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Bioconjugate Chem. 1993, 4, 268-274
Forskolin Photoaffinity Probes for the Evaluation of Tubulin Binding Sites Ashok J. Chavan,? Stewart K. Richardson,* Hyuntae Kim,t Boyd E. Haley,*J and David S. Watt’?* Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, and Department of Chemistry, College of Arts and Sciences, University of Kentucky, Lexington, Kentucky 40506. Received March 8, 1993
A photoactive C-7 derivative of forskolin, [12511 -7-forskolinyl2-(3-azido-5-hydroxy-4-iodophenoxy)acetate, enhanced GTP-promoted tubulin polymerization, preferentially bound the P-subunit, exhibited competition with the noniodinated version of the probe, and displayed saturation of labeling consistent with the presence of a forskolin binding site or sites on tubulin.
INTRODUCTION Forskolin, a diterpene isolated from Coleus forskohlii exerts a positive inotropic effect on the heart, lowers blood pressure (1,2),and holds some potential as an antithrombic agent (3) or an antitumor agent (4). These intriguing effects of forskolin led to biochemicalstudies that indicated this compound, and certain analogs, were potent activators of most mammalian adenylate cyclases (5-7) and speculation that this stimulation was at the heart of its macroscopic effects. The discovery that forskolin, like other natural products including taxol and colchicine, had an effect on the polymerization of tubulinlled to the search for biologically active, photoaffinity probes of forskolin for the delineating of the specific forskolin binding domain(s) on tubulin.’I2 EXPERIMENTAL PROCEDURES
General Chemical Procedures. Chemicals were purchased from Aldrich or Sigma and sodium [l25I]iodide was purchased from Amersham. Infrared spectra were determined on a Perkin-Elmer 337 spectrometer. The abbreviation TF denotes thin film. NMR spectra were determined on a Varian 400-MHz or Gemini 200-MHz spectrometer. Mass spectra were determined on a VG ZAB mass spectrometer. Elemental analyses were performed by Atlantic Microlabs, Atlanta, GA. Column chromatography using Macherey Nagel silica gel 60 is referred to as “chromatography on silica gel”, preparativelayer chromatography on Macherey Nagel silica gel F254 is referred to as “chromatography on a silica gel plate”, and the drying of an organic phase over anhydrous magnesium sulfate is simply indicated by the phrase
* Authors to whom correspondenceshould be addressed David S. Watt, Department of Chemistry, University of Kentucky, Lexington, KY 40506. Telephone: (6061-257-5294. FAX: (606)257-4000. Boyd E. Haley, 058Markey Cancer Center,University of Kentucky, Lexington, KY 40536. Telephone: (606)-257-4481. FAX (606)-258-2074. t Division of Medicinal Chemistry and Pharmaceutics. t Department of Chemistry. 1 Part of this work was disclosedat the 40th Southeast Regional Meeting of the American Chemical Society, Atlanta, GA, November 1988 (Synthesis and Application of a Forskolin Photoaffinity Probe to a Tubulin Binding Site. H.-W. Lee, D. S. Watt, H. Kim, and B. E. Haley). Part of this work was disclosed at the Joint Meeting of the American Society for Cell Biology and the American Society for Biochemistryand MolecularBiology,San Francisco,CA,January 1989 (Detection of a Forskolin Binding Site Using a Photoaffkity Probe. D. S. Watt, H.-W. Lee, H. Kim, and B. E. Haley). 1043-1802/93/2904-0268$04.00/0
“dried”. 2-(3-Azido-5-methoxyphenoxy)acetic acid (4a) and 4-(3-azido-5-methoxyphenoxy)butyric acid (4b)were prepared as described previously (9). I-( tert-Butyldimethylsily1)forskolin(2). To a solution of 10 mg (0.024 mmol, 1.1equiv) of forskolin (1)in 0.4 mL of anhydrous CHzClz was added 6.2 pL (5.7 mg, 0.054 mmol, 2.2 equiv) of 2,6-lutidine and 6.1 fiL (7 mg, 0.027 mmol, 1.1equiv) of TBSOTP (8). The mixture was stirred at 25 “C for 5 h, and the product was chromatographed on a silica gel plate using 3:7 EtOAc-hexane to give 10.8 mg (84%) of 2: mp 58-61 OC; IR (KBr) 3480, 2960,2870,170Ocm-1;lH NMR (CDC13)6 0.14 (s,3, SiCH3), 0.87 (8, 9, SiC(CH&, 1.04, 1.25, 1.33, 1.46, 1.65 (5 s, 15, CH3), 2.17 (8,3, OCOCHB),2.35 (d, Jgem = 15 Hz, 1,(2-12 H), 3.28 (d, Jgem= 15 Hz, 1,C-12 H), 4.44-4.50 (br m, 1, C-6a H), 4.56-4.63 (br m, 1,C-lp H), 4.91 (dd, J = 1and 10 Hz, 1, C-15E vinylic H), 5.16 (dd, J = 17 and 1 Hz, 1, C-15Z vinylic H), 5.56 (d, J = 4 Hz, 1, C-7a H), 6.02 (dd, J = 10 and 17 Hz, 1, C-14 vinylic H). Anal. Calcd for C28H40,Si: C, 64.08; H, 9.22. Found: C, 64.08; H, 9.25. 