Localizable and Highly Sensitive Calcium Indicator Based on a

Jun 30, 2010 - prepared its O6-benzylguanine (BG) derivative, BOCA-. 1-BG, which can be covalently and specifically linked to SNAP-tag fusion proteins...
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Anal. Chem. 2010, 82, 6472–6479

Localizable and Highly Sensitive Calcium Indicator Based on a BODIPY Fluorophore Mako Kamiya and Kai Johnsson* Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fe´de´rale de Lausanne, CH 1015, Lausanne, Switzerland We introduce here a new class of BODIPY-based Ca2+ indicators which can be derivatized with biological ligands that permit the localization of the indicators in living cells. The underivatized BODIPY-based Ca2+ indicator (BOCA-1) shows a 250-fold increase in fluorescence intensity upon Ca2+ binding. We also prepared its O6-benzylguanine (BG) derivative, BOCA1-BG, which can be covalently and specifically linked to SNAP-tag fusion proteins in living cells. The indicator retains its properties as a highly sensitive Ca2+ indicator after conjugation to proteins, displaying a 180-fold increase in fluorescence intensity upon Ca2+ binding. We further demonstrated that BOCA-1-BG through reaction with localized SNAP-tag fusion proteins can be used to sense changes in Ca2+ concentrations in the nuclei and in the cytosol of live CHO-K1 cells. The high sensitivity of the indicator together with the possibility to selectively couple it to proteins of interest makes it a powerful tool for measuring local changes in Ca2+ concentrations in living cells. The role of calcium (Ca2+) as a second messenger relies on the precise spatial and temporal control of its concentration in cells.1 Current approaches to measure fluctuations in Ca2+ concentration ([Ca2+]) mostly utilize either autofluorescent protein-based Ca2+ indicators such as cameleon and GCamP22,3 or synthetic Ca2+ indicators such as Fura-2 and Fluo-4.4-7 Autofluorescent protein-based sensors can be genetically targeted to the area of interest in cells, but have slower response time and lower sensitivity than synthetic indicators. In contrast, synthetic indicators lack the ability to be specifically localized in cells. In order to measure [Ca2+] with high spatiotemporal resolution, synthetic Ca2+ indicators that can be covalently and selectively * Author to whom correspondence should be addressed. E-mail: [email protected]. (1) Clapham, D. E. Cell 2007, 131, 1047–1058. (2) Miyawaki, A.; Llopis, J.; Heim, R.; McCaffery, J. M.; Adams, J. A.; Ikura, M.; Tsien, R. Y. Nature 1997, 388, 882–887. (3) Tallini, Y. N.; Ohkura, M.; Choi, B.-R.; Ji, G.; Imoto, K.; Doran, R.; Lee, J.; Plan, P.; Wilson, J.; Xin, H.-B.; Sanbe, A.; Gulick, J.; Mathai, J.; Robbins, J.; Salama, G.; Nakai, J.; Kotlikoff, M. I. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4753–4758. (4) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 3440– 3450. (5) Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 8171–8178. (6) Gee, K. R.; Brown, K. A.; Chen, W-N. U.; Bishop-Stewart, J.; Gray, D.; Johnson, I. Cell Calcium 2000, 27, 97–106. (7) Takahashi, A.; Camacho, P.; Lechleiter, J. D.; Herman, B. Physiol. Rev. 1999, 79, 1089–1125.

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coupled to proteins of choice in living cells have been developed.8,9 This hybrid approach potentially combines the superior sensitivity and kinetics of synthetic indicators with the possibility to localize them through the coupling to appropriately targeted proteins. However, the localizable synthetic Ca2+ indicators introduced so far are less sensitive than the corresponding nonlocalizable indicators.4-9 This lower sensitivity limits their practical value. Therefore, new synthetic Ca2+ indicators which are highly sensitive and can be selectively coupled to proteins in living cells are still needed. Here we introduce synthetic Ca2+ indicators based on the BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, borondipyrromethene) fluorophore that show a large fluorescence increase upon Ca2+ binding and which can be chemically derivatized without compromising their sensitivity. We demonstrate how these sensors can be specifically localized in living cells through reaction with SNAP-tag fusion proteins and permit to probe changes in local [Ca2+]. These BODIPY-based, localizable Ca2+ indicators should become valuable tools to visualize [Ca2+] with high spatiotemporal resolution in cells. EXPERIMENTAL SECTION Materials and General Instrumentation. General chemicals were of the best grade available, supplied by Sigma-Aldrich, Fluka, Acros, Armar Chemicals, Reactolab, or Invitrogen, and were used without further purification. HPLC solvents were gradient grade. SNAP-tag/CLIP-tag substrates were obtained from New England Biolabs. 1H NMR and 13C NMR spectra were recorded on a AVIII-400 (Bruker) or DPX-400 (Bruker) instrument. Chemical shifts (δ) were reported in ppm relative to the solvent residual signals of CD3OD (3.31 ppm) and CDCl3 (7.26 ppm) and coupling constants were reported in Hz. High resolution mass spectra (HRMS, ESI-TOF) were recorded on a Q-Tof Ultima spectrometer (Micromass). HPLC analysis and purification were performed on a reversed-phase column, Nova-Pak C18column (60 Å 7.8× 300 mm) (Waters), fitted on a Dionex Summit system, equipped with P680 HPLC pump, and UVD 170U UV-detector. All experiments were carried out at 298 K, unless otherwise specified. Synthesis of BOCA-1 and BOCA-1-BG. The synthetic strategy employed to obtain the new indicators is outlined in Scheme 1. (8) Tour, O.; Adams, S. R.; Kerr, R. A.; Meijer, R. M.; Sejnowski, T. J.; Tsien, R. W.; Tsien, R. Y. Nat. Chem. Biol. 2007, 3, 423–431. (9) Bannwarth, M.; Correˆa Jr, I. R.; Sztretye, M.; Pouvreau, S.; Fellay, C.; Aebischer, A.; Royer, L.; Rı´os, E.; Johnsson, K. ACS Chem. Biol. 2009, 4, 179–190. 10.1021/ac100741t  2010 American Chemical Society Published on Web 06/30/2010

