Sioconjugafe Chem. 1995, 6, 1 15-1 22
115
Evaluation of a Highly Efficient Aryl Azide Photoaffinity Labeling Reagent for the Progesterone Receptor Philip R. Kym, Kathryn E. Carlson, a n d John A. Katzenellenbogen" Department of Chemistry, University of Illinois, Urbana, Illinois 61801. Received August 10,1994@
16a,17a-[(R)-1'-(4-Azidophenyl)ethylidenedioxy]pregn-4-ene-3,20-dione (7) was prepared in high specific activity tritium-labeled form (20 CYmmol) and shown to bind to the progesterone receptor with a n affinity (& = 0.80 nM) that is 47% of that of L3H1-R 5020 (Kd = 0.38nM). L3H1Progestinaryl azide 7 exhibits high photoattachment efficiency (60% at 1 h) compared to the commonly used progesterone receptor photoaffinity labeling reagent [3H]-R5020 (2.2% a t 1 h) and is the most efficient progesterone receptor photoaffinity labeling reagent prepared to date. The photoattachment observed with 7 proceeds in a time-dependent fashion, with most of the attachment occurring within the first 10 min of photolysis. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of two adducts of molecular weight 108 500 f 800 and 87 000 f 1 500 (n = 31, the same species as labeled by [3H]-R 5020. The ratio of progesterone receptor subunits A B was determined to be 3.3:lwith both r3H]progestin azide 7 and [3H]-R 5020. Information on the specific amino acid(s) that attach to the ligand during photolysis awaits further analysis of the covalently bound ligand-protein adduct.
INTRODUCTION
Recent advances in our understanding of the mechanism of action of the progesterone and other steroid hormone receptors have led to renewed interest in the development of synthetic probes that can bind to and modulate their function (1). Despite considerable progress in this area, many aspects of the interaction of progesterone with its receptor remain only poorly understood. Most of the work in this area has focused on establishing structure-function relationships of various portions of the receptor and uncovering details of events occurring after the hormone has bound to the receptor. The cellular processes leading up to hormone binding and determination of critical hormone-receptor interactions have received only limited attention. In the absence of crystallographic data for the steroid receptors, we are left with direct chemical probe methods such as affinity labeling and indirect approaches such as mutagenesis to define critical binding interactions. Photoaffinity labeling (2) (PAL) utilizes a masked reactive functionality on the molecular probe that reacts upon photolysis to reveal a reactive intermediate that can covalently attach to the receptor. Incubation of the PAL reagent with a steroid receptor in the dark, followed by photolysis of the receptor-ligand complex, increases the probability of labeling a residue within the steroid binding site. The reactive species, typically a carbene or a nitrene, can react with the receptor through nonspecific carbon-hydrogen bond insertion reactions. A variety of functional groups have been used as the photoreactive species, including azides (acyl, aroyl, aryl), diazirines, diazo ketones, conjugated enone systems, benzophenones, and nitroanisole derivatives (3). The search for efficient and selective photoafhity labeling (PAL)reagents for the progesterone receptor (PR) has relied primarily on progestins containing A,Bring dienone systems as the photoreactive functionality (4-6). While some of these compounds bind with high affinity to the progesterone receptor, none of them demonstrate the required combination of high receptor @Abstractpublished in Advance ACS Abstracts, January 1, 1995. 1043-1802/95/2906-0115$09.00/0
R 5020
1 RBA = 100% PAE = 2-5%
RU 486 2 RBA = 170% PAE = 0-2% HQC GHJ=O OAc
F ING
3 RBA = 78% P A E = 4%
DU 41165 4 RBA = 158% PAE = 57%
Figure 1. Photoaffinity labeling (PAL) reagents for the progesterone receptor. RBA is receptor binding affinity; PAE is photoattachment efficiency.
binding affinity (RBA), good photocovalent attachment efficiency, and low nonspecific binding necessary for efficient and selective labeling of the PR. The RousselUclaf compound, promegestone (R 5020, Figure 11, is currently the most widely used PAL reagent for the PR, in spite of displaying photocovalent attachment efficiencies (PAE) of only 2-5% (4a). While low photoattachment efficiencies may be sufficient for identifying some sites of ligand-receptor contact, more efficient photocovalent attachment may be necessary to identify other residues in the PR hormone binding domain that are critical for ligand binding. Protio- and tetrafluoroaryl azides incorporated into a 3-aroyl-2-arylbenzo[b]thiophenemolecular scaffold (Figure 2) have recently been shown to have relatively high photocovalent attachment efficiencies (20-30%) in pho0 1995 American Chemical Society
Kym et ai.