1-(tert-Butyldimethylsilyl)-7-deacetylforskolin (3). To a solution of 10.8 mg (0.02 mmol, 1 equiv) of 2 in 0.8 mL of MeOH and 0.2 mL of water was added 4 mg (0.1 mmol, 5 equiv) of NaOH. The mixture was stirred for 5 h at 25 “C, and the solution was diluted with EtOAc. The solution was washed with brine and dried. The residue was chromatographed on a silica gel plate using 1:l EtOAchexane to give 9.6 mg (97%) of 3: IR (TF) 3500, 3220, 2960,1705 cm-l; lH NMR (CDCl3) 6 0.04 and 0.16 (2 s, 6, SiCH3), 0.97 (s, 9, SiC(CH3)3), 1.06, 1.25, 1.41, 1.43, 1.61 (5 S, 15, CH3), 2.42 (d, Jgem= 16 Hz, 1, C-12 H), 3.22 (d, Jgem = 16 Hz, 1, C-12 H), 4.15-4.21 (br m, 1, C-7a H), 4.46-4.56 (br m, 1, C-6a H), 4.60-4.66 (br m, 1, C-lp H), 4.95 (d, J = 10 Hz, 1, C-15E vinylic H), 5.12 (d, J = 17 Hz, 1, C-15Z vinylic H), 6.18 (dd, J = 10 and 17 Hz, 1, C-14 ~~
Abbreviations used: AD, Alzheimer’s disease; buffer A, 100 mM MES, 1 mM magnesium chloride, 1 mM EGTA (pH 6.7); buffer B, 10mM sodium phosphate, 1mM magnesium chloride, 1 mM EGTA (pH 6.7); DCC, N,”-dicyclohexylcarbodiimide; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol bis(&aminoethyl ether) N,N,”,”-tetraacetic acid; EtOAc, ethyl acetate; EtOH, ethanol; [126I]APTG, monoanhydride of guanosine 5‘(trihydrogendiphosphate) tris(triethy1a”onium) salt and [‘%I]N-(4-azidophenyl)-2-phosphoramido-3-(4-hydroxy-3-iodophenyl)-
propionamide; MeOH, methanol; MES, 2-(N-morpholino)ethanesulfonic acid; NaOAc, sodium acetate; 8-NaGTP, 8-azidoguanosine 5’-triphosphate tris(triethy1a”onium) salt; SDS, sodium dodecylsulfate;TBSOTf,tert-butyldimethylsilyl triflate; THF, tetrahydrofuran; TLC, thin-layer chromatography; TRIS, tris(hydroxymethy1)aminomethane. 0 1993 American Chemical Society
Forskoiin Photoafflnlty Probes for Tubulin
vinylic H). Anal. Calcd for Cz6H46OeSi: C, 64.69; H, 9.61. Found: C, 64.64; H, 9.65. 6-(3-Azido-5-methoxyphenoxy)caproicAcid (4c). Phloroglucinol (Aldrich) was converted to 5-aminoresorcinol, and 5-aminoresorcinol was diazotized and treated with sodium azide to afford 5-azidoresorcinol according to the procedure of Richardson (9). The alkylation of 5-azidoresorcinol with methyl 6-bromocaproate and saponification of the resulting ester according to the procedure of Richardson (9) afforded 4c: IR (KBr) 3360 (br), 3050,2200 (weak), 2110,1715,1600~m-~; lH NMR (CDCl3) 6 1.45-1.95 (m, 6, CHZ),2.40 (t, J = 8 Hz, 2, CHzCO), 3.75 (s,3, OCH3),3.95 (t,J = 8 Hz, 2, CHZOAr), 6.12-6.30 (m, 3, ArH); exact mass spectrum calcd for C13H17N304 279.1220, found 279.1220.
2-(3-Azido-4-iodo-5-methoxyphenoxy)acetic Acid (4d). To 200 mg (0.84 mmol) of methyl 2-(3-azido-5methoxyphenoxy)acetate(91,265mg (1.77mmol, 2.1 equiv) of NaI in 5 mL of acetonitrile, and 1 mL of water was added 200 pL (1.77 mmol, 2.1 equiv) of tert-butyl hypochlorite (IO). The mixture was stirred at 25 "C for 8 h and quenched with 10% sodium thiosulfate solution. The product was extracted with EtOAc and dried. The product was chromatographed on silica gel using 1:4 EtOAc-hexane to afford 40 mg (13%) of methyl 2-(3-azido-4-iodo-5-methoxyphenoxy)acetate: lH NMR (CDCl3) 6 3.79 (s, 3, ArOCH3), 3.87 ( 8 , 3, COZCH~), 4.68 (s, 2, OCHzCO), 6.10 (m, 1, ArH), 6.25 (d, J = 2 Hz, 1, ArH); exact mass spectrum calcd for CIOHIOO~N~I 362.9716, found 362.9715. To a solution of 53 mg (0.16 mmol) of methyl 2- (3-azido-4-iodo-5-methoxyphenoxy)acetate in 1.8 mL of EtOH and 0.3 mL of water was added 24.3 mg (0.434 mmol, 2.8 equiv) of KOH. The mixture was stirred at 25 "C for 2 h, acidified to pH = 1with 2 M HC1 solution, and extracted with EtOAc. The organic layers were dried and concentrated to give 37 mg (74%)of 4d: IR (KBr) 3450, 2920,2110,1730,1580 cm-'; 'H NMR (CDJOD) 6 3.90 (s, 3, OCH3), 4.73 (s, 2, OCHzCO), 6.17-6.20 (m, 1, ArH), 6.28-6.30 (m, 1, ArH); exact mass spectrum calcd for CgH804N31 348.9560, found 348.9560.