Scheme 1. Synthesis of BOCA-1 and BOCA-1-BGa

a (a) (i) TFA (cat.), DCM, rt; (ii) tetrachloro-p-benzoquinone, rt; (b) BF3-OEt2, DIEA, DCM, rt, 25% (in two steps); (c) 1 N NaOH aq, MeOH, rt, 43%; (d) (i) TFA (cat.), DCM, rt; (ii) tetrachloro-p-benzoquinone, rt; (e) BF3-OEt2, DIEA, DCM, rt, 32% (in two steps); (f) iodine monochloride, MeOH/DCM, rt, 89%; (g) acrylic acid benzyl ester, Pd(OAc)2, Et3N, MeCN, 60 °C, 82%; (h) H2, Pd/C (10%), MeOH/DCM, rt, 15%; (i) O6-(4aminomethyl-benzyl)guanine, EDC-HCl, HOBt, Et3N, DMF, 0 °C f rt; j) 1N NaOH aq, MeOH, rt, 35% (in two steps).

Compound 3. 5-Formyl-5′-methyl-BAPTA ethyl ester (1) and 3-(2-methoxycarbonylethyl)-2,4-dimethylpyrrole (2) were synthesized according to published procedures.4,10,11 To a solution of 1 (140 mg, 0.22 mmol) and 2 (100 mg, 0.55 mmol) in dry dichloromethane (DCM) (20 mL), 1 drop of trifluoroacetic acid (TFA) was added. The reaction mixture was stirred at room temperature (rt) under nitrogen overnight. After addition of tetrachloro-p-benzoquinone (66 mg, 0.27 mmol), the mixture was further stirred for 15 min. The reaction mixture was washed with water and saturated NaCl aq., dried over Na2SO4 and the solvent was evaporated. The residue was chromatographed on aluminum oxide with DCM-MeOH (100:0-100:2) as the eluent to give a brown powder. The obtained compound was dissolved in dry DCM (10 mL), and N,N-diisopropylethylamine (DIEA) (3 mL, 17.1 mmol) was added. Boron trifluoride etherate (BF3-OEt2) (1.0 mL, 7.9 mmol) was added slowly under nitrogen and stirred for 1 h. The reaction mixture was washed with water and saturated NaCl aq., dried over Na2SO4 and the solvent was evaporated. The residue was chromatographed on silica gel with hexane-AcOEt (2:1-1:1) as the eluent to give compound 3 as a brown powder (56 mg, 25%). 1H NMR (400 MHz, CDCl3): δ 6.88 (d, 1H, J ) 8.4 Hz), 6.80 (d, 1H, J ) 8.0 Hz), 6.76 (d, 1H, J ) 1.6 Hz), 6.75 (dd, 1H J ) 8.4 Hz, 1.6 Hz), 6.70-6.66 (m, 2H), 4.30-4.22 (m, 4H), 4.21 (s, 4H), 4.11 (s, 4H), 4.11 (q, 4H, J ) 7.0 Hz), 4.09 (q, 4H, J ) 7.0 Hz), 3.66 (s, (10) Sa´nchez-Martı´n, R. M.; Cuttle, M.; Mittoo, S.; Bradley, M. Angew. Chem., Int. Ed. 2006, 45, 5472–5474. (11) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357–3367.