116 Bioconjugafe Chem., Vol. 6,No. 1, 1995
J=8.5Hz,2H),6.99(d,J=8.5Hz,2H),4.03-4.05(m, 2 H), 3.75-3.78 (m, 2 HI, 1.64(s,3H); I3C NMR 6 140.16, 139.53,126.84,118.76,108.50, 64.45,27.56. MS (CI, CH4) m l z 206 (M l), 178, 163, 151, 134, 87; HRMS calcd for C10H1202N3 206.1597,found 206.1592. 2-(2'-(Diethylamino)-3'H-azepin-5-yl)-2-methyl-l,3-dioxolane (IO). Aryl azide 9 (0.210g, 1.02 mmol) was HO dissolved in 2 mL of freshly distilled diethylamine in a 5 R=H photolysis tube and the solution degassed by three 6 R=F freeze-pump-thaw cycles before being flame-sealed under vacuum. The photolysis was carried out with a 450 W medium-pressure Hanovia mercury vapor arc lamp equipped with a saturated CuSO4 filter (effective wavelength > 315 nm). The photolysis was stopped after 2 h, the photolysis tube was cracked open, and the crude reaction mixture was poured into a mixture of EtOAc (25 mL) and water (25mL). The organic layer was extracted and dried over Na2S04. Removal of solvent in uacuo afforded 0.187 g of a brown oil. Purification by flash 7 R=H chromatography (6:1 hexane:EtOAc) provided starting 8 R=F material (0.125g, 59%), substituted azepine 10 (0.045g, Figure 2. Aryl azide-based photoaffinity labeling (PAL) re18%), and two more polar products (0.005g). The yield agents for the ER and proposed aryl azide PAL reagents for of 10 corrected for recovered starting material was 43%: the PR. IR (CHCl3) u 1686;'H NMR 6 7.09 (d, J = 8.1 Hz, 1 H), 5.80 (d, J = 8.1 Hz, 1 H), 5.23(t,J = 7.3 Hz, 1 H), 3.91toaffinity labeling studies with the estrogen receptor (7). 3.96 (m, 2 H), 3.74-3.79 (m, 2 H), 3.31-3.35 (m, 4 H), We have previously reported the incorporation of protio2.2-2.8(broad s, 2 H), 1.46 (s, 3 H), 1.05-1.10 (m, 6 H); and tetrafluoroaryl azides onto the progestin backbone 13CNMR 6 146.26,141.69,141.20,108.54,108.25,107.52, through a 16a,l7a-dioxolane ketal link (Figure 2) (8). 65.77,64.20,43.13,30.43,26.09. MS (EI, 70 eV) m l z These molecules were found to bind to the PR with 250 (M+, 53), 235 (37),221 (22),207 (26),179 (29),164 affinity comparable to the natural ligand, progesterone (221,135 (12),107 (25),84(1001,72 (31),43(74);HRMS (RBA of 7 = 15%, 8 = 14%, progesterone = 13%), and calcd for C14H22N202 250.1681,found 250.1681. exhibit high photoinactivation effiencies of the PR (PIE Radiochemical Synthesis and Purification. The of 7 = 80%, 8 = 80%). We now report the preparation of procedure for formation of L3H1-7by tritiolysis of iodoaryl the protioaryl azide 7 in high specific activity tritiumazide 11 was adapted from the procedure for hydrolabeled form and the evaluation of [3Hl-7 as a photoafgenolysis of 11 (8). A sample of iodo azide 11 (0.010g, finity labeling reagent for the PR. 0.016 mmol) and the 5% Paalumina (0.015g) was sent to Amersham Corp. (Arlington Heights, IL) to be radioEXPERIMENTAL PROCEDURES labeled with carrier-free tritium gas according to the General. Reaction progress was monitored by analytifollowing instructions. To a solution of iodoaryl azide 11 cal thin-layer chromatography using 0.25mm silica gel in 2 mL of THF add 0.015 g of 5% Paalumina and 7.0 glass-backed plates with F-254indicator (Merck). Flash ,uL of triethylamine. Expose the rapidly stirring solution chromatography was performed with Woelm 32-63 pm to 10 Ci of carrier-free tritium gas for 30 min a t 25 "C silica gel packing. Visualization was accomplished by under atmospheric pressure. Filter the solution through phosphomolybdic acid or ninhydrin spray reagents, ioa 1 cm plug of Celite to remove Pd catalyst and wash dine, o r UV illumination. with benzene (3 x 1 mL). Remove exchangeable tritium Proton (lH NMR) and carbon (13C NMR) magnetic using standard solvent exchange-evaporation cycles. resonance spectra were recorded a t 500 and 125 MHz, Dissolve the tritium-labeled