Bioconjugate Chem., Vol. 4, No. 4, 1993 269
using 1:l EtOAc-hexane to give a 2:l ratio of C-7 to C-6 isomers from which 7 (yield not determined) was separated: IR (TF) 3440,3000,2940,2860,2110,1730,1695, 1590 cm-l; lH NMR (CDCl3) 6 1.04, 1.25, 1.31, 1.42, 1.63 (5 S, 15, CH3), 2.47 (d, Jgem = 17 Hz, 1,C-12 H), 3.23 (d, Jgem = 17 Hz, 1,C-12 H), 3.79 ( 8 , 3, OCH3), 4.38-4.48 (br m, 1,C-6a H), 4.54-4.62 (br m, 1, C-lpH), 4.64 (s,2,OCHzCO), 4.99 (d, J = 10 Hz, 1,C-15E vinylic H), 5.29 (d, J = 16 Hz, 1, C-15Z vinylic H), 5.61 (d, J = 4 Hz, 1, C-7a H), 5.95 (dd, J = 16 and 10 Hz, 1,C-14 vinylic H), 6.10-6.36 (m, 3, ArH). 7-Forskolinyl 2-(3-Azido-5-methoxyphenoxy)acetate (7) from 6. The procedure described for the preparation of 5 was repeated using 10.6 mg (0.029 mmol) of desacetylforskolin (6)(II),6.4 mg (0.029 mmol, 1equiv) of 4a (9),6.8 mg (0.033 mmol, 1.15 equiv) of DCC, and 0.8 mg (0.005mmol, 0.2 equiv) of 4-pyrrolidinopyridinein 0.4 mL of CHzClZ to afford, after chromatography on a silica gel plate using 1:lEtOAc-hexane, 9.3 mg (57 5% ) of 7 having spectral data identical to that described above. 7-Forskolinyl 4-(3-Azido-5-methoxyphenoxy)butyrate (8). The procedure described for the preparation of 5 was repeated using 10 mg (0.027 mmol) of desacetylforskolin (6)(II), 6.8 mg (0.027 mmol, 1 equiv) of 4b (9), 6.4mg (0.031 mmol, 1.15 equiv) of DCC, and 0.8 mg (0.005 mmol, 0.2 equiv) of 4-pyrrolidinopyridine in 0.55 mL of CHZC12 to afford,after chromatography on a silica gel plate using 1:3EtOAc-hexane, 9.4mg (57 % ) of 8: IR (TF) 3460, 3020,2960,2890,2130,1710,1595cm-l; lH NMR (CDC13) 6 1.04, 1.26, 1.30, 1.45, 1.72 (5 S, 15, CH3), 2.46 (d, Jgem = 17 Hz, 1, C-12 H), 2.64 (t, J = 6 Hz, 2, CHzCO), 2.95 (br S, 1, OH), 3.21 (d, Jgem = 17 Hz, 1, (2-12 H), 3.79 ( 8 , 3, OCH3),4.03 (t,J = 6 Hz, 2, CHzOAr),4.44-4.52 (br m, 1, C-6a H), 4.56-4.64 (br m, 1, C-lp H), 4.97 (d, J = 10 Hz, 1, C-15E vinylic H), 5.29 (d, J = 17 Hz, 1, C-15Z vinylic H), 5.54 (d, J = 4 Hz, 1,C-7a H), 5.94 (dd, J = 10 and 17 Hz, 1, C-14 vinylic CH), 6.10-6.32 (m, 3, ArH).
7-Forskolinyl5-(3-Azido-5-methoxyphenoxy)caproate (9). The procedure described for the preparation of 5 was repeated using 10.5 mg (0.028 mmol) of de1-(tert-Butyldimethylsilyl)-7-forskolinyl-2-(3-Azi- sacetylforskolin (6)(II),8 mg (0.028 mmol, l equiv) of 4c, 6.8 mg (0.033 mmol, 1.15 equiv) of DCC, and 1.0 mg (0.007 do-5-methoxyphenoxy)acetate(5). To a solution of 8 mmol, 0.24 equiv) of 4-pyrrolidinopyridine in 0.6 mL of mg (0.017 mmol, 1equiv) of 3 and 3.9 mg (0.018 mmol, 1.1 CHzClz to afford, after chromatography on a silica gel plate equiv) of 2-(3-azido-5-methoxyphenoxy)aceticacid (4a)
(9) in 0.15 mL of anhydrous THF at 25 "C under a Nz atmosphere was added a mixture of 4 mg (0.019 mmol, 1.15 equiv) of DCC and 0.52 mg (0.0035 mmol, 0.2 equiv) of 4-pyrrolidinopyridine in 0.1 mL of anhydrous THF. The solution was stirred for 2 h at 25 "C, and the product was chromatographedon a silica gel plate using 1:3EtOAchexane to give 7.4 mg (65%) of 5: IR (TF) 3550, 3320, 2980, 2120, 1740, 1700, 1595 cm-l; 'H NMR (CDC13) 6 -0.02 and 0.14 (2 s, 6, SiCH3),0.86 (s,9, SiC(CH&), 1.02, 1-22?1.27, 1.42, 1.54 (5 9, 15, CH3), 2.32 (d, Jgem = 16 Hz, 1, C-12 H), 3.26 (d, Jgem = 16 Hz, 1, (2-12 H), 3.77 (9, 3, OCH3), 4.40-4.48 (br m, 1, C-6a H), 4.55-4.66 (br m, 1, C-lp H), 4.72 (s,2, OCHzCO), 4.89 (dd, J = 1and 10 Hz, 1, C-15E vinylic H), 5.14 (dd, J = 1 and 17 Hz, 1, C-15Z vinylic H), 5.64 (d, J = 4 Hz, 1,C-7a H), 6.01 (dd, J = 10 and 17 Hz, 1, (2-14 vinylic H), 6.20-6.36 (m, 3, ArH). Anal. Calcd for C35H~309N3Si:C, 61.11; H, 7.77. Found: C, 61.04; H, 7.82. 7-Forskolinyl 2-(3-Azido-5-methoxyphenoxy)acetate (7)from 5. To a solution of 7.4 mg (0.011 mmol, 1 equiv) of 5 in 0.5 mL of anhydrous THF was added 11p L of 1 M (0.011 mmol, 1 equiv) tetra-n-butylammonium fluoride solution (8) in THF. The solution was stirred at 25 OC for 15min and chromatographed on a silica gel plate
using 1:3 EtOAc-hexane, 8.1 mg (45 % ) of 9: IR (TF) 3480, 3300,2120,1720,1600 cm-1; 1H NMR (CDCl3) 6 1.05,1.25, 1.33, 1.43, 1.70 (5 s, 15, CH3), 1.68-1.86 (m, 6, CHZ),2.60 (d, J,,, = 17 Hz, 1,C-12 H), 2.60 (t,J = 8 Hz, 2, CHzCO), 3.22 (d, Jgem= 17 Hz, 1, C-12 H), 3.78 (s, 3, OCH3), 3.90 (t,J = 8 Hz, 2, CHZOAr), 4.25-4.50 (m, 1, C-6a H), 4.524.60 (m, 1, C-lp H), 4.97 (d, J = 10 Hz, 1, C-15E vinylic H), 5.30 (d, J = 17 Hz, 1, C-15Z vinylic H), 5.52 (d, J = 4 Hz, 1, C-7cu H), 5.93 (dd, 1, J = 10 and 17 Hz, 1,(2-14 vinylic H), 6.05-6.27 (m, 3, ArH).
