Hexestrol Diazirine Photoaffinity Labeling Reagent for the Estrogen

Raghoottama S. Pandurangi, Przemyslaw Lusiak, Robert R. Kuntz, Wynn A. ... Gagik G Melikyan , Steve Sepanian , Bobby Riahi , Ferdinand Villena , John ...
3 downloads 0 Views 1MB Size
Download PDF
Bioconjugate Chem. 1994, 5, 141-150

141

Hexestrol Diazirine Photoaffinity Labeling Reagent for the Estrogen Receptor Kathryn E. Bergmann, Kathryn E. Carlson, and John A. Katzenellenbogen' Department of Chemistry, University of Illinois, Urbana, Illinois 61801. Received October 4, 1993'

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (l),a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. K d : estradiol = 0.19 nM; (1) = 0.98 nM, respectively). I t is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at >315 nm. The tritium-labeled diazirine [3H]-1undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90 % Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species a t M , 65 000, the same species that is labeled by F3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormonebinding domain of ER.

.

INTRODUCTION Affinity labeling is a technique that can be used to obtain structural information about binding sites in biomolecules, through the use of photochemicallyreactive or electrophilic derivatives of ligands that are capable of covalent attachment to the proteins (1). This technique has been used to label many binding proteins, such as hormone receptors, enzymes, and immunoglobins (2);affinity labels have also been used to investigate membrane structures ( 3 ) . We have developed molecular probes to study the ligandbinding domain of the estrogen receptor (ER) through the use of photoaffinity labels containing a wide variety of photoreactive functionalities: diazo ketones ( 4 ) , aryl (I, 4a, 5), acyl (6) or alkyl (7) azides, a,&unsaturated ketones (8),aryl halides, (9)and nitro compounds ( 1 , 4 b ) . A photosensitive group that has not previously been employed in photoaffinity labels for ER is the diazirine heterocycle. Diazirines have shown favorable characteristics in many other photoaffinity labeling studies (10). Diazirines absorb a t 350-380 nm, well away from the absorption maxima of proteins (280 nm) and nucleic acids (260 nm) ( 1 2 ) . (On the other hand, aryl azides, the most widely used photoactivatable group to date, absorb maximally at 250 nm (12).) Irradiation of diazirines generates carbenes that have sufficient lifetime to undergo C-H bond insertion reactions (11b ) , forming stable covalent bonds with many types of functional groups found in proteins. Diazirines can also undergo photochemical rearrangement to diazo compounds, which upon protonolytic diazotization give carbocations that can form covalent bonds by nucleophile capture (11a,c). Certain other photoaffinity reagents which undergo covalent attachment to proteins do not give reagent-receptor bonds that are stable during the chemical manipulations required to identify the site of attachment (e.g., proteolytic and chemical cleavage to peptides and Edman degradation) (12). Photoaffinity labels with a diazirine functional group, on the other hand,

* Abstract published in Advance ACS Abstracts, February 1, 1994. 1043-1802/94/2905-0141$04.50/0

have been shown to modify amino acids in several proteins in such a manner that these proteins do not undergo significant loss of label during the residue identification processes (13). Both steroidal and nonsteroidal ligands for ER have been modified with photochemically reactive or electrophilic groups (I, 8, 14). Hexestrol (2), a nonsteroidal estrogen with a 3-fold higher affinity for ER than estradiol, has been functionalized with reactive groups as a biological probe for the ER, e.g., 3-azidohexestrol (3) (4, 15) and ketononestrol aziridine (4) (16). 3-Azidohexestrol(3)was found to have low photocovalent attachment to ER (ca. lo%),and its labeling selectivity was poor ( 4 ) . Ketononestrol aziridine (41, an electrophilic labeling agent, is limited to reaction with highly reactive nucleophilic amino acid residues (17). Recently, Kuhn et al. (18)described the preparation of a photolabile thioglycoside containing a dialkyldiazirine ( 5 ) as an analog of N-acetylhexosaminides and used it to specifically label both subunits of the enzyme 8-hexosaminidase. On the basis of this favorable approach, we undertook the preparation of a hexestrol derivative substituted on the side chain with the same diazirine functionality. We report here the synthesis of the hexestrol diazirine photoaffinity label in unlabeled (1) and in high specific-activity tritium labeled forms (ISH]-1). We have found that this hexestrol diazirine demonstrates highly efficient and selective photocovalent attachment to ER. EXPERIMENTAL PROCEDURES A. Chemical Procedures. Materials. The preparation of 2(R*),3(S*)-bis[4- [ (tert-butyldimethylsilyl)oxy] phenyllpentanethiol (6) was reported previously (16b). 3-Azibutanol and its toluenesulfonate ester 7a were prepared according to the procedures of Church (19).Dry tetrahydrofuran (THF) was obtained by distillation from sodium benzophenone ketyl. All other chemicals and reagents were purchased from Aldrich Chemical Co. and were used without further purification. Methods. All reactions were carried out under nitrogen atmosphere. In most cases, a standard procedure for 0 1994 American Chemical Society

142

Bioconjugate Chem., Vol. 5, No. 2, 1994

product isolation and purification was used. Isolation consisted of quenching the reaction mixture in water or an aqueous solution, extracting with an organic solvent, drying over an anhydrous salt, filtering, and evaporating the solvent under reduced pressure. This is indicated in the text by the phrase "product isolation", which is followed by a list of the components in parentheses. The usual method for purification of reaction products consisted of flash chromatography performed according to standard methods (20) using Woelm 32-63-pm silica gel. This is indicated in the text by the phrase "purification", with the eluting solvent given in parentheses. Elemental analyses were performed by the Microanalytical Laboratory a t the University of Illinois and are within f0.4% of theory. High-performance liquid chromatography (HPLC) was performed isocratically for the unlabeled hexestrol diazirine using column A (a Whatman Partisil10 silica gel preparative column (30 cm X 4.6 cm)) with a UV variable-wavelength detector set at 254 nm; the solvent system used was 35% CHZC12-i-PrOH (95:5) and 65% hexane at 5 mL/min. HPLC of [3H]hexestrol diazirine was performed isocratically using column B (a Supelcosil LC-CN semipreparative column (5 pm, 25 cm X 10 mm) from Supelco, Inc., with a guard column packed with pellicular CN from Alltech) with a UV wavelength detector set at 254 nm and a Packard Flo-one p radiomatic radiochromatography detector; the solvent systems used were 8% (2% MeOH-EtOAc) and 92% hexane (normal phase conditions using Flo-Scint I cocktail) and 30% or 40 % water-MeOH (reversed phase conditions using FloScint I1 cocktail). Chemical Synthesis of 3-Azibutyl 2,3-Bis(4-hydroxypheny1)pentyl Sulfide. 3-Azi-1 -[(methylsulfony1)oxylbutane (7b). 3-Azi-1-butanol, as well as its toluenesulfonate ester 7a, were prepared as previously reported (19). The methanesulfonate ester 7b was prepared as follows: 3-Azi-1-butanol (371 mg, 3.706 mmol) was dissolved in 7 mL of dry T H F and cooled to 0 "C. Et3N (1.03 mL, 7.390 mmol) was added to the solution, followed by slow addition of CH3SOzCl (440 pL, 5.569 mmol). A precipitate formed, and the solution turned a pale yellow. After the solution was stirred a t 0 "C for 1 h, product isolation (H20, EtOAc, MgS04) and purification (50% EtOAc/Hex) afforded 578 mg (88%)of the methanesulfonate ester 7b as a clear oil: lH NMR (CDCl3) 6 1.11(s,3H, CN~CHS), 1.80 (t, 2H, J = 6.2 Hz, CHZCN~), 3.07 (s, 3H, OS02CH3),4.14 (t, 2H, J = 6.3 Hz, CH20S02); mass spectrum (CI) mlz (relative intensity) 178 (8), 152 (4), 111(5), 109 (6),97 (7),67 (7),55 (100). The molecular ion was too weak to be observed a t high resolution. 3-Azibutyl (2R*,35'*)-2,3-Bis[4-[(tertbutyldimethylsilyl)oxy]phenyl]pentyl Sulfide (8). The oil was removed from -40 mg of KH (60% in oil, 5-10 fold excess) with dry THF, and the solid was cooled to -78 "C. erythroPentanethiol 6 (51.5 mg, 0.0996 mmol) and toluenesulfonate 7a (30.7 mg, 0.1207 mmol) were dissolved in 1 mL of dry THF, and the solution was added to the KH slowly. The mixture was allowed to warm to room temperature slowly. After the mixture was stirred for an additional 5 h, product isolation (5 5% aqueous Na2HP04, EtOAc, MgS04) and purification (5% EtOAc/Hex) afforded 44.9 mg (75 % ) of the protected pentyl butyl sulfide 8 as an oily white solid: mp 44-49 "C; lH NMR (CDC13) 6 0.21 (s, 12H, (CH&Si), 0.53 (t,3H, J = 7.3 Hz, CH~CHS), 0.90 (5, 3H, CNZCH~), 0.98 (s, 9H, (CH3)3C),0.99 (s, 9H, (CH3)3C),1.2-1.5 (m, 4H, CH2CH3 and C H ~ C H Z C N1.97 ~), (t, 2H, J = 7.9 Hz, C H ~ C H ~ C N Z2.4-2.47 ), (m, 2H, CHCHzS), 2.54 (dt, l H , J = 3.3, 10.6 Hz, CHCHzS), 2.79