sample in 25 mL of 9:l respectively, and chemical shifts are reported as ppm benzenelethanol and store a t -20 "C. downfield from an internal tetramethylsilane standard The total amount of tritium incorporated into the (6 scale). The data are reported in the following form: sample was 43 mCi (10% of theoretical). The 43 mCi chemical shift (multiplicity, coupling constant in Hz (if sample returned from Amersham was assayed by TLC applicable), number of protons). Only characteristic and HPLC. From coelution with unlabeled standards, infrared (IR) bands are reported (as cm-'1. Electron the crude material was found to be a mixture of [3Hlaryl ionization (EI) mass spectra data were obtained a t 70 azide 7 (50%), L3H1aryl amine 12 (40%), L3H1-base line eV and are reported in the following form: mlz (intensity material (lo%), and unlabeled iodoaryl azide 11. Flash relative to base peak = 100). 4'-Azidoacetophenone and 16a,17ad(R)-lf-(4-Azido-3- chromatography (silica gel, 2.5:1 hexane:EtOAc) of 18 mCi of the crude mixture efficiently separated the [3Hliodophenyl)ethylidenedioxy]pregn-4-ene-3,2O-dionewere aryl azide 7 from the other tritium-labeled products. prepared as previously described (8). While some of the unlabeled iodoaryl azide (4.0mg) was Chemical Synthesis.2-(4'-AzidophenyZ)-2-methyl-1,3recovered, a large amount was not separated from the dioxoZane (9). Aryl azide 13 (0.510g, 3.17 mmol) was [3H]aryl azide 7. Final purification of [3H]-7 was acheated at reflux in 50 mL of benzene with 5 mL of complished by preparative normal phase HPLC (0.9cm ethylene glycol and a catalytic amount of p-toluenex 50 cm, Whatman Partisil M-9 Si02 column) eluting sulfonic acid for 16 h; water was removed by azeotropic with 18% EtOAc in hexanes at a rate of 3 mumin. distillation using a Dean-Stark apparatus. Product Under these HPLC conditions, the I3H1-7had a retention isolation (aqueous NaHC03, EtOAc, brine, Na2S04) aftime of 38 min and the unlabeled iodo azide 11 had a forded 9 as a brown oil. Purification by flash chromaretention time of 42 min. After multiple injections, 4.3 tography (€91hexanes:EtOAc) provided 9 as a light yellow mCi of [3H]aryl azide 7 was isolated in pure form. oil (0.474g, 73%): IR (CHC13)u 2136;'H NMR 6 7.46(d, N3&R
P A \
I/
+
Bioconjugate Chem., Vol. 6,No. 1, 1995 117
Aryl Azide Photoaffinity Labeling Reagent Scheme 1 H3C
Pd/alumina triethylamine
3H,, 38,
n
12
"
10% radiochemical yield
Table 1. Comparison of Biological Data for Photoaffinity Labeling Reagents for the PR
compd no. 7 1 2
3 4
compd name PAA 7 R 5020 RU 486 ING DU 41165 progesterone
receptor binding affinity (RBA)" (%) PR (rat)b PR (humany (R5020 = 100) 15 100 170 78 158 13
(R5020 = 100)
photoinactivation efficiency (PIE) (%)at 60 min
photoattachment efficiency (%)
81 66
60 2-5
ndd nd
1.0% are the average of two or more determinations, which are generally reproducible to within 30% (relative error). Cytosol preparations were from estrogen-primed immature rat uterus, with PH1-R 5020 as tracer. Cytosol preparations were from human T 4 7 ~breast cancer cells, with [3H]-R5020 as tracer. nd = not determined. e na = not applicable. a
The specific activity of L3H1-7was determined by HPLC analysis to be 20 Ci/mmol. The UV detector sensitivity was calibrated by injecting a series of known quantities of unlabeled standard and determining the peak area; simultaneous measurement of peak area and radioactivity on an injected sample of [3H]-7 enabled the specific activity to be determined. The chemical and radiochemical purity were evaluated by reinjection onto HPLC and by normal phase TLC analysis. HPLC analysis showed a single peak by UV and radiometric analysis, a t the same retention time as our authentic unlabeled standard; TLC analysis (2:l hexanes:EtOAc) indicated a single peak of radioactivity with