7-Forskolinyl2-(3-Azido-4-iodo-5-methoxyphenoxy)acetate (10). The procedure described for the preparation of 5 was repeated using 30 mg (0.081 mmol) of desacetylforskolin (6)(II), 28.5 mg (0.081mmol, 1equiv) of 4d,19.3 mg (0.094 mmol, 1.15 equiv) of DCC, and 2.4 mg (0.016 mmol, 0.2 equiv) of 4-pyrrolidinopyridinein 2 mL of CHzClz to afford, after chromatography on silica gel plates using 3:7 EtOAc-hexane, 22 mg (81%) of 10: IR (TF) 3550, 3010, 2960, 2880, 2130, 1730, 1700, 1580 cm-l; lH NMR (CDC13) 6 1.03, 1.24, 1.25, 1.42, 1.56 (5 S, 15, CH3), 2.44 (d, J,,, = 17 Hz, 1, (2-12 H), 2.84 (br s, 1, OH), 3.22 (d, J,,, = 17 Hz, 1, C-12 H), 3.90 (s, 3, OCH3),4.41-4.50 (br m, 1, C-6a H), 4.55-4.65 (br m, 1, C-10 H), 4.83 (d, J = 2 Hz, 2, OCHzCO),4.98 (d, J = 10 Hz, 1, C-15E vinylic
270
Bioconjugate Chem., Vol. 4, No. 4, 1993
H), 5.29 (d, J = 17 Hz, 1, C-15Z vinylic H), 5.62 (d, J = 4 Hz, 1, C-7a H), 5.90-6.04 (m, 1,C-14 vinylic H), 6.136.35 (m, 2, ArH). Anal. Calcd for C ~ Q H ~ ~ O Q C,N49.72; ~I: H, 5.61. Found: C, 49.66; H, 5.66. 7-Forskolinyl 2-(3-Azido-5-hydroxyphenoxy)acetate (12). The procedure described for the preparation of 5 was repeated using 48.9 mg (0.133 mmol) of desacetylforskolin (6) (11),36.9 mg (0.266 mmol, 2 equiv) of bromoacetic acid, 31.5 mg (0.266 mmol, 2 equiv) of DCC, and 3.9 mg (0.027 mmol,O.2 equiv) of 4-pyrrolidinopyridine in 1 mL of CHzClz to afford, after chromatography on silica gel plates using 1:3 EtOAc-hexane, 7-forskolinyl 2-bromoacetate (yield not determined): IR (KBr) 3450, 3300,1720 cm-l; lH NMR (CDCl3) 6 1.04,1.26,1.35,1.45, 1.75 (5 S, 15, CH3), 2.48 (d, Jgem= 17 Hz, 1,C-12 H), 3.22 (d, Jgem= 17 Hz, 1, C-12 H), 3.94 (d, J = 2 Hz, 2, CHzBr), 4.45-4.55 (m, 1,C-6aH),4.55-4.65 (m, l,C-l@H),4.99(d, J = 10 Hz, 1,C-15E vinylic H), 5.28 (d, J = 17 Hz, 1, C-15Z vinylic H), 5.53 (d, J = 4 Hz, 1, C-7a H), 5.95 (d, J = 10 and 17 Hz, 1,C-14 vinylic H). To 14.7 mg (0.030 mmol) of 7-forskolinyl2-bromoacetatein 0.25 mL of anhydrous acetone under aN2 atmosphere were added 21.4 mg (0.142 mmol, 4.7 equiv) of 5-azidoresorcinol(9) and 20.7 mg (0.150 mmol, 5 equiv) of KzCO3. The mixture was stirred for 0.5 h at 60 "C. The solution was evaporated and chromatographed on a silica gel plate in 1:9 EtOAeCH2C12to afford 9.5 mg (57 % ) of 12: IR (KBr) 3400 (br), 2920,1735,1710, 1595 cm-l; lH NMR (CDCl3) 6 0.97, 1.07, 1.23, 1.35, 1.37 (5 9, 15, CH3), 2.53 (d, Jgem = 17 Hz, 1,C-12 H), 2.82 (br 9, 1,OH), 3.18 (d, Jgem = 17 Hz, 1, (2-12 H), 4.34-4.40 (m, 1,C-6a H), 4.62 (8, 2, CHzOAr), 4.65-4.75 (m, 1, (2-18H), 5.01 (d, J = 10 Hz, 1,C-15E vinylic H), 5.21 (d, J = 17 Hz, 1, C-15Z vinylic H), 5.95-6.55 (m, 5, C-7a, C-14 H and ArH). 7-Forskolinyl2-(3-Azido-5-hydroxy-4-iodophenoxy)acetate (13). To 12.1 mg (0.022 mmol) of 12 and 6.5 mg (0.043 mmol, 2 equiv) of NaI in 150 pL of acetonitrile and 38 pL of water was added 5.2 pL (0.043 mmol, 2 equiv) of tert-butyl hypochlorite (10). The mixture was stirred at 25 "C for 2.5 hand quenched with 10% sodium thiosulfate solution. The product was extracted with EtOAc and dried. The product was chromatographed on silica gel using 1:l EtOAc-hexane to afford 3.3 mg (27%) of unreacted 12 and 3.2 mg (22%) of 13: lH NMR (CDCl3) 6 0.95, 1.07, 1.23, 1.37, 1.39 (5 8, 15, CH3), 2.49 (d, Jgem = 17 Hz, 1, C-12 H), 2.94 (br S, 1,OH), 3.16 (d, Jgem = 17 Hz, 1,(2-12 H), 4.30-4.33 (m, 1, C-6a H), 4.60 (s,2, CH20Ar), 4.65-4.69 (m, 1, C-18 H), 4.92 (d, J = 10 Hz, 1, C-15E vinylic H), 5.17 (d, J = 17 Hz,1, C-15Z vinylic H), 5.956.55 (m, 4, C-7a, C-14 H and ArH). [1~I]-7-Forskolinyl 2-(3-Azido-5-hydroxy-4-iodophenoxy)acetate (14). The following procedure was performed exclusively in a fume hood with all appropriate precautions to minimize exposure to the investigator. To a vial containing 18 p L (9 mCi) of [1261]NaIwas added in succession 20 pL of 1 M NaOAc buffer (pH 3.9), 5.2 pL of 8.6 mM 7-forskolinyl 2-(3-azido-5-hydroxyphenoxy)acetate (12) in acetonitrile, 15 p L of acetonitrile, and 5.3 pL of tert-butyl hypochlorite. The mixture was vortexed brieflyand allowed to stand for 2.5 h a t 25 OC. The reaction was quenched by adding 20 pL of 5 % sodium thiosulfate solution. The mixture was applied to an analytical, glassbacked Merck silica gel F254 plate (10 cm X 20 cm) that had been predeveloped in EtOAc and dried. The plate was eluted with 1:l EtOAc-hexane, and the location of the product was determined by autoradiography. The silica gel was carefully scraped from the plate (in fume hood) and loaded into a disposable glass pipet with a glass
Chavan et ai.