Bergmann et al.

(dt, l H , J = 4.9,9.7, CHCH~CH3),6.75-7.05(m, 8H,ArH); MS (FAB) m/z (relative intensity) 599 (M+ + 1, 2), 586 (2), 483 (2), 349 (5), 279 (23), 261 (35), 249 (50), 223 (38), 221 (25). Anal. Calcd for C ~ ~ H ~ ~ N Z OC,Z 66.17; S S ~ ~H,: 9.08; N, 4.68. Found: C, 66.32; H, 9.25; N, 4.62. 3-Azibutyl(2R*,3S*) -2,3-Bis(4-hydroxyphenyl)pentyl Sulfide (1). From the Bis(sily1)ether (8). To the solution of silyl-protected hexestrol diazirine 8 (17.7 mg, 0.0295 mmol) dissolved in 5 mL of T H F was added 120 pL of (n-Bu)4NF (0.120 mmol). After the solution was stirred for 30 min, a 10-fold excess of TsOH (56 mg, 0.294 mmol) was added to quench the phenolic ammonium salts. After being stirred 5 min, the solution was passed through a Si02 plug, and the solvent was dried with MgS04 and then was removed under reduced pressure. Purification (40% EtOAc/Hex) afforded 10.6 mg (97 % ) of the deprotected diazirine 1 as a white solid. Further purification by HPLC was accomplished using column A (tR = 20.8 min) before submission for binding analysis: mp 132-134 "C dec; lH NMR (acetone-d6) 6 0.50 (t, 3H, J = 7.3 Hz, CH2CH3),0.87 (s, 3H, CNZCH~), 1.2-1.5 (m, 4H, CHZCHB and C H Z C H ~ C N 2.0 ~ ) , (t, 2H, J = 7.8 Hz, C H ~ C H ~ C N Z ) , 2.45-2.5 (m, 2H, CHCHzS), 2.59 (dt, l H , J = 3.6,10.8 Hz, CHCHzS), 2.81 (dt, l H , J = 5.0, 8.6, CHCH2CH3), 6.757.15 (m, 8H, A r m , 8.15 (s, 2H, ArOH); IR (KBr pellet) cm-l3302,2955,1599,1512,1236; UV (MeOH) nm (e) 206 (26 500), 230 (20 goo), 282 (3900);MS (FAB) m/z (relative intensity) 371 (M+ + 1, 12), 279 (6), 261 (3), 250 (3), 235 (3),195 (4), 169 (8);HRMS calcdfor C Z I H Z ~ N Z371.1793, O~S found 371.1785. Anal. Calcd C, 68.08; H, 7.07; N, 7.56; S, 8.65. Found: C, 68.04; H, 7.15; N, 7.52; S, 8.54. From Reaction with 3-Azi-1- [(methylsulfonyl)oxylbutane (7b). The oil was removed from -15-20 mg KH (60% in oil, 5-10 fold excess) with dry THF, and the solid was cooled to -78 "C. erythro-Pentanethiol 6 (20.8 mg, 0.0402 mmol) and methanesulfonate 7b (36 mg, 0.202 mmol) were dissolved in 1mL of dry THF, and the resulting solution was added to the KH slowly. The mixture was allowed to warm to room temperature. After the mixture was stirred for an additional 24 h, product isolation (5% aqueous Na2HP04, EtOAc, MgS04) and purification (40 % EtOAc/Hex) afforded 5.4 mg (36%) of the deprotected pentyl butyl sulfide 1 as a white solid. From the tetraiodohexestrol (9). The starting tetraiodide 9 (3.7 mg, 0.0042 mmol) was dissolved in 0.5 mL EtOAc. To the solution was added 5% Pd on alumina (22.5mg) and Et3N (5 pL). The reaction was placed under hydrogen atmosphere. After 2.5 h, the solution was passed through a plug of Celite, and the solvent was removed in vacuo. Purification (403'% EtOAciHex) afforded 1.4 mg (89%) of the expected protio product 1 and 0.6 mg of the corresponding ketone 13 (obtained by over reduction and hydrolysis of the diazirine). 3-Azibutyl (2R*,3S*)-2,3-Bis(3,5-diiodo-4-hydroxypheny1)pentyl Sulfide (9). The starting bisphenol 1(11.4 mg, 0.31 mmol) and molecular iodine (60 mg, 0.236 mmol) were dissolved in 2 mL of benzene. Morpholine (50 pL, 0.573 mmol) was added to the solution, which turned from orange to clear. The mixture was allowed to stir at room temperature overnight. Product isolation (5 % HC1, EtOAC, 20 % aqueous Na2S203, MgS04) and purification (30% EtOAc/Hex) afforded 21 mg (78%) of the tetraiodo product 9 as a pale yellow solid: lH NMR (acetone-de) 6 0.55 (t, 3H, J = 7.4 Hz, CH~CHS), 0.92 (s, 3H, CNZCH~), 1.2-1.4 (m, 4H, contains (1.36, t, 2H, J = 7.3 Hz, CH2CH2CN2) and CHZCH~), 2.13 (t, 2H, J = 7.7 Hz, CHZCHZCN~), 2.546 (AB quartet, l H , J = 13.2 Hz, AV = 21.07 Hz, CHCHHS), 2.558 (AB quartet, l H , J = 13.3 Hz, Av =