a n Rf of 0.51, identical to the Rf of a n unlabeled standard run on the same plate. Biological Procedures. Materials. Radioligands were obtained from the following sources: lea-ethyl-21-hydroxy-19-nor[6,7-3H]pregn-4-ene-3,20-dione (ORG 2058, 58 Ci/mmol), (Amersham Corp., Arlington Heights, IL); [17a-methyl-3Hlpromegestone(R 5020),86 Ci/mmol, (DuPont New England Nuclear, Boston, MA). Unlabeled ligands: promegestone (DuPont New England Nuclear, Boston, MA), ORG 2058 (kindly supplied by Dr. F. Zeelen, Organon Corp., Oss, The Netherlands), and estradiol (Sigma Chemical Co., St. Louis, MO). Preparation of Cytosol. The progesterone receptor (PR) levels in the uteri of immature rats were induced by estrogen treatment. Immature female Sprague-Dawley rats (19 days, 60 g) were given three daily subcutaneous injections of 5 pg of estradiol in 0.1 mL of sunflower seed oil-ethanol, prepared fresh daily. The cytosol was prepared 24 h after the last injection as previously reported (9). All cytosol for the progesterone receptor was prepared in PR buffer (0.01 M Tris-HC1:0.0015 M EDTA 0.02% sodium azide:20 mM sodium molybdate:0.012 M mercaptoethanol:20% glycerol, pH 7.4 a t 25 "C) and stored in liquid nitrogen. In all studies, glucocorticoid receptor sites in the cytosol preparations were saturated by the addition of 1pM hydrocortisone. Scatchard Assay. Uterine progesterone cytosol was incubated a t 0 "C for 4 h with various concentrations of 3H-ligand in the absence or presence of a 100-fold excess of unlabeled ORG 2058. Aliquots of the incubation solution were counted to determine the concentration of total 3H-steroid. The incubation solutions were then
treated with charcoal-dextran and the bound 3H-steroid determined. Data were processed according to the method of Scatchard (10). Photolysis. Photolysis was routinely carried out at '315 nm (450 W mercury vapor lamp, Hanovia L679A, surrounded by a solution filter of saturated aqueous copper(I1) sulfate) a t 2-4 "C employing Pyrex reaction vessels as previously described (11). Photoattachment Assay. Rat uterine cytosol was incubated for 1h a t 0 "C with 25 nM [3H]-labeledligand in the presence or absence of a 100-fold excess of nonphotoreactive blocking agent, ORG 2058, and photolyzed a t '315 nm. Covalent binding of labeled ligands was measured directly by a filter disk assay described previously for the estrogen receptor (12). Electrophoresis. SDS electrophoresis samples and gels were prepared as previously reported (13)with standard proteins of phosphorylase B (MW 97 4001, bovine serum albumin (MW 67 000), ovalbumin (MW 446001, and carbonic anhydrase (MW 29 000). RESULTS
Radiochemical Synthesis. The preparation of [3H]7 was accomplished by a palladium-catalyzed hydrogenolysis of a n iodoaryl precursor using carrier-free tritium gas (Scheme 1). Our preparation of iodoaryl azide 11 and development of exchange reaction conditions using hydrogen gas have been previously reported (8).A sample of the aryl azide iodoprogestin 11 and the palladium on alumina catalyst that we had used in the hydrogen exchange reaction were sent to Amersham Corp. to be used in the labeling experiment with carrierfree [3H]H2 gas. Amersham carried out the tritiolysis using the same conditions that we had used in the hydrogenolysis. Under these reaction conditions, tritium incorporation proceeded in 10% radiochemical yield. The radiolabeled sample returned from Amersham was assayed by TLC and HPLC and found to be a mixture of L3H1aryl azide 7, [3H]arylamine 12, and unlabeled aryl azide iodoprogestin 11. Purification of the crude mixture by flash chromatography and normal phase HPLC (see Experimental Section for details) afforded pure L3H1aryl azide 7. The specific activity of [3H]-7 was determined
118 Sioconjugate Chem., Vol. 6 , No. 1, 1995
Kym et al.