wool plug. The product 14 was eluted from the silica gel with EtOAc and evaporated under a stream of dry Nz. The product was characterized by TLC comparison with nonradioactive, iodinated forskolin derivative 13. The product was stored in absolute MeOH at -20 OC and had a specific activity of 25 mCi/pmol. Although the anticipated specific activity based upon using carrier-free ['%I] NaI was expected to be higher than this value, the low activity obtained was presumably due to our inability to separate the small amounts of the radioiodinated product 14 from small amounts of starting material 12 that contaminated the product. Tubulin Preparations. Microtubule protein was isolated from fresh bovine or ovine brain by two cycles of the reversible assembly method of Shelanski (12). Microtubules obtained from the second cycle of polymerization were stored at -70 "C until needed. Prior to use, pellets were thawed and purified by gel filtration on Sephadex G-25 (medium) using either buffer A for the polymerization assay or buffer B for the photolabeling experiments. Human Brain Homogenate. Normal and AD brain homogenates were prepared from 400-600-mg pieces of respective brain tissue collected on autopsy by the Sanders-Brown Center for Aging at the University of Kentucky. The samples were collected as soon as possible after death and immediately frozen. Homogenized samples (20% w/v) were prepared using a Potter-Elvehjem all-glass homogenizer. Polymerization Assay. Microtubule formation was followed by the light-scattering assay using a Beckman Model 25 spectrophotometer equipped with a cuvette chamber heated to 37 OC (13). The change in absorbance was monitored at 350 nm. Photoaffinity Labeling. Photolabeling of tubulin was optimized for irradiation time, preincubation time, concentration of DMSO, protein concentration, salt concentration, type of buffer, and pH of the solution. Tubulin solutions (5 pg) in buffer B in Eppendorf tubes were incubated with the radioiodinated forskolin derivative 14 in 30 pL of the final volume for 2 min at 4 "C, photolyzed for 2 min at 4 "C with a hand-held UV lamp with the glass face removed (4600 pW/cm2,Model UVS-11,Ultra-Violet Products, Inc.) at a distance of 4 cm. Upon completion of photolabeling, the samples were denatured by the addition of a "protein-solubilizing mixture" containing 25% sucrose, 2.5% SDS,lO.25 mg/mL pyronin Y, 25 mM TRIS-HC1 (pH 8.0), and 15.4 mg/mL dithiothreitol. The a-and 8-tubulin monomers were separated on 10% SDSPAGE. Protein bands were stained with Coomassie Brilliant Blue, destained, dried on a slab gel dryer, subjected to autoradiography, and cut from the gel to determine the radioactivity in Minaxi Gamma Counter 5000 series (Packard Instruments Co.). Photolabeling of denatured tubulin was performed as described in Figure 4. The results in Figure 4 differ from the above experiment in that the tubulin was denatured (heat, 1%SDS, or 8 M urea) prior to photolabeling. RESULTS
Chemical Synthesis. The route shown in Scheme I, in which the C-1 hydroxyl of forskolin (1) was protected as a TBS ether (2), was designed to avoid initial concerns about the competitive acylation of the C-1 and C-7 positions in 1. The saponification of the C-7 acetate in 2 and the acylation of the C-7 hydroxyl group in 3 with 2-(3-azido-5-methoxyphenoxy)aceticacid (4a) (9)provided the TBS-protected derivative 5. However, removal of the
Forskolln Photoafflnlty Probes for Tubulln
BloconJugate Chem., Vol. 4, No. 4, 1993
Scheme I. Synthetic Routes. HO
od
TESQ
,'\
OH
0.15
OH 2 R=COCH 3 R = H
'\
,
E
H
l e HQ
0.20
,'\
---t
OH
A
od
/ - -
OCOCH3 i
271
TESO
0 m
3
A
0.10
a
4. \ od,
0.05
0.00 6 OH
I
5
H 0 2 C ( C H 2 ) n , o P Y
OCH3
0.15
#'c"
$
0.10
m 0
A ( C H 2 ) n , 0 p N 3
3 4
0.05 OH
X OR
4a 4b 4c 46
n=l, n=3. n=5 n=l,
X=H X=H X=H X=l
7 n=l, R=CH~X . =H 8 n = 3 . R=CH3. X=H 9 n=5. R=CH3. X = H 1 0 n = l . R=CH3, X=l
11 12 13 14
n=l. n.1, n=l, n=l.
R = C H ~ ,x.125 R=H, X=H R=H, X = l R=H, X.125
0.00
-3
a Reagents: (a) TBSOTf, 2,b-lutidine; (b) NaOH, aqueous MeOH; (c) 4a, 4b,4c, or 4d, DCC, 4-(pyrrolidino)pyridine;(d) (n-Bu)dF,THF(e)see refs 11,14;(f) NaI, t-BuOC1;(g) [TINaI, t-BuOC1.