Hexestrol Diazirine Photoaffinity Labeling Reagent

36.99 Hz, CHCHHS), 2.65-2.8 (m, l H , CHCHzS), 2.98 (dt, l H , J = 3.8,10.6 Hz, CHCHzCHs), 7.73 (s, 2H, ArH), 7.76 (5, 2H, ArH); IR (KBr pellet) cm-I 3456, 2924, 1456; UV (MeOH) nm (e) 224 (56 goo), 292 (59001, 300 (5800); MS (FAB) mlz (relative intensity) 875 (M+ + 1, lo), 748 (4), 419 (lo), 387 ( l l ) , 359 (13), 345 (lo), 223 (25), 195 (100); HRMS calcd for CzlH23NzOzSI4 874.7659, found 874.7632. Radiochemical Synthesis of [3H]-3-Azibutyl 2,3Bis(4-hydroxypheny1)pentyl Sulfide ([3H]-1). The conditions for tritiolysis of the tetraiodohexestrol diazirine 9 were based on those used for the hydrogenolysis of 9, with the exception that the reaction time was doubled (to 90 min) in the hopes of ensuring complete reduction. A sample of the Irhexestrol diazirine 9 (47.9 mg, 0.0548 mmol), purified by flash chromatography, was sent to Amersham Corp. (Arlington Heights, IL) where the radiochemical incorporation was performed according to the reported procedure. "To a solution of 14-hexestrol 9 (47.9 mg, 0.0548 mmol) in 5 mL EtOAc was added 72 pL Et3N (0.514 mmol) and 118 mg Pd on alumina (576, 0.05545 mmol Pd). The solution was exposed to carrier-free tritium gas for 90 min at 25 "C and atmospheric pressure. The solution was then filtered through a 2 cm plug of Celite to remove the Pd catalyst and the residue was washed with 60% EtOAc/ hexane (3 X 2 mL). The solvent was removed in vacuo using low heat (tepid bath). Exchangeable tritium was removed using standard solvent exchange-evaporation cycles, but avoided warming above 40 "C. The resulting crude product was dissolved in ethanol and stored a t -20 "C. The total amount of radioactivity produced was 1.75 Ci (28% of theoretical)." The 100-mCi sample, obtained from Amersham, was examined by TLC and HPLC. Radio-TLC was difficult to interpret, and the reaction product was later determined to be unstable on silica. Analysis of the crude I3H1hydrogenolysis products by normal and reversed-phase HPLC using cyanopropyl-substitutedsilica packing showed the presence of all iodinated intermediates, plus a small amount ( 45 min), and most of the solvent was removed under reduced pressure using low heat. The activity was taken up in 3 mL of EtOAc, and to the solution was added 5 mg of P d catalyst (5% on alumina) and 10 pL of Et3N. The solution was placed under a hydrogen atmosphere in the dark for 60 min, monitoring the reaction by HPLC (every 20 min) for disappearance of the radioactive iodinated species. The reaction was worked up by passing the mixture through a plug of Celite atop of silica, using 50% EtOAc/hexane as the eluting solvent. The solution was concentrated in vacuo, and the product was purified using the same conditions as before (cyanopropyl column, 8 7% (5 % MeOH/EtOAc) in 92% hexane as the eluent), giving -25 mCi, which corresponds to a radiochemical yield of -80 5%. The specific activity of [3H]hexestrol diazirine 1, obtained from the tetraiodo precursor 9 by this two-stage tritiolysis-hydrogenolysis procedure, was determined to

Bioconjugate Chem., Vol. 5, No. 2, 1994 143

be 32 Ci/mmol by Scatchard analysis (21). The radiochemical purity was evaluated by reinjection onto HPLC using both normal- and reversed-phase conditions; these analyses indicated a single peak of radioactivity (>go%) with a retention time of 37 min for normal phase (same conditions) and 14 min for reversed phase (40% H2O in MeOH), identical to the retention time of the unlabeled standard. B. Biological Procedures. Materials. Biochemicals were obtained from the following sources: tritium-labeled estradiol ([6,7-3HlE2) (estra-l,3,5(10)-triene-3,17/3-diol), 48 Ci/mmol, and PHItamoxifen aziridine (TAZ) ([ring3Hl42)-[l-[4- [2-(N-aziridinyl)ethoxylphenyll I - 1,2-diphenyl-1-butene), 20 Ci/mmol, were from Amersham Corp.; dextran, grade C, from Schwarz/Mann; 2-mercaptoethanol, ethylenedinitrilotetraacetic acid tetrasodium salt (EDTA), acrylamide, Photo-Flo 200, and N,N'diallyltartardiamide were from Eastman Kodak Co.; Triton X-114 was from Chem Central-Indianapolis;bromophenol blue, N,N,N',N'-tetramethylethylenediamine (TEMED), sodium azide, and 1,4-bis(5-phenyloxazol-2-yl)benzene (POPOP) were from Aldrich Chemical Co.; sodium dodecyl sulfate (SDS) was from Matheson, Coleman, and Bell; ammonium peroxydisulfate and N,N-dimethylformamide (DMF) were from Fisher Scientific; periodic acid was from G. Frederick Smith Chemical Co.; unlabeled estradiol, leupeptin, phenylmethylsulfonyl fluoride (PMSF), activated charcoal, Trizma base, ovalbumin (MW 44 600), bovine serum albumin (MW 67 000), phosphorylase B (MW 97 400), andp-galactosidase (MW 116 000) were from Sigma Chemical Co.; Coomassie Brilliant Blue R-250 was from Colab Laboratories, Inc.; and 2,5-diphenyloxazole (PPO) was from Research Products International Corp. Rat and lamb uterine cytosols were prepared and stored as previously described (22). All experiments were done in TEA buffer (0.01 M Tris-HC1, 0.0015 M EDTA, and 0.02% sodium azide, pH 7.4 a t 25 "C). Leupeptin (0.1 mg/mL) and PMSF (1mM) were added to the cytosol to prevent proteolysis in samples for electrophoresis. The charcoal-dextran slurry used to remove unbound ligand was prepared as previously reported (15a) and was used a t 1 part to 10 parts of cytosol solution. Methods. Relative Binding Affinity ( R B A ) . Assays were performed as previously reported using lamb or rat uterine cytosol diluted to 1.5 nM receptor (15a). Cytosol was incubated with buffer or several concentrations of unlabeled competitor together with 10 nM [3Hlestradiol as tracer at 0 "C for 18-24 h. The unlabeled competitor was prepared in 1:l dimethylformamide (DMF)/TEA to ensure solubility. Scatchard Assay. Rat uterine cytosol was incubated a t 0 "C for 4 h with various concentrations of [3Hlligand in the absence or presence of a 100-fold excess of unlabeled estradiol. Aliquots of the incubation solution were counted to determine the concentrations of total [3H]steroid.The incubation solutions were then treated with charcoaldextran, and the bound l3H1steroidwas determined. Data were processed according to the method of Scatchard (23). Specific Actiuity. A Scatchard plot for the [SHIdiazirine ligand plotting Ci/mL was employed to determine the specific activity (21). The X-intercept gave 6.347 X lo4 Ci/mL. From the parallel incubation assay of [3H]estradiol with the same receptor preparation, it was determined that there were 1.938 nM receptor sites. If one assumes that both the hexestrol diazirine and estradiol bind to the same sites, this corresponds to a specific activity of 32 Ci/mmol. Photolysis. Photolysis was routinely carried out at >315

-

144

Bergmann et ai.