2
fi]-Azlde 7
0
tots,
in
nMFree
A
28 --
I
non-specific
0
10
0
nM Free
\
Scatchard Plot
1
20
C
\ \t[3HJ-R 5020
0.4
0.8
1
nM Bound Figure 3. Direct binding curves for [3H]protioaryl azide 7 and L3HI-R 5020. Rat uterine cytosol was incubated a t 0 "C for 4 h with various concentrations of tritium-labeled ligand in the absence and presence of 100-fold excess of unlabeled ORG 2058. Aliquots of the incubation solution were counted to determine the concentration of total tritium-labeled ligand present. The incubation solutions were then treated with charcoal-dextran, and the concentration of the bound tritium-labeled ligand was determined. Data are presented as direct (panels A and B) or Scatchard (panel C ) binding plots.
by HPLC analysis to be 20 Ci/mmol, which is 70% of the theoretical maximum for incorporation of one tritium atom per molecule of azide.
Progesterone Receptor Binding Properties of Progestin Ketal 7. Progesterone Receptor Binding Studies. The relative binding affinity (RBA) (14) of the 16a,l7a-(methylenedioxy)progesteronesfor the progesterone receptor (PR) was previously determined at 0 "C by a competitive radiometric binding assay using [3Hl-R 5020 as tracer (8). The RBA values of the progestin aryl azide 7 and several other progestins are shown in Table 1. The data are reported relative to R 5020 which is assigned a value of 100%. The RBA of progestin aryl azide 7 was found to be 15% in rat uterine cytosol, comparable to the natural hormone, progesterone, which has a n RBA of 13%. In human PR, the binding affinity was somewhat higher (RBA = 71%). Direct Binding Assay of L3H1Progestin Ketal 7. The binding affinity of [3Hlprogestin aryl azide 7 was determined in rat uterine cytosol using a charcoal adsorption assay to remove free ligand. The direct binding plots (Figure 3, panels A and B) show that the [3H]progestin aryl azide 7 binds to the same number of specific PR sites
as does [3H]-R5020, but with somewhat reduced selectivity (more nonspecific binding). A comparison of the specific binding curves for 7 and [3H]-R5020 is presented as a Scatchard (10) plot (Figure 3, panel C). The 3Hlabeled aryl azide 7 has a n affinity (Kd = 0.80 nM) for the PR that is 47% of the affinity of L3H1-R 5020 (Kd = 0.38 nM). This is a somewhat higher affinity than measured by the RBA assay. Many progestins with high affinity for PR, like R 5020, associate slowly with the PR (15).The direct assay, incubated for only 4 h, may not have allowed the progestins to reach complete equilibrium, as seen after 18 h with the RBA assay. Photoreactive Properties of Progestin Aryl Azide 7. Photoinactivation Assay. In our earlier study (8),we reported on a preliminary investigation of the photochemical behavior of nonradiolabeled 7 in the active site of the PR. Its photoinactivation efficiency (PIE) for PR was determined by photolysis of the PR complex formed by incubation of 7 with rat uterine cytosol preparations of PR for 4 h a t 0 "C (12). Irradiation of the receptorligand complexes was conducted at 254 nm or >315 nm, and the loss of reversible binding capacity of the PR was measured by an exchange assay using L3H1-R 5020. Control experiments in which the receptor sites were blocked with the nonphotoactive progestin ORG 2058 demonstrated high levels of PR decomposition when irradiation was carried out at 254 nm (22). Irradiation at '315 nm did not cause this protein damage and resulted in highly specific photoinactivation of the PR. By this assay, progestin aryl azide 7 demonstrated the highest photoinactivation efficiency (80%)of any potential photoaffinity labeling reagent for the PR tested to date by this method (Table 1, column 5, and ref 8). Furthermore, the ability of 7 to inactivate the receptor upon photolysis was shown to be completely specific for the PR binding site. Blocking of the PR binding site with ORG 2058 prior to incubation with the photolabeling ligand 7 eliminated any photoinactivation of PR reversible binding capacity (data not shown, see ref 8). Photolabeling of the Progesterone Receptor with [3H]Progestin Ketal 7. [3HlProgestin aryl azide 7 and 3[Hl-R 5020 were photolyzed a t >315 nm in rat uterine cytosol preparations, and the percent of specific and nonspecific attachment was examined by a filter disk-solvent extraction assay (12). The PR specificity of the covalent photolabeling can be determined by a simultaneous measurement of the nonspecific photolabeling done in the presence of a n excess of unlabeled, nonphotoreactive blocking agent, ORG 2058. The difference between the labeling in the absence or presence of ORG 2058 is the PR specific labeling. The photoattachment efficiency is defined as the amount of PR covalently labeled as a percent of PR which was occupied reversibly by the synthetic progestin; the photoattachment selectivity is defined as the amount of specific PR labeled as a percent of the total protein labeled after photolysis. The time course of specific photoattachment is illustrated in Figure 4. [3HlProgestinaryl azide 7 exhibits high photoattachment efficiency (60% at 1 h). It is, in fact, the most efficient progesterone receptor PAL reagent prepared to date (see Table 1, column 6), being much higher than the commonly used PR PAL reagent [3H]-R 5020 (2.2% a t 1 h; Figure 4A). The photoattachment with 7 proceeds in a time-dependent fashion, with most of the attachment occurring within the first 10 min of photolysis. The attachment selectivity of 7 is relatively high a t early time points (65% at 2 min) but falls due to increasing levels of nonspecific attachment as the photolysis experiment proceeds (30% a t 1 h; Figure 4B).