6H), 2.63 (t, 4H, J ) 8.0 Hz), 2.53 (s, 6H), 2.35 (t, 4H, J ) 8.0 Hz), 2.25 (s, 3H), 1.40 (s, 6H), 1.20 (t, 6H, J ) 7.0 Hz), 1.18 (t, 6H, J ) 7.0 Hz); 13C NMR (101 MHz, CDCl3): δ 173.0, 171.3, 171.2, 153.9, 150.8, 150.2, 140.7, 140.1, 139.4, 137.0, 132.2, 131.1, 129.0, 128.4, 122.2, 121.2, 119.7, 119.1, 115.2, 113.2, 67.5, 67.1, 60.8, 60.5, 53.7, 53.6, 51.6, 34.1, 20.8, 19.3, 14.2, 14.1, 12.5, 12.0; HRMS (ESI+, m/z): calcd for [M+H]+, 1021.4802; found, 1021.4793. BOCA-1. To a solution of 3 (10 mg, 9.8 µmol) in DCM/ methanol (1 mL/2 mL), 1 N NaOH aq. (0.5 mL) was added. The reaction mixture was stirred at rt for 4 h. After neutralization with 1 N HCl aq., the solvent was evaporated and the residue was purified by semipreparative reversed-phase HPLC using eluent A (CH3CN with 0.1% TFA and 1% H2O) and eluent B (H2O with 0.1% TFA and 1% CH3CN) (A/B ) 5/95-100/0 in 24 min) and lyophilized to give compound BOCA-1 as an orange powder (3.7 mg, 43%). 1H NMR (400 MHz, CD3OD): δ 6.98 (d, 1H, J ) 8.0 Hz), 6.94 (d, 1H, J ) 1.6 Hz), 6.86 (d, 1H, J ) 8.4 Hz), 6.84 (d, 1H, J ) 1.2 Hz), 6.78 (dd, 1H, J ) 8.4 Hz, 1.6 Hz), 6.67 (dd, 1H, J ) 8.0 Hz, 1.2 Hz), 4.33 (s, 4H), 4.19 (s, 4H), 4.06 (s, 4H), 2.65 (t, 4H, J ) 7.6 Hz), 2.49 (s, 6H), 2.34 (t, 4H, J ) 7.6 Hz), 2.25 (s, 3H), 1.45 (s, 6H); 13C NMR (101 MHz, CD3OD): δ 176.6, 175.3, 175.0, 155.1, 152.2, 152.0, 142.6, 141.5, 141.0, 136.8, 135.1, 132.4, 130.6, 129.6, 123.0, 122.5, 121.3, 120.0, 116.0, 115.5, 69.2, 68.5, 56.1, 55.3, 35.3, 21.1, 20.4, 12.8, 12.5; HRMS (ESI+, m/z): calcd for [M+H]+, 881.3235; found, 881.3270. Compound 4. To a solution of 1 (300 mg, 0.48 mmol) and 2,4-dimethylpyrrole (100 mg, 1.1 mmol) in dry DCM (50 mL), 1 Analytical Chemistry, Vol. 82, No. 15, August 1, 2010

