receptor purifications of up to lOOO-fold.) We also compared E, and DHT beads diluted IO-fold with un-derivatized Sepharose-6B beads. The results presented in Table 1 show considerable receptor uptake even by the diluted beads. The same volume of Sepharose-6B alone retained little if any receptor in the controls, but the values in the table have nevertheless been corrected for this. The 83% uptake of AR by the DHT beads compared with the 64% uptake of ER by the E, beads suggests that the DHT beads should perform at least as well for AR purification as the E2 beads do for ER, although with DHT beads, ER and PgR will have to be blocked (e.g. with DES and triamcinolone acetonide) to achieve maximum AR specificity. This new affinity chromatography reagent thus appears to show promise for purification of androgen receptor, and this is being pursued by Tindall et al. in Houston. It has also already been applied in the

Sopharosr

17cc-epoxypropyl-DHT

and

Thiopropyl-

study of specific androgen binding protein (ABP) in the monkey by Keeping et al. in Pittsburgh[9], and other such applications seem possible as well. Acknowledgements-This work was supported by the U.S. NIH, grants (CA-29971, HD-10202 and CA-30195 GCC; RR 08194, PLS), Robert A. Welch Foundation (AX-637, PLS) and was carried out while S.R. was in receipt of a World Health Organization Research Training Grant. We gratefully acknowledge the technical assistance of Letitia C. Fulcher. REFERENCES

1. Greene G. L. and Jensen E. V.: Monoclonal antibodies as probes for estrogen receptor detection and characterization. J. steroid Biochem. 16 (1982) 353-359. 2. Fieser L. F. and Fieser M.: Reagents for Organic Synthesis. John Wiley, New York, Vol. 1 (1968) p. 142. B. S.: 3. Schmidt W. N. and Katzenellenbogen Androgen-uterine interactions: an assessment of androgen interaction with the testosterone- and estrogenreceptor systems and stimulation of uterine growth and progesterone receptor synthesis. Molec. cell. Endocr. 15 (1979) 91-108. 4. Chang C. H. and Tindall D. J.: Physicochemical characterization of the androgen receptor in rat uterine cytosol. Endocrinology 113 (1983) 14861493. 5. Counsel1 R. E., Klimstra P. D., Elton R. L. and Nutting E. F.: Chemical and biological properties of some 17-substituted estradiol derivatives. J. med. Chem. 9 (1966) 689-692, and references cited therein. 6. Kirk D. N. and Hartshorn M. P.: Steroid Reaction Mechanisms. Elsevier, Amsterdam (1968). 7. Salman M., Reddy B. R., Ray S., Stotter P. L. and Chamness G. C.: 17a-Ally1 estradiol analogues as candidates for development of high-affinity fluorescein-estradiol conjugates. J. steroid Biochem. 24 (1986). 539-548. 8. Ojasso T. and Raynaud J. P.: Unique steroid congeners for receptor studies. Cuncer Res. 38 (1978) 418&4198. 9. Keeping H. S., Winters S. J. and Troen P.: Identification of androgen-binding protein from testis cytosol and Sertoli cell culture medium of the cynomolgus monkey, Mucucu fusciculuris. Endocrinology 117 (1985) 1521-1529. Note udded in proof: The PgR affinity data for epoxides 4a

and 4b were obtained with human uterine PgR. We have since found to our surprise that rut uterine PgR shows somewhat greater affinity for 4b than 4~; this and other differences between rat and human PgR are discussed in a forthcoming manuscript (Salman, Ruiz, Stotter and Chamness, submitted to J. sreroid Biochem.).