Bioconjugate Chem., Vol. 6,No. 1, 1995 119
Aryl Azide Photoaffinity Labeling Reagent "1
*0 z
A
.
[3HJ-ProgestinAzide 7
A
Aryl Azide
2000.
P a W a
20-
u
B
400.
.
R5020 R ~ O ~ O + P
[3H]-R 5020
A
Total
5
E
10
15
20
25
30
GEL SLICE
0
w
Nonspecific
a
L
0
'IL
. I
3
Specific
1
I 0
20
40
60
60
MINUTES OF PHOTOLYSIS Figure 4. Time-course of photoattachment of [3Hlprogestin azide 7 with the PR. Rat uterine cytosol was incubated for 1h a t 0 "C with 25 nM [3Hlprogestin azide 7 or 25 nM [3H]-R 5020 with or without a 100-fold excess of unlabeled blocking agent ORG 2058 and then photolyzed at > 315 nm for the times indicated. Covalent attachment was measured by an ethanol disk assay (22).Shown in panel B are the total (no ORG 2058 blocking) and the nonspecific (ORG 2058 blocked) attachment; the specific attachment is the difference between these values. A direct comparison of the efficiency of the specific photoattachment efficiencies of L3Hlprogestin azide 7 and PH1-R 5020 is shown in panel A.
At the concentration of [3H]progestin azide 7 used in the experiment shown in Figure 4B (25 nM),the selectivity of covalent attachment is lower than might be expected on the basis of the selectivity of reversible binding shown in Figure 3A. Part of this difference arises from the fact that only 60% of the reversibly bound receptor complexes undergo covalent attachment, but other proteins as well are being labeled by free or dissociated photolabel. Nevertheless, covalent labeling selectivity of 30% in an unfractionated receptor preparation (rat uterine cytosol) is quite high. The level of nonspecific attachment with [3H]progestin azide 7 is comparable to that seen with R 5020, and the nonspecific labeling is spread among many different proteins (cf. Figure 5AB below). Control experiments were performed to demonstrate that the attachment of 7 with the PR was a chromophoredependent, photoactivated process. First, incubation of 7 with the PR in the dark results in no covalent attachment. Prephotolysis of the L3H1progestinazide 7 for 15 min prior to incubation with the PR-rich cytosol resulted in a dramatic decrease in attachment efficiency to 4%. We also demonstrated that the blocking agent ORG 2058 (no photoactivatible chromophore) did not attach to the PR upon photolysis.
10'
0.0
0.2
0.4
0.6
0.8
1.0
Rf Figure 5. SDS-polyacrylamide gel electrophoresis of photoattached [3Hlprotio azide 7 (panel A) and c3H1-R 5020 (panel B). The calibration curve of standard proteins is shown in panel C. The standard proteins are phosphorylase B (Phos B),bovine serum dbumin (BSA), ovalbumin (OV),and carbonic anhydrase (CA). "he coefficient of the line is 0.996 by linear regression.