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drop of TFA was added. The reaction mixture was stirred at rt under nitrogen overnight. After tetrachloro-p-benzoquinone (120 mg, 0.49 mmol) was added, the mixture was further stirred for 15 min. The reaction mixture was washed with water and saturated NaCl aq., dried over Na2SO4 and the solvent was evaporated. The brown crude solid was dissolved in dry DCM (20 mL), and N,N-diisopropylethylamine (DIEA) (3 mL, 17.1 mmol) was added. BF3-OEt2 (1.0 mL, 7.9 mmol) was added slowly under nitrogen and stirred for 1 h. The reaction mixture was washed with water and saturated NaCl aq., dried over Na2SO4 and the solvent was evaporated. The residue was chromatographed on silica gel with DCM-MeOH (100:3) as the eluent to give compound 4 as a brown powder (130 mg, 32%). 1H NMR (400 MHz, CDCl3): δ 6.88 (d, 1H, J ) 8.8 Hz), 6.78-6.66 (m, 5H), 5.97 (s, 2H), 4.28-4.22 (m, 4H), 4.21 (s, 4H), 4.10 (q, 4H, J ) 7.2 Hz), 4.09 (s, 4H), 4.07 (q, 4H, J ) 7.2 Hz), 2.5 (s, 6H), 2.24 (s, 3H), 1.48 (s, 6H), 1.19 (t, 6H, J ) 7.2 Hz), 1.18 (t, 6H, J ) 7.2 Hz); 13C NMR (101 MHz, CDCl3): δ 171.4, 171.3, 155.3, 150.8, 150.3, 143.1, 141.6, 140.2, 137.1, 132.3, 131.7, 128.1, 122.3, 121.2, 121.1, 119.8, 119.1, 115.4, 113.2, 67.6, 67.3, 60.9, 60.6, 53.7, 20.9, 14.6, 14.2, 14.1; HRMS (ESI+, m/z): calcd for [M+H]+, 849.4066; found, 849.4097. Compound 5. To a solution of 4 (40 mg, 47 µmol) in methanol/DCM (4 mL/2 mL), iodine monochloride (15 mg, 92 µmol) in methanol (2 mL) was added dropwise. The reaction mixture was stirred at rt for 5 min. After confirming the consumption of starting material by TLC, water was added and the product was extracted with DCM. The organic layer was washed with water and saturated NaCl aq., dried over Na2SO4 and the solvent was evaporated. The residue was chromatographed on silica gel with hexane-AcOEt (2:1-1:1) as the eluent to give compound 5 as a red powder (46 mg, 89%). 1H NMR (400 MHz, CDCl3): δ 6.89 (d, 1H, J ) 8.8 Hz), 6.77-6.66 (m, 5H), 4.27-4.21 (m, 4H), 4.22 (s, 4H), 4.14-4.04 (m, 8H), 4.08 (s, 4H), 2.63 (s, 6H), 2.25 (s, 3H), 1.50 (s, 6H), 1.20 (t, 6H, J ) 7.0 Hz), 1.19 (t, 6H, J ) 7.0 Hz); 13C NMR (101 MHz, CDCl3): δ 171.1, 171.0, 156.4, 150.8, 150.1, 145.2, 141.1, 140.5, 136.9, 132.3, 131.5, 127.4, 122.2, 120.9, 119.7, 119.0, 115.3, 112.8, 85.4, 67.5, 67.1, 60.8, 60.5, 53.5, 20.9, 15.9, 15.1, 14.1, 14.0; HRMS (ESI+, m/z): calcd for [M+H]+, 1101.1998; found, 1101.1990. Compound 6. Acrylic acid benzyl ester was synthesized according to published proceedures.12 A solution of 5 (10 mg, 9 µmol), acrylic acid benzyl ester (3.7 mg, 23 µmol), palladium(II) acetate (0.1 mg, 5 mol %), triethylamine (1 µL, 14 µmol) in dry acetonitrile (5 mL) was stirred at 60 °C under nitrogen overnight. The solvent was evaporated and the residue was chromatographed on silica gel with hexane-AcOEt (2:1-1:1) as the eluent to give compound 6 as a red powder (8.7 mg, 82%). 1H NMR (400 MHz, CDCl3): δ 7.63 (d, 2H, J ) 16 Hz), 7.41-7.33 (m, 10H), 6.90 (d, 1H, J ) 8.8 Hz), 6.77-6.65 (m, 5H), 6.11 (d, 2H, J ) 16 Hz), 5.23 (s, 4H), 4.28-4.22 (m, 4H), 4.22 (s, 4H), 4.11 (q, 4H, J ) 7.2 Hz), 4.07 (s, 4H), 4.05 (q, 4H, J ) 7.2 Hz), 2.70 (s, 6H), 2.23 (s, 3H), 1.58 (s, 6H), 1.20 (t, 6H, J ) 7.2 Hz), 1.16 (t, 6H, J ) 7.2 Hz); 13C NMR (101 MHz, CDCl3): δ 171.2, 171.1, 167.1, 157.0, 150.9, 150.2, 142.6, 140.8, 136.1, 135.8, 132.4, 132.0, 128.6, 128.3, 128.2, 127.1, 126.2, 122.4, 121.3, 119.8, 119.2, 118.1, 115.4, (12) Fu, H.; Lam, Y. J. Comb. Chem. 2005, 7, 734–738.