Bioconjugate Chem., Voi. 5, No. 2, 1994

Scheme 1 KH, THF, 5 h

-70 "C to RT (75%with 7a)

N=N

TBSO

7a R =Ts

6

TBSO 0

7b R = MS

\

KH, THF, 24h -70 "C to RT

2) TsOH 97%

1

Scheme 2

12,

Morpholine (76%) I

,

* Ilr\

nm, using a 450-W mercury vapor lamp, Hanovia L679A, surrounded by a solution filter of saturated aqueous copper(I1) sulfate at 2-4 "C, employing Pyrex reaction vessels, as previously described (4b). Inactiuation Assay. Covalent binding of nonradioactive ligands was estimated by a photolysis-exchange assay previously described for the estrogen receptor (4b). Attachment Assay. Covalent binding of labeled ligands was measured directly by a filter disk assay previously described (24). Electrophoresis. SDS electrophoresis samples and gels were prepared as previously reported (5c) with standard proteins of 6-galactosidase (MW 116 000), phosphorylase B (MW 97 4001, bovine serum albumin (MW 67 000), and ovalbumin (MW 44 600) to establish a molecular weight curve. RESULTS

Synthesis of Hexestrol Diazirine (1). The thioether linkage in the hexestrol diazirine derivative (1) could conceivablybe formed from either side of the sulfur atom. However, since the protected erythro-pentanethiol6 was readily available (16b) and the 3-azibutanol derivative 7a had been previously synthesized (19),we selected the route shown in Scheme 1. Due to the low yields encountered in the preparation of 3-azi-1-[ (p-tolylsulfonyl)oxylbutane (7a), we also prepared the methanesulfonate derivative, 7b. Activation of the alcohol was accomplished by reaction with methanesulfonyl chloride (MsC1) in Et3N to form 7b in high yield (88%1. Coupling of the pentanethiol 6 with either of the activated azibutanols using KH as the base (Scheme 1)afforded either the protected thioether product 8 (75% yield from toluenesulfonate 7a) or the deprotected product 1 (35% yield from the less reactive methanesulfonate, 7b), the simultaneous deprotection in this case resulting from hydrolysis of the aryl silyl ethers during the more vigorous thioether synthesis. Deprotection of the aryl silyl ethers in 8 was accomplished by treatment with tetra-n-butylammonium fluoride (TBAF) to give the bisphenol diazirine 1 in 97% yield. Synthesis and Hydrogenation of Tetraiodohexestrol Diazirine (10). The aromatic rings were selected

as sites for radiolabeling of the hexestrol diazirine. Iodination of both unprotected phenolic rings in 1 using iodine in morpholine (25)gave the tetraiodo hexestrol 10 in good yield (Scheme 2). During the course of the reaction (after 1 h), only one intermediate was observed by TLC analysis; it most likely contained a mixture of iodinated hexestrols (11-13stage). Complete iodination to the 14 stage was achieved after 8 h. Palladium-catalyzed hydrogenolysis of aryl iodides is a standard method for tritiation of aromatic rings (16u,17b,d, 26). However, in our system, the potential sensitivity of the diazirine to reduction was a concern (10). Hydrogenation of the tetraiodo diazirine under standard conditions (5% palladium on alumina with Et3N to scavenge HI) afforded the protiodiazirine 1 in good yield within 1 h. Extended reaction times or increased quantities of catalyst led to the formation of the methyl ketone 13. In a time course study in which 1mol % of palladium on alumina catalyst was used, the optimum reaction time for complete reduction of aryl iodide 9 without overreduction of the product diazirine 1 was 45 min. A possible mechanism for the transformation of the diazirine to the ketone is outlined in Scheme 3. The diazirine is first reduced to the diaziridine 10, which is cleaved to form the unstable a-diamine 11 that would undergo hydrolysis to form the ketone 13. Although hydrogenation with palladium on carbon is known to produce the alkyl amine and ammonia (27), we did not observe formation of the alkylamine 14 under our reduction conditions, indicating that the proposed imine intermediate 12 undergoes hydration in preference to hydrogenation. Radiochemical Synthesis and Purification of [3H]Hexestrol Diazirine ([3H]-1). A sample of the tetraiodohexestrol diazirine derivative and the palladium on alumina catalyst we had used were sent to Amersham Corp. to be labeled with carrier-free I3H1H2. While a 45-min reaction time had been optimal in our hands for hydrogenolysis, we recommended a 90-min reaction time for tritiolysis, in an attempt to compensate for an anticipated tritium isotope effect (26). The radiolabeled sample returned from Amersham was

Bioconjugate Chem., Voi. 5, No. 2, 1994

Hexestrol Diazirine Photoaffinity Labeling Reagent

145

Scheme 3

schIII

14

HO

13

13

Table 1. Relative Binding Affinities and Inactivation Efficiencies (PIE) of Some Hexestrol Affinity Labels and Related Compounds

R"

HO

HO

compd no. 1 15d

- s ( c H ~ ~ N ~

16be 16ce 178 18e 3h

19" 4e

20e 21dj 22'

RBA" Ez = 100%

substituents R = S(CHz)zCNzCH3 = S(CH&CH3

16ae

HO

n=2 n=3 n=4

= CHzOCOCHNz

= COCHNz

17 (20)' 37 5.8 1.8 0.30 55 2.8

inactivation efficiencyb (%) 31 (29Y 0

60-100 NDf ND ND

R' = H , R " = H 3 R' = CHzCOCHNz, R" = H

69 1.8

9, 15 15

R"'

8.3 44 10.5 38

48-70

.

(cH~,N~

= S(CH2)3CH3

= CHNz = CHzN3

40 ND

a The receptor binding affinity (RBA) was determined in a competitive radiometric binding assay with [3H]estradiol (Ez)as the tracer. For further details, see the individual references designated. The inactivation was determined by an exchange assay. The aziridines inactivated the estrogen receptor via nucleophilic addition, whereas the others were photoactivated. The numbers in parentheses represent covalent photoattachment of the [3Hl-labeled derivative. K. E. Bergmann and J. A. Katzenellenbogen, unpublished results. e For preparation, see ref 16b. f ND = not determined. J. T. Park and J. A. Katzenellenbogen, unpublished results. h For preparation, see ref 15a,b. i Values for photoinactivation at >315 and 254 nm, respectively. j S. W. Landvatter and J. A. Katzenellenbogen, unpublished results.

assayed by HPLC and was found to contain a mixture of all possible hydrogenolysis products: these include unreacted tetraiodide starting material (I4), iodinated intermediates (Il-13, seven of them are possible), and asmall amount of the expected tetratritiohexestrol diazirine product [3H]-1(9a). No significant amount of the reduced diazirine was observed. In order to obtain the desired product with the highest possible specific activity, the 11I3 species and the IOor 3H4 species (with a small amount of 11) were isolated as two separate fractions. Each of these fractions was exposed to hydrogen to complete the hydrogenolysis. The fraction with the mixture of 11-13 species, which contained most (ca. 90%) of the total activity, was reduced smoothly, and the desired product was purified with high efficiency, by careful normal-phase HPLC on derivatized silica gel (see below). The fraction with the highest specific activity (10-11) (ca. 5% of the total activity), however, decomposed upon hydrogenation, and no expected product could be isolated. Traditional methods for determination of specific activity involve HPLC analysis. However, this could not be readily used for the [3H]hexestrol diazirine as its UV absorption a t long wavelengths is too low to quantitate readily. The specific activity, determined indirectly by receptor binding analysis (see below), was 32.8 Ci/mmol, indicating an average of about one tritium atom per hexestrol molecule. Estrogen Receptor Binding Studies on Hexestrol Diazirines 1 a n d [3H]-1. Relative binding affinities

(RBA) of the hexestrol diazirine 1, four similar hexestrol aziridines (16a-c, 41, and some structurally related side chain-substituted photolabile hexestrols (diazoketones 17, 18, and 21 and P-ketomethyl azide 22) for the estrogen receptor (ER) were determined by a competitive radiometric binding assay with PHI estradiol as the radiotracer (15~).The data given in Table 1 are expressed relative to estradiol (100%,Kd= 0.19 nM) (28). Hexestroldiazirine 1has a moderately high RBA of 17% ' ,which is higher than the hexestrol aziridines (16 and 4) with similar structures (0.3-8.3 % ). The photolabile hexestrol derivatives (all except 15, 16, 4, and 20) exhibit a wide range of binding affinities, ranging from the diazomethyl ketone 18 (2.8%) to the more extended diazoacetate 17 (55 % 1. Placing a keto functionality a t the C-2 position of hexestrol (29)has been shown to enhance binding affinity (butyl thioester 20 = 44% vs butyl sulfide 15 = 37 % and C-2 diazomethyl ketone21 = 10.5%vs C-3 diazomethyl ketone 18 = 2.8%). However, even though a thioester linkage might increase the binding affinity of the hexestrol diazirine for the receptor, their hydrolytic lability compared to the sulfide linkage makes them less desirable. The binding affinity of [3H]hexestrol diazirine 1 to the estrogen receptor was determined directly using a binding titration assay. The direct binding plots for the PHIhexestrol diazirine (Figure 1, panelB) show moderate levels of nonspecific binding. This is consistent with the higher lipophilicity of this compound (measured as the octanolwater partition coefficient, log P ) compared to estradiol:

146

Bergmann et ai.