TBS group in 5 using tetra-n-butylammonium fluoride (8)led to scrambling of the aryl azide-containingacyl group (II), and a separable 2:l mixture of the C-6 and C-7 esters was obtained. A similar acyl scrambling occurred in forskolin (1) itself when exposed to basic alumina (11)or tetra-n-butylammonium fluoride. The direct C-7 acylation of desacetylforskolin (6) with 2-(3-azido-5-methoxyphenoxy)acetic acid (4a) provided a mixture of the C-6 and C-7 analogs in which the desired C-7 derivative 7 was the principal product. Acylation at C-1 was not observed. The C-7 derivatives 8 and 9 with longer "spacers" between the aryl azide and forskolin subunits were also prepared using 4-(3-azido-bmethoxyphenoxy)butyricacid (4b) (9) and 6-(3-azido-5-methoxyphenoxy)caproic acid (4c), respectively. The direct iodination of the forskolin derivative 7 failed to provide the iodinated derivative 10, presumably as a consequence of the deactivating influence of the azide group. A marginally useful synthesis of 10 involved the iodination of 4a in order to obtain 4d and the subsequent coupling of 4d and desacetylforskolin (6) in order to obtain 10 in low yield. Efforts, however, to adapt this procedure to the preparation of a radioiodinated analog 11 led to a probe with low specific activity (10 mCi/mmol). Since a hydroxyl group on the aryl azide moiety would facilitate the iodination reaction better than a methoxy group, desacetylforskolin (6) was converted first to the 7-bromoacetylderivative and then subjected to an s N 2 reaction with 3-azidoresorcinol(9)to furnish the resorcinol derivative 12. The iodination or radioiodination of 12 provided the forskolin photoaffinity probes 13 and 14, respectively, having, in the case of the latter derivative, the necessary high specific activity (25 mCi/pmol). Polymerization Assay. Microtubule formation was followed by the light-scattering assay described by Gaskin (13) using a Beckman Model 25 spectrophotometer equipped with a cuvette chamber heated to 37 "C. The change in absorbance was monitored at 350 nm and indicated that forskolin (1) and the derivative 12 enhanced
10
0
20
30
Time (mi.)
Figure 1. Effect of forskolin (1) and probe 12 on GTP-promoted tubulin polymerization: (A) tubulin (1mg) was incubated at 37 OC in a 1-mL final volume of 10% DMSO in buffer A in the presence of either 1mM GTP alone (- -) or in the presence of 1 mM GTP and 10 pM, 100 pM, or 1mM forskolin (1) (-). The change in absorbance was monitored at 350 nm; (B) same as A using 1 mM GTP alone (- -) or 1 mM GTP and 100 pM probe (12)
(3.
V
5.0-
*-e A-A
?/A
@-Subunit a-Subunit
0.0 0
10
20
30
40
50
3
Forskolin Probe 14 (pU)
Figure 2. Saturation of photolabeling. Tubulin (5 pg) was exposed to varying concentrations of probe 14 in buffer B (final volume 30 pL), incubated (2 min, 4 "C), irradiated (2 min, 4 "C), and analyzed on 10% SDS-PAGE gels that were subsequently sliced and radioactivity determined using a Minaxi y-counter. the GTP-promoted polymerization of tubulin as indicated in Figure 1. Saturation Experiments. The tubulin solutionswere incubated with increasing concentrations of forskolin probe 14 and photolyzed to produce the results shown in Figure 2. The @-tubulinshowed saturation of labeling at a concentration of approximately 25 pM of forskolin probe 14 with an apparent Kd of 8-10 pM. The a-subunit was also labeled with approximately the same saturation concentration and apparent Kd; however, the labeling in the a-subunit was 35 % less than that in the @-subunitat the saturation concentration. Competition Experiments. Tubulin solutions (5 pg) in buffer B in an Eppendorf tube were incubated successively with various concentrations of non-iodinated probe 12 (10 min, 4 "C) and 5 p M radioiodinated probe
272
Scheme 11. Selected Forskolin Photoaffinity Probes
100
.-0C
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Chavan et at.
Bloconlugate Chem., Vol. 4, No. 4, 1993
E l a-subunit
75
0
a 0
-
-
50
Lo
7 N
8? 25
0
3
2
1
4
Figure 3. Competitionexperiments with probe 14. Tubulin (5 pg) was exposed to 1000p M of probe 12 in buffer B (final volume
30 pL), incubated (10 min, 4 "C), exposed to probe 14, incubated (2 min, 4 "C), irradiated (2 min, 4 "C), and analyzed on 10% SDS-PAGE gels as in Figure 2: column 1, photolabeling of a-tubulin in the absenceof probe 12; column 2, photolabeling of a-tubulin in the presence of probe 12; column 3, photolabeling of @-tubulinin the absence of probe 12; and column 4, photolabeling of @-tubulinin the presence of probe 12.