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113.3, 110.1, 110.0, 67.7, 67.2, 66.4, 61.0, 60.6, 53.7, 21.0, 14.4, 14.2, 13.4; HRMS (ESI+, m/z): calcd for [M+H]+, 1169.5117; found, 1169.5106. Compound 7. To a solution of 6 (105 mg, 90 µmol) in dry DCM/methanol (1 mL/1 mL), palladium activated on carbon (Pd/ C, 10%) was added. The reaction mixture was stirred at rt under hydrogen overnight. After removing Pd/C through filtration, the solvent was evaporated and the residue was chromatographed on silica gel with DCM-methanol-TFA (100:10:0-100:20:0.1) as the eluent. The obtained crude product was further purified by semipreparative reverse-phase HPLC using eluent A (CH3CN with 0.1% TFA and 1% H2O) and eluent B (H2O with 0.1% TFA and 1% CH3CN) (A/B ) 20/80-100/0 in 24 min) to give compound 7 as an orange powder (13 mg, 15%). 1H NMR (400 MHz, CDCl3): δ 6.88 (d, 1H, J ) 8.4 Hz), 6.79-6.66 (m, 5H), 4.28-4.24 (m, 4H), 4.20 (s, 4H), 4.14-4.05 (m, 8H), 4.08 (s, 4H), 2.67-2.62 (m, 4H), 2.53 (s, 6H), 2.43-2.38 (m, 4H), 2.25 (s, 3H), 1.41 (s, 6H), 1.19 (t, 6H, J ) 7.2 Hz), 1.18 (t, 6H, J ) 7.2 Hz); 13C NMR (400 MHz, CDCl3): δ 177.7, 171.6, 171.3, 153.9, 150.8, 150.3, 140.8, 140.1, 139.6, 137.0, 132.3, 131.1, 128.8, 128.4, 122.2, 121.3, 119.8, 119.1, 115.1, 113.3, 67.6, 67.1, 61.0, 60.7, 53.7, 34.0, 20.9, 19.1, 14.2, 12.6, 12.2; HRMS (ESI+, m/z): calcd for [M+H]+, 993.4489; found, 993.4433. BOCA-1-BG. O6-(4-Aminomethyl-benzyl)guanine was synthesized according to published procedures.13 To a solution of 7 (5.6 mg, 5.6 µmol) and O6-(4-aminomethyl-benzyl)guanine (1.5 mg, 5.6 µmol) in dry dimethylformamide (DMF), 1-hydroxybenzotriazole (HOBt) (2.1 mg, 5.5 µmol), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) (1.1 mg, 5.7 µmol), and triethylamine (14 µL, 9.9 µmol) were added at 0 °C. The reaction mixture was stirred at rt overnight. The mixture was extracted with AcOEt, washed with saturated NaCl aq., dried over Na2SO4 and the solvent was evaporated. The residue was purified by semipreparative reversed-phase HPLC using eluent A (CH3CN with 0.1% TFA and 1% H2O) and eluent B (H2O with 0.1% TFA and 1% CH3CN) (A/B ) 20/ 80-100/0 in 24 min) to give an intermediate ethyl ester. In order to cleave the ester, the compound was dissolved in methanol and 1 N NaOH aq. (0.5 mL) was added to the solution. After stirring at rt for 4 h, the solution was neutralized with 1 N HCl aq. and the solvent was evaporated. The residue was purified by semipreparative reversed-phase HPLC using eluent A (CH3CN with 0.1% TFA and 1% H2O) and eluent B (H2O with 0.1% TFA and 1% CH3CN) (A/B ) 5/95-100/0 in 24 min) and lyophilized to give compound 9 as an orange powder (2.2 mg, 35%). 1H NMR (400 MHz, CD3OD): δ 8.34 (s, 1H), 7.30 (d, 2H, J ) 8.0 Hz), 7.04 (d, 2H, J ) 8.0 Hz), 6.89 (d, 1H, J ) 8.0 Hz), 6.80 (d, 1H, J ) 1.2 Hz), 6.77 (d, 1H, J ) 8.0 Hz), 6.65 (dd, 1H, J ) 8.0 Hz, 1.2 Hz), 6.59 (dd, 1H, J ) 8.0 Hz, 2.0 Hz), 6.53 (d, 1H, J ) 2.0 Hz), 5.39 (d, 1H, J ) 12.0 Hz), 5.35 (d, 1H, J ) 12.0 Hz), 4.34 (d, 1H, J ) 15.2 Hz), 4.29 (d, 1H, J ) 15.2 Hz), 4.29-2.26 (m, 2H), 4.16 (s, 4H), 4.11-4.07 (m, 2H), 3.99 (s, 4H), 2.74-2.69 (m, 2H), 2.58-2.55 (m, 2H), 2.54 (s, 3H), 2.48 (s, 3H), 2.38 (t, 2H, J ) 6.8 Hz), 2.31 (t, 2H, J ) 7.6 Hz), 2.24 (s, 3H), 1.41 (s, 3H), 1.40 (s, 3H); HRMS (ESI+, m/z): calcd for [M+2H]2+, 567.2220; found, 567.2238. (13) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86–89.