Characterization of the Progesterone Receptor Covalently Labeled with [SHIProgestinAryl Azide 7. PR covalently labeled with [3Hlprogestin azide 7 in the absence and presence of an excess of unlabeled ORG 2058 was analyzed by SDS-polyacrylamide gel electrophoresis (Figure 5). For comparison, similar procedures were used to covalently label PR with [3H]-R5020. We found that the [3H]progestin azide 7 successfully labeled two proteins with high specificity. These two proteins appear to be the same as those labeled with L3H1-R 5020 and have molecular weights similar to those reported for the two subunits of PR. Our results indicate labeling of proteins of molecular weight 108 500 f 800 and 87 000 f 1500 ( n = 3). The higher molecular weight protein corresponds to literature values for subunit B of PR (109600 f 1200) ( 4 ~ 1and , the lower molecular weight protein corresponds to literature values of subunit A of PR (85 600 f 1200) (4a). The ratio of the PR subunits A:B was determined to be 3.3:l with both L3H1progestin azide 7 and [3H]-R5020. This is in agreement with other reports of the rat uterine A B ratios (4a,d). Interestingly, the ratios of subunits A B in human breast cells (5)and chick oviduct (6) are quite different. In addition to the intact subunits, the gels show several proteolytic fragments of PR that retain specific attachment of protioaryl azide 7, and some free aryl azide 7 appears with the
Kym et al.
120 Sioconjugate Chem., Vol. 6,No. 1, 1995
Scheme 2 hu, 2 h. 25 "C I
o
m
H3C'
13
N
,
ethylene glycol TsOH, benzene *
"
reflux, 16 h
73%
*
tar
+
SM 9
toluene
9
10 43% based on recovered SM
tracking dye. The molecular weight curve of the standard proteins is shown in Figure 5C. Solution Photolysis Experiment. In an attempt to provide evidence for the type of covalent link formed upon photolysis of the ligand-receptor complex, a solution photolysis experiment was performed using a model aryl azide ketal that mimicked the protio aryl azide 7 (Scheme 2). Ketal 9 was prepared in good yield (73%)by heating aryl azide 13 under reflux in the presence of ethylene glycol and a catalytic amount of p-toluenesulfonic acid while removing water as a benzene azeotrope. The photochemical behavior of 9 was evaluated in two solvents: diethylamine to evaluate attachment by nucleophilic addition of the amine to the ring-expanded dehydroazepine and toluene to evaluate covalent attachment by a nonspecific C-H bond insertion mechanism. The samples were degassed by three freeze-thaw cycles prior to photolysis; the photolysis was carried out with a Hanovia mercury arc lamp equipped with a saturated CuSO4 filter (effective wavelength > 315 nm). Photolysis of aryl azide 9 in toluene resulted in an uncharacterizable tar and starting material. The major product from photolysis of 9 in diethylamine was the diethylamine adduct of the ring-expanded dehydroazepine (10)(43% corrected for recovered starting material). Two other products that accounted for less than 5%of the total mass recovered were not identified. Stability of the Covalently Attached I3H1-7-PR Adduct. We examined the covalently attached L3H1-7PR complex under a variety of conditions that are used in protein digestion and N-terminal sequencing. We found that the covalent link was stable to the protein buffer conditions that are typically used in trypsin, chymotrypsin, and V-8 protease digests (16).We found that it was also stable to 0.1% trifluoroacetic acid, which is commonly added to solvents used in reversed-phase HPLC of proteins. At higher concentrations of acid, however, the covalent [3H]progestin azide 7-receptor complex was not as stable. Incubation of the complex with 70% formic acid for 1 h a t room temperature (conditions used in cyanogen bromide digests) resulted in a loss of 30% of the specific attachment. Treatment of the complex with 10% trichloroacetic acid, 10% trifluoroacetic acid, or 100% trifluoroacetic acid a t 0 "C or room temperature for 10 s completely destroyed the specific attachment. DISCUSSION
The photoaffinity labeling (PAL) reagents that have been developed for the PR have, up until now, all relied on a n A,B-ring dienone functionality as the photoreactive moiety, but the very inefficient attachment of these probes to the PR has been a major limitation in the uses of these PAL reagents. The [3H]progestin aryl azide 7 attaches to the PR with 60% efficiency, compared to the attachment efficiency of 5-7% for the most efficient A,Bring dienone based PAL reagent, DU 41165 (4) (4d)