Bioconjugate Chem., Vol. 5, No. 2, 1994 100

Kd (1) = 0.98 nM Kd (E2) = 0.19 nM Kd (E,)/Kd (1) = 20%

80

I

ae

v

P5 60 5U 40

5

n

20

0 0.0

3.5

,

0.2

0.4

I

0.8

0.6

nM Bound I

',O~ 2.5

0.0

v

0

I'/

1.0

1.2

I

1.4

I

Non-specific

I

I

I

I

5

10

15

20

n M Free Figure 1. Binding assay for [3H]hexestroldiazirine1.Rat uterine cytosol was incubated at 0 "C for 4 h with various concentrations of tritium-labeledligand ([3H]-1or [3H]-E2)in the absence (for total binding) or presence (for nonspecific binding) of a 100-fold excess of unlabeled estradiol. Aliquots of the incubation solution were counted to determine the concentration of total tritiumlabeled ligand present. The incubation solutionswere then treated with charcoal-dextran, and the concentration of the bound tritium-labeled ligand was determined. Specific binding is the difference between total and nonspecific binding. Data are presented as Scatchard (panel A) or direct (panel B) plots. log P (estradiol) = 3.52; log P (1) = 4.63. A direct comparison of the specific binding curves for the PHIhexestrol diazirine and for [3H]estradiol are presented as a Scatchard (23) plot (Figure 1, panel A). From this plot [3H]hexestrol diazirine 1 has an affinity of & = 0.98 nM for ER, or 20% that of [3H]estradiol. This value compares closely to that observed in the competitive binding assay (RBA = 17%). Solution Photolysis and Estrogen Receptor Photoinactivation by the Hexestrol Diazirine 1. A preliminary investigation of the time course of photolysis of the hexestrol diazirine in solution was effected by irradiation with a mercury arc lamp through an aqueous CuSO4 filter (effective X > 315 nm) ( 4 b ) . Hexestrol was added as the internal standard, and the progress of the photolysis was monitored by HPLC a t 254 nm (Figure 2). Under these conditions, more than 70% of the diazirine has undergone photolysis by 5 min, with only 10% remaining a t 15 min. Two minor products which increased with time of photolysis (to 25-30% of the expected maximum) had

0

5

10 15 PHOTOLYSIS (min)

20

Figure 2. Time course of photolysis of hexestrol diazirine 1 (lo4 M in 5% DMF/TEA buffer). Photolysis was at >315 nm at 0 O C ; aliquots were removed at various time points and extracted with EtOAc, and the quantity of hexestrol diazirine remaining was determined by reference to the internal standard (hexestrol)by HPLC.

retention times similar t o the hexestrol butenes that might be produced from the carbene intermediate by a 1,2hydrogen shift ( 1 1 ) ,but these were not identified definitively. No other significant products were observed. However, products that might have formed from insertion into water or buffer components might have been too polar to be observed under the normal-phase HPLC conditions we employed. T o investigate the photoinactivation efficiency (PIE) ( 4 b ) of the hexestrol diazirine 1 in the binding site of the estrogen receptor (ER), we photolyzed (at >315 nm) a receptor cytosol preparation that had been preincubated with the hexestrol diazirine. Upon photocovalent attachment, the receptor-ligand complex loses its capability for reversible binding. This is measured by a radiotracer exchange assay together with certain controls: (1) to monitor the stability of ER under photolysis conditions, (2) to monitor the nonspecific component of photoinactivation, and (3) to ensure no photoinactivation results from the photolysis products. As shown in Figure 3, hexestrol diazirine reaches 31 ?4 photoinactivation within 30 min of photolysis, and the receptor is stable under these photolysis conditions for up to 6 h. No photoinactivation is observed when the receptor is incubated with either estradiol or the prephotolyzed hexestrol diazirine. Photolysis for shorter periods showed that most of the photoinactivation occurs within the first 5 min. (In order to determine the extent of photocovalent attachment of hexestrol diazirine to the ER and the extent of nonspecific attachment, it is necessary to study the photolysis with [3H]hexestrol diazirine 1; see below.) Inactivation efficiencies for hexestrol diazirine 1 and related hexestrol derivatives are given in Table 1. The percent inactivation of ER by hexestrol diazirine 1 (31 7%) was greater than the inactivation with the other photoactivatable derivatives (aryl azide 3 (9-1576) and diazo ketone 19 (15%)),although it was not as high as the inactivation by the electrophilic aziridines (4 and 16b;50100%) * Photolabeling of the Estrogen Receptor with Hexestrol Diazirine [3H]-l. [3HIHexestrol diazirine 1 was photolyzed at >315 nm in rat uterine cytosol preparations, and the percent of specific and nonspecific attachment

Bioconjugate Chem., Vol. 5, No. 2, 1994 147

A

l o o t - 7--m- - - -

-v---

0.8 L

n

25

0.6

a 40

0.2

20

-

0.4

Non-specific 0

1

2

3

4

PHOTOLYSIS (h)

5

6

b I

7

B

"

I

401

0 Reversible Binding

20

01 0

2.5

I

I

I

I

I

I

5

10

15

20

25

30

t

Attachment

35

PHOTOLYSIS (min) Figure 3. Photoinactivation of ER by hexestroldiazirine 1during 6 h (panel A) or 30 min (panel B) of photolysis. Cytosol was incubated with diazirine 1 in the presence or absence of a 100fold excess of estradiol for 1 h at 0 "C and then photolyzed at >315 nm for various times. Followingcharcoal-dextran treatment to remove free ligand, the cytosol was exchanged at room temperature for 20 h against [3H]estradiol.Photoinactivation of reversible binding is seen as a loss of exchangeable sites and plotted as a percent of initial binding before photolysis. The percent specific photoinactivation is shown as a vertical double headed arrow, with a value given in parentheses. Control experimenb included incubation with E2 alone to measure photodestruction of the protein and incubation with prephotolyzed 1 to measure inactivation by photoproducts. was examined by a filter disk-solvent extraction assay (24). The binding was done in the presence of DMF (5%) (5c, 30), and the sample was pretreated with charcoal t o remove excess free reagent before photolysis. Low concentrations of DMF reduce the extent of nonspecific binding of lipophilic ligands, thereby increasing the fraction of the ligand that is available for binding to the ER (30);this increases the extent and selectivity of ER labeling (5c). Photoattachment efficiency is defined as the amount of ER covalently labeled after photolysis as a percent of ER occupied reversibly by the affinity reagent; photoattachment Selectivity is defined as the amount of ER labeled as a percent of the total protein covalently labeled upon photolysis (5c). The time course of specific covalent attachment is shown in Figure 4. The specific attachment rises with time of photolysis and levels off after - 5 min. Diazirine 1exhibits

g

L

'H]-(l)

Prephotolyzed [ HI-(l)

Figure 5. Binding properties of [3H]hexestrol diazirine 1 and prephotolyzed [3H]hexestrol diazirine 1. A sample of [3H]hexestrol diazirine 1 was prephotolyzed for 30 min at >315 nm in ethanol. Rat uterine cytosol was incubated for 1 h at 0 "C in the dark with [3Hl hexestrol diazirine 1 and the prephotolyzed sample in the presence and absence of a 100-fold excess of unlabeled estradiol. After charcoal treatment to remove reversible binding, a portion was photolyzed at >315 nm for 30 min and assayed for photoattachment by ethanol disk assay.

moderate photoattachment efficiency (29 % ) and high photoattachment selectivity (90% ) under these optimal conditions (5% DMF and charcoal treatment prior to photolysis). The attachment efficiency is comparable to the rate and extent predicted by the photolysis time course (Figure 2) and the photoinactivation assay (Figure 3,31%), respectively. The extent of ER binding and photolabeling by the photoproducts of [3Hlhexestrol diazirine 1 was also examined (Figure 5 ) . A prephotolyzed sample, prepared by irradiating [3H]hexestrol diazirine 1 in EtOH for 30

148

Bergmann et al.