Plasmids used and Protein Purification. SNAP-tag and CLIP-tag used in this work are 182-amino-acid mutants of the wildtype human O6-alkylguanine-DNA alkyltransferase in which the last 25 amino acids were deleted and the following mutations were introduced: for SNAP-tag,14 K32I, L33F, C62A, Q115S, Q116H, K125A, A127T, R128A, G131K, G132T, M134L, R135S, C150Q, S151G, S152D, G153L, A154D, N157G, and S159E, and for CLIP-tag,15 K32I, L33F, M60I, C62A, Y114E, Q115S, Q116H, A121V, K125A, A127T, R128A, G131N, G132T, M134L, R135D, C150Q, S151G, S152D, G153S, A154D, N157P, E159L. Plasmids for the expression of SNAP-NLS3 and SNAP-MEK1 in mammalian cells were previously described.15,16 CLIP-GPI for mammalian expression was constructed by replacing SNAP-tag in perviously described plasmid for expression of SNAP-GPI.14 For in vitro experiments, SNAP-tag was expressed as a hexahistidine fusion protein in Escherichia coli BL21 (DE3) and purified using a Ni-NTA (Qiagen) agarose column according to the manufacturer’s protocol. In Vitro Fluorescence Properties and Determination of Fluorescence Quantum Yield. Fluorescence emission spectra were recorded on an Infinite M1000 spectrofluorometer (TECAN) using black 96-well plates (FALCON). Excitation wavelength was set to 515 nm, and the excitation and emission bandwidths were set to 5 nm. The spectra were measured with a step size of 1 nm. 200 µM solutions of BOCA-1 and BOCA-1-BG in DMSO were prepared, and 1 µL was added to a well containing 200 µL of buffer at varying Ca2+ concentrations (0-39 µM). For charactierzation of protein-bound BOCA-1, recombinant SNAP-tag (140 µM) was incubated with 50 µM of BOCA-1-BG for 15 min. We confirmed that BOCA-1-BG quantitatively reacts with SNAP-tag protein to form BOCA-1-SNAP conjugate by incubating SNAP-tag with varying concentrations of BOCA-1-BG and analyzing the reaction by SDS-PAGE and in-gel fluorescence scanning (Figure S1 in the Supporting Information (SI)). For the fluorescence measurements, 4 µL of BOCA-1-SNAP prepared as described above were added to a well containing 200 µL of buffer at varying Ca2+ concentrations (0-39 µM). To control Ca2+ concentrations, a calcium calibration buffer kit (Molecular Probes), which contains 30 mM MOPS/KOH, pH 7.2, 100 mM KCl and 10 mM EGTA or 10 mM CaEGTA, was used. Apparent dissociation constant (KD) for Ca2+ were calculated from the fluorescence spectra using plots of log [(F - Fmin)/(Fmax - F)] against log [Ca2+]free (mol/L) as previously described (F ) fluorecence intensity, Fmax ) maximum fluorescence intensity at saturating [Ca2+], Fmin ) minimum fluorescence intensity at zero [Ca2+]).4,5 To determine fluorescence quantum yields, absorption spectra were recorded on a Lambda10 UV/vis spectrometer (PerkinElmer) and fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrometer (VARIAN), using quartz Ultra-Micro Cells (10 × 4 mm) (Hellma). Excitation wavelength was set to 490 nm, and the excitation and emission slits were set to 5 nm. (14) Gronemeyer, T.; Chidley, C.; Juillerat, A.; Heinis, C.; Johnsson, K. Protein Eng., Des. Sel. 2006, 19, 309–316. (15) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128–136. (16) Gautier, A.; Nakata, E.; Lukinavicˇius, G.; Tan, K.-T.; Johnsson, K. J. Am. Chem. Soc. 2009, 131, 17954–17962.

Fluorescein in 0.1 mol L-1 NaOH aq. (Φfl ) 0.85) was used as a fluorescence standard.17 Cell Culture and Transient Transfection. Adherent Chinese hamster ovary (CHO)-K1 cells were cultured in Ham’s F12 (Lonza) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), on 25 cm2 tissue culture flask at 37 °C in a 5% CO2/95% air incubator. For transfection and fluorescence imaging, the CHOK1 cells were seeded on a µ-Dish (Ibidi), and were transiently transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Fluorescence Microscopy. Twenty-four hours after cotransfection of cells with SNAP-NLS3 or SNAP-MEK1 plasmid together with CLIP-GPI plasmid, the cells were loaded with BOCA-1-BG using bead-loading according to published proceedures.18 Briefly, the medium was removed from the dish, 100 µL of 50 µM BOCA-1-BG solution in Ham’s F12 medium containing 10% FBS and 1% DMSO was added to the middle of the dish. Glass beads (Sigma), which were neutralized by 1 N NaOH aq., washed and dried beforehand, were added to the dish. The dish was then swirled a few times to achieve the loading of the sensor in the cell. Afterward, the cells were washed several times with culture medium to remove the beads, and the cells were incubated for 30 min, 45 min, 1.5 h at 37 °C to remove excess dye from cells. Then, the cells were incubated with 5 µM solution of BC-Cy5 (CLIP-surface 647) in Ham’s F12 medium containing 10% FBS and 0.5% DMSO for 15 min. After washing with physiological salt solution (PSS), pH 7.4, containing 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 0.1% glucose, 900 µL of PSS was added to the dish. To stimulate cells to induce a Ca2+ signal, 100 µL of 1 mM of adenosine 5′-triphosphate (ATP) solution in PSS was added to the dish. A time delay in the stimulation of the cells after ATP addition resulted from the diffusion of ATP in the bathing solution (final ATP concentration: 100 µM). Images were captured during the ATP stimulation. All images were acquired using a confocal microscope (TCS SP5X; Leica) equipped with a white laser and an objective lens (HC PL APO 20x/0.70; Leica). Excitation and emission wavelengths were set to 525 nm, 535-620 nm for BODIPY fluorescence, and 631 nm, 650-747 nm for Cy5 fluorescence, respectively. The microscope was controlled by LAS AF software. RESULTS AND DISCUSSION BODIPY-based fluorophores are known to be highly fluorescent, resistant to photobleaching, and relatively insensitive to solvent polarity and pH.19,20 Furthermore, a variety of BODIPY derivatives have found applications in the fluorescence labeling of proteins and DNA.21-24 We therefore focused on BODIPY(17) Paeker, C. A.; Rees, W. T. Analyst 1960, 85, 587–600. (18) McNeil, P. L.; Warder, E. J. Cell Sci. 1987, 88, 669–678. (19) Karolin, J.; Johansson, L. B. A.; Strandberg, L.; Ny, T. J. Am. Chem. Soc. 1994, 116, 7801–7806. (20) Wittmershaus, B. P.; Skibicki, J. J.; McLafferty, J. B.; Zhang, Y. Z.; Swan, S. J. Fluoresc. 2001, 11, 119–128. (21) Haugland, R. P. The Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes: Eugene, OR. (22) Yee, M.; Fas, S. C.; Stohlmeyer, M. M.; Wandless, T. J.; Cimprich, K. A. J. Biol. Chem. 2005, 280, 29053–29059. (23) Castro, A.; Williams, J. G. Anal. Chem. 1997, 69, 3915–3920. (24) Metzker, M. L.; Lu, J.; Gibbs, R. A. Science 1996, 271, 1420–1422.