(Table 1). In addition, photoattachment of all A,B-ring dienone based PAL reagents would likely result in labeling of similar amino acid residues in primarily one area of the PR hormone binding site. The aryl azide 7 is expected to attach to amino acids in a different area of the hormone binding domain and provide new information on amino acids that are in close contact with the ligand. The relative binding affinity (RBA) for the rat PR of the aryl azide 7 is somewhat lower than the RBAs for the A,B-ring dienone based PAL reagents, but it is still slightly higher than the natural ligand, progesterone. The binding affinity determined directly from Scatchard analysis of [3Hlprogestin aryl azide 7 was 47% of the affhity of L3H1-R 5020 for the PR. Its affinity for the human PR is even higher (RBA = 71%)) so that it is almost comparable to that of several of the A,B-ring dienone based PAL reagents. Although the sequences of the rat and human PRs are very similar in the hormone binding domain (17, 18),it is not uncommon to observe significant differences in binding affinities between a probe and the same receptor from different species (19,20). Literature precedent suggests that the most likely mechanism for photoattachment for [3Hlprogestin azide 7 would involve attack of a nucleophilic residue on the ring-expanded dehydroazepine (21). This would lead to covalent attachment of the molecular probe 7 with the PR through a potentially acid-labile aminal or thioaminal linkage. The nature of the covalent link is of importance because the most commonly used N-terminal sequencing method, Edman degadation (22),is typically performed using trifluoroacetic acid as solvent in one step of the cycle. The results of the photolysis experiment with the model aryl azide 9 (Scheme 2) indicated, as expected, that the preferred photochemical pathway for protioaryl azide ketals is ring expansion to the dehydroazepine, followed by covalent attachment by a nucleophilic species. These results are consistent with the extensive photochemical work that has been done on phenyl azide, which undergoes ring expansion to the complete exclusion of C-H insertion chemistry (211. In our initial report on the progestin aryl azide derivatives (8)) we also prepared a tetrafluoro azide analog 8. Our interest in this analog was based on the reports by Platz and Keana indicating that the fluoroaryl nitrene photoproduct might have a higher likelihood of forming the more stable C-H bond insertion products (23),rather than the acid labile links resulting from ring expansion and nucleophilic trapping of a dehydroazepine. The tetrafluoroaryl azide 8 had comparable binding affinity and photoinactivation efficiency for PR. While it is possible that it might also form more acid-stable covalent links to PR, the full substitution of the arene ring in this analog does not make it amenable to tritium labeling by the method we have used here. An arene
Sioconjugate Chem., Vol. 6, No. 1, 1995 121
Aryl Azide Photoaffinity Labeling Reagent
analog we have not yet synthesized, having only fluorine substituents in the two critical positions ortho to the azide (241, could, in principle, be tritium-labeled at the remaining two free positions. Although the limited stability of the covalent link between [3H]-7 and the PR may preclude sequence analysis by Edman degradation, newly developed methods for sequencing peptides by mass spectrometry may provide a n alternative method for obtaining the desired information. Our current strategy for determining the amino acids involved in covalent attachment of L3H1-7 to PR involves initial digestion of the photoattached [3H]7-PR complex with trypsin, chymotrypsin, or V-8 protease to obtain a labeled peptide, followed by analysis by electrospray ionization mass spectrometry. Our efforts a t sequencing the [3H]-7-PR complex will be published elsewhere. CONCLUSION
16a,17a-[(R)-1’-(4-Azidophenyl)ethylidenedioxylpre~4-ene-3,20-dione(7)was prepared in tritium-labeled form by a palladium-catalyzed tritiolysis of a n aryl iodide precursor. The L3H1-labeled progestin aryl azide 7 was then evaluated as a photoaffinity labeling reagent for the PR. Scatchard plot analysis of the reversible binding of this compound revealed it to bind to the PR with 47% of the affinity of R 5020. Photolysis of L3H1-7 with the PR resulted in a specific covalent attachment effkiency of 60%. The attachment efficiency of this compound far exceeds the efficiencies normally observed for A,B-ring dienone-based PAL reagents for the PR. The photoattached protein was analyzed by SDS-polyacrylamide gel electrophoresis and found to label two proteins that correspond to the two subunits of the PR. From this labeling experiment, it was determined that the two subunits of the PR were present in a 3.3:l ratio of subunit A:subunit B. Attempts at sequencing the covalently labeled PR by protein digestion methods and electrospray ionization mass spectrometry are underway. ACKNOWLEDGMENT
We are grateful for support of this research through a grant from the National Institutes of Health (PHS 5R37 DK15556). NMR spectra a t 300 and 400 MHz were obtained on instruments supported by a grant from the National Institutes of Health (PHS l S l 0 RR02299) and the National Science Foundation (CHE 90001438 EQ), respectively; mass spectra were obtained on instruments supported by a grant from the National Institutes of Health (GM27029). P.R.K. also thanks the University of Illinois and E. I. DuPont de NeMours & Co. for a graduate fellowship. LITERATURE CITED
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