Bioconjugate Chem., Vol. 5, No. 2, 1994

1000

2000

800

1500 600

E 1000

E

4

0

400

500 200 0 0

5

10

15

20

25

0

30

0

5

10

Slice Number

15

20

25

30

Slice Number l

o

O

o

:

~

~

.

I

D

m

zx

8

z

100:

10

min in a Pyrex tube a t X > 315 nm, showed reversible ER binding equivalent to [3Hlestradiol and PHI hexestrol diazirine 1 but gave negligible photoattachment. Characterization of the ER Covalently Labeled with [3H]HexestrolDiazirine. SDS-polyacrylamide gel electrophoretic analysis of ER covalentlylabeled with PHIhexestrol diazirine 1 in the presence and absence of excess estradiol, and with and without pretreatment with charcoal, is shown in Figure 6. The major peak migrates with a MI of -65 000 and corresponds to ER; a minor peak, a t a MI of -53 000, probably represents a proteolysis fragment of ER (31)(panel A). Similar results have been observed with PHItamoxifen aziridine, which is a well characterized electrophilic affinity label for the ER (panel B) ( 1 7u,b). The labeling of both fragments is blocked by addition of excess unlabeled estradiol. In the experiment in panel A, excess [ 3 H ] h e x e ~ t rdiazirine ~l was removed by treatment with charcoal prior to photolysis. If free ligand is not removed, the extent of nonspecific labeling is much greater (panel C). DISCUSSION

We have made a significant effort to develop affinity labeling agents for the estrogen receptor (ER) that will be efficient and selective in their labeling and will provide information about the composition of the ligand binding site of the receptor (4,5,8,15-17). Such information is of great importance in elucidating structure-function

I

'

~

~

1: I3.P'

ul

E

~

:

I

.

,

.

,

.

relationships of these receptors and has proven to be very useful in the development of three-dimensional models for hormone binding domains of these receptors (32). The most successful affinity labeling agent for ER, prepared to date, is tamoxifen aziridine, an electrophilic derivative of an estrogen antagonist that labels ER with an efficiency approaching 100% and with selectivity as high as 90% ( 1 7u,b). The most promising photoaffinity label is a benzothiophene azide recently prepared by our group, which demonstrates high receptor binding affinity (66 9% ), high specific photoinactivation efficiency (55%), and good photocovalent attachment (25 % ) (5c). Despite this promise, the photocovalent link of this reagent with ER appears to be chemically unstable (K. E. Carlson, J. A. Katzenellenbogen, unpublished results). In this report, we present the synthesis and evaluation of a hexestrol-based photolabeling agent with a photosensitive diazirine group. Diazirines were initially developed to overcome some of the deficiencies of nitreneyielding aryl azides (12),and their increasing use reflects their synthetic accessibility, their chemical and thermal stability, and their efficient photolabeling reaction via highly reactive carbene intermediates ( 1 1 ) . We chose to attach the diazirine function to the nonsteroidal estrogen hexestrol because several hexestrol derivatives, functionalized on the side chain, still exhibit relatively high affinity for ER (23, 29, 33). Hexestrol diazirine 1 showed a good binding affinity to

Hexestrol Dlaririne Photoaffinity Labeling Reagent ER (17% that of estradiol) that was slightly less than a protioanalog (butylpentylsulfide 15,37 %),butwas higher than the aziridines of similar structure (16a-c,; 5.8%,1.8%, 0.396,respectively). The specific binding of PHI-1 to ER, relative to that of I3H1estradiol, determined by Scatchard analysis, was comparable a t 20 % The photolysis of diazirine 1 proceeds smoothly and rapidly at long wavelengths (>315 nm), where ER appears to be very stable to photodegradation. The photoattachment efficiency of 13Hldiazirine 1to ER (29?G ), determined directly, is almost exactly the same as the photoinactivation efficiency (31% 1, determined indirectly on unlabeled diazirine 1 by a photolysis exchange assay. While, in principle, these efficiencies may be equal, we have rarely found the attachment efficiency of a photoaffinity labeling agent to be equal to its photoinactivation efficiency, since many processes other than covalent attachment may contribute to the loss of exchangable sites in the indirect photoinactivation assay (4b, 34). The 30% efficiency for I3H1-1 is also relatively high for steroid receptor photolabeling agents, and with proper experimental protocols (charcoal pretreatment), the labeling of ER is also very selective, approaching 9076, in rat uterine cytosol preparations, despite the relatively high lipophilicity of diazirine 1. While competition for covalent labeling of ER by pretreatment with unlabeled estradiol is generally considered a good criterion for receptor specific labeling, the charcoal pretreatment protocol compromises this and requires additional verification of this selectivity. SDSpolyacrylamide gel electrophoretic analysis of PHI diazirine 1 labeled ER shows a pattern equivalent to that obtained when ER is labeled with the well characterized electrophilic affinity labeling agent tamoxifen aziridine (17u,b). Although photoaffinity labeling agents have been used to identify binding site residues in the progesterone receptor and the glucocorticoid receptor (351, in ER the only sites identified have been two cysteine residues labeled by the electrophilic agent tamoxifen aziridine (17~). The aziridine function in this reagent is weakly electrophilic, so the reagent labels only the most nucleophilic residue, cysteine. In the development of diazirine 1 and its use to label ER, we hope to be able to identify additional residues in the ligand binding site. Such work is currently underway.

.

ACKNOWLEDGMENT We are grateful for support of this research through a grant from the National Institutes of Health (PHS 5R37 DK15556). High-resolution mass spectra were obtained on instruments supported by the National Institutes of Health (GM 27029), and 'H NMR were obtained on a VarianQE 300 MHz instrument supported by the National Institutes of Health (PHS l S l 0 RR 02299). LITERATURE CITED (1) (a) Katzenellenbogen, J. A., Kilbourn, M. R., and Carlson, K. E. (1980)Photosensitive Steroids as Probes of Estrogen Receptors Sites. Ann. N . Y.Acad. Sci. 346,18-30.(b)Knowles, J. R. (1972)Photogenerated Reagents for Biological ReceptorSite Labeling. Acc. Chem. Res. 5,155-60.(c) Bayley, H. (1983)

Photogenerated Reagents in Biochemical and Molecular Biology, Elsvier, Amsterdam. (2) (a) Plapp, B. V. (1982)Application of Affinity Labeling for Studying Structure and Function of Enzymes. Methods Enzymol. 87,469-99. (b) Shih, L. B., and Bayley, H. (1985) ACarbene-YieldingAmino Acid for Incorporation into Peptide Photoaffinity Reagents. Anal. Biochem. 144, 132-41.