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Figure 1. (A) Structure of newly developed BODIPY-based Ca2+ indicator (BOCA-1) in the absence and presence of Ca2+. (B) Structure of BOCA1-BG. (C) Labeling of a SNAP-tag fusion protein (X represents a protein with defined localization) with BOCA-1-BG and its subsequent use for local Ca2+ sensing. (D) Use of BOCA-1-BG and a nuclear localized SNAP-tag fusion protein for Ca2+ sensing in the nuclei of living cells.

based fluorophores as a core structure for the generation of a highly sensitive fluorescent Ca2+ indicator suitable for linking to proteins. BODIPY-based Ca2+ indicators have been reported previously.25-27 However, these indicators showed relatively modest fluorescence increases upon Ca2+ binding (1.5-fold higher) and CalciumGreen-1 (>10-fold higher) as well as green fluorescent protein-based GCamp2 (>30-fold higher) or the recently introduced GCamp3 (>15-fold higher)34 (Table 1). Next, we focused on the use of BOCA-1-BG for the specific labeling of SNAP-tag fusion proteins in living cells. BAPTA-based Ca2+ indicators in general are not membrane permeable but can be made membrane permeable by transforming them into the corresponding acetoxymethyl (AM) esters.4,5 The AM esters are then hydrolyzed in the cell by esterases to release the functional Ca2+ indicator. To test if this strategy can also be applied to our BODIPY-based sensor, we first synthesized the acetoxymethyl (AM) ester of BOCA-1, AM-BOCA-1 (Scheme S1 in the SI). However, this compound showed nonspecific compartmentalization when applied to cells (data not shown), probably due to its hydrophobic nature. These experiments suggested that the AM ester of BOCA-1-BG might also show unspecific binding. Therefore, we focused on bead-loading of BOCA-1-BG for the specific labeling of SNAP-tag fusion proteins in cultured mammalian cells. Bead-loading is achieved by rolling glass beads over cultured cells in the presence of the molecule of interest.18 The method has been shown to be generally benign (34) Tian, L.; Hires, S. A.; Mao, T.; Huber, D.; Chiappe, M. E.; Chalasani, S. H.; Petreanu, L.; Akerboom, J.; McKinney, S. A.; Schreiter, E. R.; Bargmann, C. I.; Jayaraman, V.; Svoboda, K.; Looger, L. L. Nat. Methods 2009, 6, 875–881.

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for cultured cells and is experimentally simple. To test if the approach could be used for the labeling of SNAP-tag fusion proteins with BOCA-1-BG, we transiently coexpressed nucleartargeted SNAP-tag (SNAP-NLS3) together with cell surfacetargeted CLIP-tag (CLIP-GPI) in CHO-K1 cells (Figure 1D). We chose nuclei as a first target of localization as it is known that small molecules can be delivered from cytosol through nuclear pore complex. CLIP-tag is an another self-labeling protein tag that can be specifically labeled with benzylcytosine (BC) derivatives.15 CLIP-GPI, which was labeled with BC-Cy5, served as a transfection marker in these experiments. BOCA-1-BG was then introduced into CHO-K1 cells by bead-loading, resulting in the nonselective and heterogeneous staining of transfected and nontransfected cells. In agreement with previous data,18 beadloading did not result in any obvious change of cellular morphology. In the first hour after loading, we observed fluorescence signals both in nuclei and in cytosol of transfected and nontransfected cells (Figure S3 in the SI). However, after incubating the cells for more than 1.5 h after the bead-loading, fluorescent signal exclusively from the nuclei of transfected cells could be observed (Figure 3, Figure S3 in the SI). These data suggest that (i) BOCA1-BG can be introduced into CHO-K1 cells using bead-loading, (ii) the sensor reacts with nuclear-localized SNAP-tag, and (iii) excess BOCA-1-BG is washed out of cells during a simple incubation step after bead-loading. It has been observed previously