a subunit-
p subunit-
U
Heat (lOO°C/10 min) 1% SDS
8M urea
-
+
-
-
+
-
Figure 4. Effect of denaturation. Tubulin (5 pg) was denatured in various ways and exposed to 10 p M of probe 14, incubated (2
min, 4 "C), irradiated (2 min, 4 "C), and analyzed on 10% SDSPAGE gels. Autoradiography was performed for 8 h. 14 (2 min, 4 "C) in 30 p L of the final volume, photolyzed, and analyzed to give the results shown in Figure 3. Relatively high concentrations of the noniodinated probe were required to afford protection of labeling. At 1000 p M concentration of the competing noniodinated probe, approximately 65% of labeling in the @-subunit was protected whereas 54% of labeling in the a-subunit was protected. Specificity. Denatured tubulin failed to bind the radioiodinated analog 14 as indicated by the autoradiogram of the SDS-PAGE gels in Figure 4. Human brain homogenate (10 p L of 20% from either normal or AD brains) in buffer R in an Eppendorf tube was incubated with 10 pM of probe 14 either alone or with 100 pM of various competitors for 2 min at 0 "C, photolyzed, and analyzed to give the autoradiogram of the SDS-PAGE gels shown in Figure 5. Five major protein bands were labeled. In addition to a- and @-tubulin,a band with M , of 25 kDa was also labeled. DISCUSSION The development of a suitable forskolin photoaffinity probe required a balance between the biological activity
OH
necessary to guarantee that the probe would mimic forskolin itself and the chemical properties necessary to achieve reasonable levels of cross-linking and detection. Although many modifications of forskolin (1) led to diminished activity (11)with respect to the stimulation of adenylate cyclase (AC), the C-7 forskolin analogs 7-9 possessed significant potency (15). Ruoho (16) and Pfeuffer (17) also reported C-7 forskolin derivatives 15 and 16, respectively, (Scheme 11) that possessed a phenyl azide attached to forskolin through a different connecting arm. The most significant difference in these probes was the presence of an activating hydroxyl group on the phenyl azide in the latter probe (16) that facilitated radioiodination. These forskolin derivatives bound the glucose transporter but did not activate adenylate cyclase. Shanahan (18) used [3H]forskolin in a direct photoaffinity labeling study of the glucose transporter. Finally, Mende and Ho (15) described another C-7 derivative (17) that also possessed a phenyl azide but included a tyrosine group, again to facilitate radiolabeling, as part of the connecting arm. The forskolin probe 17 activated adenylate cyclase but not at the same level as the C-7 forskolin derivatives 7-9. On this basis, these latter derivatives seemed the most reasonable candidates for the tubulin binding study, and after studying the problem of incorporating a radiolabel of high specific activity, the iodinated probes 10 and 13 as well as [1251]-labeledprobe 14 were prepared. Forskolin (1) and the derivative 12 enhanced the GTPpromoted polymerization of tubulin as shown in Figure 1. The increase in the extent of polymerization was saturated at 1pM of forskolin (1). The chemically denatured tubulin (using either 1% SDS or 8 M urea) showed, as expected, minimal or no forskolin-bindingability, as shown in Figure 4. Heat denaturation (Figure 4) produced diminished binding, presumably as a consequence of incomplete denaturation or the exposure of some hydrophobicdomains for which the forskolin probe had some affinity. Since the chemical denaturants were not removed prior to photolabeling, these specific or nonspecific hydrophobic binding domains were not accessibleto the probe as in the case of heat denaturation. Necessary studies validated the attributes of 14 as a forskolin photoaffinity probe: the demonstration of saturation effects; the prevention of photoinsertion by the natural compound (Le., the probe is a mimic at concentrations that agree with the known Kd of the natural compound); and the selectivity of labeling (Le., the probe only photoinserts into proteins that are known to bind the
Forskolin Photoaffinity Probes for Tubulin
Biocon/ugate Chem., Vol. 4, No. 4, 1993 273
SDS-PAGE 1
3
2
4
5
AUTORADIOGRAPHY 6
7
1
8 P-
2
3
4
S
6
7
8
M, x 10-3
31
I
r
* I
‘1 -
+
-
-
-
-
-
+ -
+
+
-
-
-
-
-I
+
-
-
+
Non-Iodinated Probe 12 GTP ATp
-
+
-
-
-
-
+
-
-
-
-
-
+
+
-
-
-
+
-
-
-
-
+
Figure 5. Photolabeling of human control (lanes 1-4) and AD brain homogenate (lanes 5-8) with probe 14. Homogenates (7 pL of 20 % ) was exposed either to 10 pM of probe 14 alone or 10 pM of probe 14 and 100 pM of various competitors in buffer B (finalvolume 30 pL), incubated (2 min, 4 OC), irradiated (2 min, 4 “C), and analyzed on 10% SDS-PAGE gels. Autoradiography was performed for 12 h.
native compound). Preliminary photolabeling studies indicated that maximal incorporation of the probe 14 into tubulin subunits occurred within 5 min of irradiation, was unaffected by long incubation times, and was maximized at a 12% DMSO-buffer B concentration. Using tubulin prepared from bovine or ovine brain (12),it was demonstrated that the forskolin probe 14 exhibited saturation of the binding sites on both the a-and @-subunitof tubulin as shown in Figure 2. Significant amounts of binding to both subunits was noted, although under the optimal conditions developed for the photolabeling experiments using a 10 mM sodium phosphate buffer with 1 mM magnesium chloride and 1mM EGTA, the @-subunitwas preferentially labeled. Competition for the binding of the radioiodinated forskolin probe 14 was demonstrated using the noniodinated probe 12. Consistent with the observations of Ruoho, relatively high concentrations of 12 were required to achieve the results in Figure 3. These observations are consistent with a low Kd value for forskolin and rapid binding of forskolin or the probes 12 and 14 to the binding site. Consistent with these suggestions,the order in which the probes were added to the tubulin preparation had a dramatic effect on the competition experiments. However, the efficiencyof cross-linkingin photolabeling experiments was judged to be too low to permit isolation of the cross-linked peptide and sequencing efforts. The photolabeling efficiency using forskolin probe 14 ranged from0.07 to 2 % in four separate experiments. This result should be contrasted with literature reports using other forskolinprobes shown in Scheme11,in which the efficiency was suggested to be “high” (16),was not reported (In,or was comparable (0.09%)(18). The low efficiency in the particular case of forskolin probe 14 was ascribed to the electron-rich nature of hydroxy- or alkoxy-substituted dehydroazepine intermediate (19, 20) generated in the photolysis of the alkoxy-substituted aryl azide and to a photodeiodination process (21)that competes effectively with the photolysis of the aryl azide. In the normal brain homogenate, forskolinprobe 14 photolabeled three protein bands including a-and @-tubulinas well as a 25 kDa Mr protein. It was interesting that a- and @-tubulinphotolabeling could be protected to some extent by GTP,
suggesting a conformation change in the presence of GTP that altered forskolin binding or perhaps the prevention of nonspecific labelingby forskolinin the presence of GTP. The forskolin probe 14 photolabeled a-and @-tubulins in AD brain homogenates in addition to three other principal bands. In comparison to photolabeling experiments with purified tubulin, it was of interest that the forskolin probe 14 exhibited slightly more selectivity for @-tubulinthan a-tubulin in AD brain homogenates. It was also of interest that photolabeling of the @-tubulin from AD brains was observed using the forskolin probe 14 but was not observed using “GTP” probes, [ T - ~ ~ P ] - ~ - N ~ GTP (22)and [1251]APTG(23). These results suggested that there is a differential binding of the forskolin probe 14 on tubulin from AD and normal brains and raised the possibility that a forskolin-modified affinity resin might permit the isolation of tubulin from AD brains reported to have aberrant polymerization properties (24). In summary, the demonstration that the forskolin 14 photolabeled tubulin and activated adenylate cyclase augured well for future plans to engineer a more successful probe that incorporates a photoactive group capable of crosslinking with higher efficiency than the phenyl azide probe in 14. ACKNOWLEDGMENT
We thank the National Institutes of Health (HL 20780) for their generous financial support. LITERATURE CITED (1) Lindner, E., Dohadwalla, A. N., and Bhattacharya, B. K.