Sioconjugate Chem., Vol. 5, No. 2, 1994

140

(3) (a) Brunner, J., and Richards, F. M. (1980) Analysis of

Membranes Photolabeled with Lipid Analogues.J.Biol. Chem. 255,3319-28.(b) Delfino,J. M., Schreiber,S. L., and Richards, F. M. (1993)Design, Synthesis, and Properties of a Photo-

activatable Membrane-Spanning Phospholipidic Probe. J.Am. 115,3458-74.(c) Lundblad, R. L., and Noyes, C. Chem. SOC. M. (1984)Chemical Reagents for Protein Modification, Vola. 1 and 2, CRC Press, Boca Raton, FL. (4) (a) Katzenellenbogen, J. A., Carlson, K. E., Johnson, H. J., and Myers, H. N. (1977)Estrogen Photoaffinity Labels 11: Reversible Binding and CovalentAttachment of Photosensitive Hexestrol Derivatives to the Uterine Estrogen Receptor. Biochemistry 16,1970-76.(b) Katzenellenbogen,J. A., Johnson, H. J., Carlson, K. E., and Myers, H. N. The Photoreactivity of Some Light Sensitive Estrogen Derivatives. The Use of as Exchange Assay to Determine Their Photoinactivation with Rat Uterine Estrogen Binding Protein. Biochemistry 13,298694. (5) (a) Katzenellenbogen, J. A. (1978)Photoaffinity Labeling of Estrogen Receptor. Fed. Proc. 37,174-78. (b) Pinney, K. G., and Katzenellenbogen, J. A. (1991)Synthesisof aTetrafluoro-

Substituted Aryl Azide and Ita Protio Analogue as Photoaffinity Labeling Reagents for the Estrogen Receptor. J.Org. Chem.56,3125-33.(c)Pinney, K. G., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1991)Efficient and Selective Photoaffinity Labeling of the Estrogen Receptor Using Two Nonsteroidal Ligands That Embody Aryl Azide or TetrafluoroarylAzide Photoreactive Functions. Biochemistry 30,242131. (6) Pinney, K. G.Design,Synthesis, and BiochemicalEvaluation of Novel Photoaffinity Labeling Reagents for the Estrogen and Progesterone Receptors. Ph. D. Thesis, University of Illinois at Urbana-Champaign, 1990. (Acyl azides were proposed as photoactivatible groups for affinity labels ((a)Sigman, M. E., Autrey, T., and Schuster, G. B. (1988)Aroylnitrenes with Singlet Ground States: Photochemistry of AcetylSubstituted Aroyl and AryloxycarbonylAzides. J.Am. Chem. SOC. 110,4297.(b) Melvin, T., and Schuster, G. B. (1990)The Photochemistry of Acetyl-Substituted Aroyl Azides: The Design of Photolabeling Agents for Inert Sites in Hydrophobic Regions. Photochem. Photobiol. 31,155);however, their lack of chemical stability greatly decreases their receptor attachment efficiency and selectivity.) (7) Link, R. P., Kutner, A., Schnoes, H. K., and DeLuca, H. F. (1987)Photoaffinity Labeling of Serum Vitamin D Binding Ds. BiochemProtein by 3-Deoxy-3-azido-25-hydroxyvitamin istry 26, 3957-64. (8) Katzenellenbogen, J. A., and Katzenellenbogen, B. S. (1984) Affinity Labeling of Receptors for Steroid and Thyroid Hormones. Vitamins and Hormones, pp 213-74,Vol. 41, Academic, New York. (9) (a) Katzenellenbogen, J. A., and Hsiung, H. M. (1975) Iodohexestrols I. The Synthesis and Photoreactivity of Iodinated Hexestrol Derivatives. Biochemistry 14, 1736-41. (b) Katzenellenbogen, J. A., Hsiung, H. M., Carlson, K. E., McGuire, W. L., Kraay, R. J., and Katzenellenbogen, B. S. (1975)Iodohexestrols 11.Characterization of the Binding and Estrogenic Activityof Iodinated Hexestrol Derivatives,In Vitro and In Vivo. Biochemistry 14, 1742-50. (10) Church, R. F. R., Kende, A. S., and Weiss, M. J. (1965) Diazirines.I. SomeObservations on the Scopeof the AmmoniaHydroxylamine-0-sulfonic Acid Diaziridine Synthesis. The Preparation of Certain Steroid Diaziridines and Diazirines.J. Am. Chem. SOC. 87,2665-71. (11) (a) Chemistry ofDiazirines (1987)(M. T. H. Liu, Ed.) CRC Press, Boca Raton, FL. (b) Modarelli, D. A., Morgan, S., and Platz, M. S. (1992)Carbene Formation, Hydrogen Migration, and Fluorescence in the Excited States of Dialkyldiazirines. J. Am. Chem. SOC. 114,7034-41.(c) Morgan, S., Jackson, J. E., and Platz, M. S. (1991)Laser Flash Photolysis Study of 113,2782-3. Adamantanylidene. J.Am. Chem. SOC. (12)Azides and Nitrenes (1984)(E. F. V. Scriven,Ed.) Academic, Orlando, FL. (13) (a) Nakayama, T. A., and Khorana, H. G. (1990)Synthesis of a New Photoactivatable Analog of 11-cis-Retinal. J. Org. Chem. 55, 4953-6. (b) Van Ceuenbroeck, J. Cl., Krebs, J.,