that BAPTA derivatives are leaked out of cells relatively rapidly,35 probably through anion channels. The stimulation of CHO-K1 cells with extracellular ATP leads to an increase in intracellular [Ca2+]i.36 When CHO-K1 cells expressing SNAP-NLS3 and labeled with BOCA-1-BG were stimulated by addition of ATP to the bathing solution, we observed a 3-5-fold fluorescence increase in the nuclei of transfected cells (Figure 3A and B, Figure S4 and Movie S1 in the SI), demonstrating that SNAP-tag-bound BOCA-1 functions as a localized Ca2+ indicator in living cells. To test if BOCA-1 can also be localized to other cellular compartments, we cotransfected CHO-K1 cells with plasmids of cytosolic kinase MEK137 as a SNAP-tag fusion protein (SNAPMEK1) and CLIP-GPI as a transfection marker. Bead-loading of BOCA-1-BG and a subsequent incubation for 1.5 h resulted in a cytosolic fluorescent signal from transfected cells, indicating a specific labeling of SNAP-MEK1 with BOCA-1-BG. BOCA-1SNAP-MEK1 also senses changes in [Ca2+]i in the cytosol: stimulation of cells by ATP resulted in a 2-3-fold increase in fluorescence intensity in the cytosol of transfected cells (Figure 3A and C, Figure S5 and Movie S2 in the SI). It was previously reported that ATP stimulation (10 µM final concentration) of nonadherent CHO-K1 cells results in a change of [Ca2+]i from 138 to 972 nM.36 Considering the fluorescence emission spectra of BOCA-1-SNAP (Figure 2C), the measured magnitude of the fluorescence increase in the cytosol and in the nuclei of the stimulated CHO-K1 cells is in agreement with the properties of BOCA-1-SNAP and the reported increase in [Ca2+]i in stimulated (35) Di Virgilio, F.; Steinberg, T. H.; Silverstein, S. C. Cell Calcium 1990, 11, 57–62. (36) Iredale, P. A.; Hill, S. J. Br. J. Pharmacol. 1993, 110, 1305–1310. (37) Burack, W. R.; Shaw, A. S. J. Biol. Chem. 2005, 280, 3832–3837. (38) Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.; Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R. F.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.; Wood, K. V. ACS Chem. Biol. 2008, 3, 373–382.

CHO-K1 cells (Figure S6 in the SI). These proof-of-principle experiments thus demonstrate how BOCA-1-BG can be used for visualizing changes in local [Ca2+] in living cells. CONCLUSIONS In summary, the here introduced BODIPY-based Ca2+ indicators (BOCAs) possess a number of important properties: First, BOCAs are highly sensitive Ca2+ indicators; the 250-fold increase in fluorescence intensity observed for BOCA-1 is the highest fluorescence increase published for any Ca2+ indicator so far. Second, a BG derivative of BOCA-1 can be coupled to SNAP-tag proteins and be used as a highly sensitive and localizable Ca2+ indicator in living cells. Third, as the sensor utilizes the well-characterized BAPTA moiety, a rational modification of the Ca2+ affinity of BOCAs without affecting their spectroscopic properties should be feasible. Fourth, BOCAs derivatives that can be coupled to other protein tags such as CLIP-tag15 and Halo-tag38 should be easily accessible by simply replacing the BG moiety with their respective ligands. Together, these properties should make BOCAs sensitive tools for studying [Ca2+]i with high spatiotemporal resolution in living cells. ACKNOWLEDGMENT We thank Prof. T. Nagai, Dr. D. Maurel, and Dr. T. Ueno for helpful discussions; Dr. A. Gautier, C. Chidley, and S. Fujishima for technical assistance; and C. Fellay for providing 5-formyl-5′methyl-BAPTA ethyl ester. This work was supported by the Swiss National Science Foundation and a JSPS stipend to MK. SUPPORTING INFORMATION AVAILABLE Characterization of fluorescence properties of BOCAs and their reaction with SNAP-tag, synthesis of AM-BOCA-1, live-cell fluorescence imaging data and movies. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 23, 2010. Accepted June 17, 2010. AC100741T

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