(1978) Positive inotropic and blood pressure lowering activity of diterpene derivatives isolated from Coleus forskohlii: forskolin. Arzneim. Forsch. 28, 284-289. (2) Seamon, K. B., Padgett, W., and Daly, J. W. (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. h o c . Natl. Acad. Sci. U.S.A. 78,3363-3367. (3) Siegel,A. M., Daly, J. W., and Smith, J. B. (1982)Inhibition of aggregation and stimulation of cyclic AMP generation in intact human platelets by the diterpene forskolin. Mol. Pharmacol. 21,680-687
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(4) Agarwal, K. C., and Parks, R. E., Jr. (1983) Forskolin: A potential antimetastatic agent. Int. J. Cancer 32, 801-804. (5) Metzger, H., and Linder, E. (1981) The positive inotropic-
actingforskolin,a potent adenylatecyclaseactivator. Arzneim. Forsch. 31, 1248-1250. (6) de Souza, N. J., Dohadwalla, A. N., and Reden, J. (1983) Forskolin: A labdane diterpenoid with antihypertensive, positive inotropic! platelet aggregation inhibitory, and adenylate cyclase activating properties. Med. Res. Reu. 3, 201219. (7) Daly, J. W. (1984) Forskolin, adenylate cyclase, and cell
physiology: An overview. Adu. Cyclic Nucleotide Protein Phosphorylation Res. 17, 81-89.
(8) Corey, E. J., and Venkateswarlu, A. (1972) Protection of
hydroxylgroupsas tert-butyldimethylsilyl derivatives. J.Am. Chem. SOC.94,6190-6191. (9) Richardson, S. K., Jeganathan, A., Mani, R. S., Haley, B. E., Watt, D. S., and Trusal, L. R. (1987) Synthesis and biological activity of C-4 and (2-15 aryl azide derivatives of anguidine. Tetrahedron 43, 2925-2934. (10) Kometani, T., Watt, D. S., Ji, T., and Fitz, T. (1985) An
improvedprocedurefor the iodination of phenolsusing sodium iodide and tert-butyl hypochlorite. J. Org. Chem. 50, 53845387. (11) Bhat, S. V., Bajwa, B. S., Dornauer, H., and de Souza, N. J. (1982) Reactionsof forskolin,a biologicallyactive diterpenoid from Coleus forskohlii. J. Chem. SOC.,Perkin Trans. 1 767771. (12) Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973)
Microtubule assembly in the absence of added nucleotides. Proc. Nut. Acad. Sci. U.S.A. 70, 765-768. (13) Gaskin, F., Cantor, C. R., and Shelanski, M. L. (1974) Turbidimetric studies of the in vitro assemblyand disassembly of porcine neurotubules. J. Mol. Biol. 89, 737-758. (14) Seamon, K. B., Daly, J. W., Metzger, H., de Souza, N. J., and Reden, J. (1983) Structure-activity relationships for activation of adenylate cyclase by the diterpene forskolin and its derivatives. J. Med. Chem. 26, 436-439.
(15) Ho, L. T., Nie, Z. M., Mende, T. J., Richardson, S., Chavan,
A., Kolaczkowska, E., Watt, D. S., Haley, B. E., and Ho, R.-J. (1988-89) Modification of adenylate cyclase by photoaffinity analogs of forskolin. Second Mess. Phosphoprot. 12, 209233. (16) Wadzinski,B. E., Shanahan, M. F.,andRuoho, A. E. (1987)
Derivatization of the human erytrocyte glucose transporter using a novel forskolin photoaffinity label. J. Biol. Chem. 262,17683-17689. (17) Pfeuffer, E., and Pfeuffer,
T.(1989) Affinity labeling of forskolin-binding proteins. Comparison between glucose carrier and adenylate cyclase. FEBS Lett. 248, 13-17. (18) Shanahan, M. F., Morris, D. P., and Edwards, B. M. (1987) [3H]-Forskolin.DirectPhotoaffiiitylabeling of theerythrocyte D-glucose transporter. J. Biol. Chem. 262,5978-5984. (19) Torres, M. J., Zayas, J., and Platz, M. S. (1986) A formal CH insertion reaction of an aryl nitrene into an alkyl CH bond. Implications for photoaffinity labelling. Tetrahedroll Lett. 27, 791-794. (20) Shields, C. J., Chrisope, D. R., Schuster, G. B., Dixon, A. J., Poliakoff, M., and Turner, J. J. (1987) Photochemistry of
aryl azides: detection and characterizationof a dehydroazepine by time-resolved infrared spectroscopy and flash photolysis at room temperature. J. Am. Chem. SOC. 109, 4723-4726. (21) Watt, D. S., Kawada, K., Leyva, E., and Platz, M. S. (1989) Exploratory photochemistry of iodinated aromatic azides. Tetrahedron Lett. 30, 899-902. (22) Kim, H., Ponstingl,H., andHaley,B. E. (1987)Identification
of the guanosine interacting peptide of the GTP binding site of @-tubulinusing 8NsGTP. Fed. Proc. 46, 2229. (23) Chavan, A. J., Kim, H., Haley, B. E., and Watt, D. S. (1990) A photoactive phosphonamide derivative of GTP for the identification of the GTP binding domain in 8-tubulin. Bioconjugate Chem. 1, 337-344. (24) Iqbal, K., Zaidi,T., Wen, G. Y.,Grundke-Iqbal, I.,Merz, P.
A., Shaikh,S. S., Wisniewski,H. M., Alafuzoff,I., and Winblad, B. (1986) Defectivebrain microtubule assemblyin Alzheimer’s disease. Lancet 421-426.