150

Bioconjugate Chem., Vol. 5, No. 2, 1994

Hanssens, I., and Van Cauwelaert, F. H. (1986) Study of a Hydrophobic Site on Bovine a-Lactalbumin by Labeling With ['25I]-TID. J . Biochem. Biophys. Res. Commun. 138,604-10. (c) White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991)The Hydrophobic Photoreagent 3-(Trifluoromethyl)3-m-([126I]iodophenyl)diazirine ([125I]TID) Is a Novel Noncompetitive Antagonist of the Nicotinic Acetylcholine Receptor. J. BioK Chem. 266, 21595-607. (14) (a) Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1988) In Affinity Labeling and Cloning of Steroid and Thyroid Hormone Receptors (H. Gronemeyer, Ed.) pp 87108, Ellis Horwood Ltd., Chichester, England. (b) Katzenellenbogen, J. A. (1977) In Biochemical Actions of Hormones (G. Litwack, Ed.) Vol. 4, Chapter 1,pp 1-84, Academic, New York. (15) (a) Katzenellenbogen, J. A., Johnson, H. J., Jr., and Myers, H. N. (1973) Photoaffinity Labels for Estrogen Binding Proteins of Rat Uterus. Biochemistry 12,4085-92. (b) Katzenellenbogen, J. A,, Myers, H. N., and Johnson, H. J., Jr. (1973) Reagents for Photoaffinity Labeling of Estrogen Binding Proteins. Synthesis of Some Azide and Diazo Derivatives of Estradiol, Estrone, and Hexestrol. J. Org. Chem. 38,3525-33. (c) Katzenellenbogen, J. A., Myers, H. N., Johnson, H. J., Jr., Kempton, R. J., and Carlson, K. E. (1977) Estrogen Photoaffinity Labels I: Chemical and Radiochemical Synthesis of Hexestrol Diazoketone and Azide Derivatives: Photochemical Studies in Solution. Biochemistry 16, 1964-70. (16) (a) Elliston, J. F., Zablocki, J. A., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1987) Ketononestrol Aziridine, an Agonistic Estrogen Receptor Affinity Label: Study of Its Bioactivity and Estrogen Receptor Covalent Labeling. Endocrinology 121,667-76. (b) Zablocki,J. A., Katzenellenbogen, J. A., Carlson, K. E., Norman, M. J., and Katzenellenbogen, B. S. (1987)Estrogenic Affinity Labels: Synthesis, Irreversible Receptor Binding, and Bioactivity of Aziridine-Substituted Hexestrol Derivatives. J. Med. Chem. 30, 829-38. (17) (a) Harlow, K. W., Smith, D. M., Katzenellenbogen, J. A., Greene,G. L., and Katzenellenbogen,B. S. (1989)Identification of Cysteine-530as the Covalent Attachment Site of an Affinity Labeling Estrogen (Ketononestrol Aziridine)and Antiestrogen (Tamoxifen Aziridine) in the Human Estrogen Receptor. J . Biol. Chem.264,17476-85. (b)Katzenellenbogen,J. A., Carbon, K. E., Heiman, D. F., Robertson, D. W., Wei, L. L., and Katzenellenbogen, B. S. (1983)Efficient and Highly Selective Labeling of the Estrogen Receptor with [SHITamoxifen Aziridine. J. Biol. Chem. 258, 3487-95. (c) Salituro, F. G., Elliston, J. F., Carlson, K. E., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (1986) [126]Iododesethyl Tamoxifen Aziridine: Synthesis and Covalent Labeling of the Estrogen Receptor with an Iodine-Labeled Affinity Label. Steroids 48, 287-313. (d) Simpson, D. M., Elliston, J. J., and Katzenellenbogen, J. A. (1987) Desmethylnafoxidine Aziridine: An Electrophilic Affinity Label for the Estrogen Receptor with High Efficiency and Selectivity. J . Steroid Biochem. 28,23345. (18) Kuhn, C.-S., Lehmann, J.,and Sandhoff, K. (1992)Efficient Photoaffinity Labeling of Human @-HexosaminidaseA. Synthesis and Application of 3-Azi-1-[(2-acetamido-2-deoxy-l-P~-glucopyranosyl)thio]-and -galactopyranosyl)thiol butane. Bioconjugate Chem. 3, 230-33. (19) Church, R. F. R., and Weiss, W. J. (1970) Diazirines. 11. Synthesis and Properties of Small Functionalized Diazirine Molecules.SomeObservations on the Reaction of A Diaziridine with Iodine-Iodine Ion System. J. Org. Chem. 35, 2465-71. (20) Still, W. C., Kahn, M., and Mitra, A. P. (1978) Rapid ChromatographicTechnique for Preparative Separations With Moderate Resolution. J . Org. Chem. 43, 2923-25. (21) Bindal, R. D., Carlson, K. E., Reiner, G. C. A,, and Katzenellenbogen, J. A. (1987) ll@-Chloromethyl[3H]estradiol-17@:A Very High Affinity, Reversible Ligand for the Estrogen Receptor. J . Steroid Biochem. 28, 361-370. (22) Carlson, K. E., Sun, L.-H. K., and Katzenellenbogen, J. A. (1977) Characterization of Trypsin-Treated Forms of the Estrogen Receptor From Rat and Lamb Uterus. Biochemistry 16, 4288-96.

Bergmann et al.

(23) Scatchard, G. (1949) The Attractions of Proteins for Small Molecules and Ions. Ann. N. Y . Acad. Sei. 51, 660-72. (24) Katzenellenbogen,J. A., Ruh, T. S.,Carbon,K. E., Iwamoto, H. S., and Gorski, J. (1975) Ultraviolet Photosensitivity of the Estrogen Binding Protein from Rat Uterus, Wavelength and Ligand Dependence. Photocovalent Attachment of Estrogens to Protein. Biochemistry 14, 2310-16. (25) Southwick,P. L., and Kirchner, J. R. (1962)The MorpholineIodophenylacetylene Adduct or Charge-Transfer Complex. Formation and Conversion to N-Styrylmorpholine. J. Org. Chem. 27, 3305-8. (26) Evans, E. A. (1974) Tritium and its Compounds, pp 318415, John Wiley and Sons, New York. (Other references which use this procedure: 17b,d and 16a.) (27) Schmitz, E., and Ohme, R. (1961) Preparation and Transformations of Diazirines. Chem. Ber. 94, 2166-73. (28) With electrophilic affinity labeling agents, such as the aziridine compounds 4,16, and TAZ, the determination of the receptor binding affinity is probably distorted by simultaneous covalent attachment of these species to the estrogen receptor. Thus, these values should be considered "apparent" RBA values. (29) (a) Landvatter, S. W., and Katzenellenbogen, J. A. (1982) Nonsteroidal Estrogens: Synthesis and Estrogen Receptor Binding Affinity of Derivatives of (3R*,4S*)-3,4-Bis(4-hydroxypheny1)hexane (Hexestrol) and (2R*,3S*)-2,3-Bis(4hydroxypheny1)pentane (Norhexestrol) Functionalized of the Side Chain. J . Med. Chem. 25,1300-7. (b) Landvatter, S. W., and Katzenellenbogen, J. A. (1981) Stereochemical Considerations in the Binding of Non-Steroidal Estrogens to the Estrogen Receptor. Molec. Pharmacol. 20, 43-51. (30) Katzenellenbogen, J. A,, Heiman, D. F., Carlson, K. E., and Lloyd, J. E. (1982) In Receptor Binding Radiotracers (W. C. Eckelman, Ed.) Vol. 1, pp 93-126. CRC Press, Boca Raton, FL. (31) (a) Sheen, Y. Y., and Katzenellenbogen, B. S. (1987) Antiestrogen Stimulation of the Production of a 37000 Molecular Weight Secreted Protein and Estrogen Stimulation of the Production of a 32000 Molecular Weight Secreted Protein in MCF-7 Human Breast Cancer Cells. Endocrinology 120,1140-51. (b) Monsma, F. J., Jr., Katzenellenbogen, B. S., Miller, M. A., Ziegler, Y. S.,and Katzenellenbogen, J. A. (1984) Characterization of the Estrogen Receptor and Its Dynamics in MCF-7 Human Breast Cancer Cells Using a Covalently Attaching Antiestrogen. Endocrinology 115, 143-53. (32) (a) Goldstein, R. A., Katzenellenbogen, J. A., LutheySchulten, A. 2. A., Seielstad, D. A., and Wolynes, P. G. (1993) Three-dimensionalModel for the Hormone Binding Domains of Steroid Receptors. Proc. Natl. Acad. Sei. U.S.A. 90,99499953. (b) Lemesle-Varloot, L., Ojasoo, T., Mornon, J. P., and Raynaud, J. P. (1992) A Model for the Determination of the 3D-Spatial Distribution of the Functions of the HormoneBinding Domain of Receptors that Bind 3-Keto-4-eneSteroids. J . Steroid Biochem. Molec. Biol. 41, 369-88. (33) Goswami, R., Harsy, S. G., Heiman, D. F., and Katzenellenbogen, J. A. (1980) Estrogen Receptor-Based Imaging Agents 2. The Synthesis and Receptor Binding Affinity of Side-Chain Halogenated HexestrolDerivatives. J. Med. Chem. 23,1002-8. (34) Cridland, N. A., Wright, C. V. E., McKenzie, E. A., and Knowland, J. (1990) Selective Photochemical Treatment of Estrogen Receptor in a Xenopus Liver Extract Destroys HormoneBindingand Transcriptional Activation but not DNA Binding. EMBO J. 9, 1859-66. (35) (a) Carlstedt-Duke, J., Stromstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J. A.; and Jornvall, H. (1988) Identification of Hormone-Interacting Amino Acid Residues Within the Steroid-Binding Domain of the Glucocorticoid Receptor in Relation to Other Steroid Hormone Receptors. J. Biol. Chem. 263,6842-6. (b) Stromstedt, P.-E., and Berkenstam, A., Jornvall, H., Gustafsson, J. A., and Carlstedt-Duke, J. (1990)Radiosequence Analysis of the Human Progesterone Receptor Changes with [3H]Promegestone.J. Biol. Chem.265, 12973-7.