Supporting Information Visualization of Phagosomal Hydrogen Peroxide Production by A Novel Fluorescent Probe That Is Localized via SNAP-tag Labeling Masahiro Abo, Reiko Minakami, Kei Miyano, Mako Kamiya, Tetsuo Nagano, Yasuteru Urano, and Hideki Sumimoto * Departments of Biochemistry and Health Sciences, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan, and the Graduate Schools of Medicine and Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan * To whom correspondence should be addressed. E-mail: hsumi@med.kyushu-u.ac.jp. Tel: 81(Japan)-92-642-6096. Fax: 81(Japan)-92-642-6103. S 1
CONTENTS Synthesis of NBzF-BG S-3 Fluorescence of NBzF-BG at various ph S-5 References S-6 S 2
Figure S1. Synthesis of NBzF-BG. (i) 1-ethynyl-4-nitrobenzene, bis(triphenylphosphine)palladium(ii) chloride, triethylamine, THF; (ii) potassium carbonate, methanol; (iii) NHS, N,N -dicyclohexylcarbodiimide, DMF, ethyl 4-piperidinecarboxylate; (iv) lithium hydroxide, methanol/water; (v) palladium(ii) chloride, DMSO; (vi) NHS, EDCI HCl, DMF, O 6 -[4-(aminomethyl)benzyl]guanine. Synthesis of NBzF-BG NBzF-BG was synthesized from 5-iodofluorescein (compound 1) as a starting material, as described below. Compound 1 was prepared according to the method by Jiao et al. 1, and compound 2 was synthesized from compound 1 as previously described 2. For synthesis of compound 3, compound 2 (1.29 g, 2.70 mmol), N-hydroxysuccinimide (NHS; 1.34 g, 11.6 mmol) and N,N -dicyclohexylcarbodiimide (2.32g, 11.2 mmol) were mixed in 10 ml of N,N-dimethylformamide (DMF) and the mixture was heated for 2 h at 60 C, affording a colorless precipitate. The precipitate was removed by filtration and washed with 10 ml of DMF. To the filtrate were added ethyl 4-piperidinecarboxylate (2.50 ml, 16.2 mmol) and triethylamine (5.00 ml, 36.1 mmol), and the mixture was stirred for 15 h at 25 C. The reaction mixture was diluted with ethyl acetate, and washed with aqueous hydrogen chloride three times and with brine once. Ethyl acetate was evaporated and the mixture was purified by column chromatography over silica gel using acetone/dichloromethane in the gradient from 40% to 100%, affording 1.36 g of an intermediate product. Lithium hydroxide monohydrate (880 mg, 21.0 mmol) in 10 ml of water was added to the intermediate product dissolved in 30 ml of methanol, and the mixture was stirred for 3 h at 25 C. Methanol was evaporated and the mixture was acidified with aqueous hydrogen chloride. The yellow precipitate obtained was filtered and washed with aqueous hydrogen chloride and with water, and then dried under vacuum, affording a product (770 mg, 1.31 mmol, 48%). 1 H NMR (400 MHz, DMSO-d 6 ) 8.32 (d, 2H, J = 8.8 Hz), 7.92 (m, 4H), 7.60 (d, 1H, J = 7.6 Hz), 7.13 (m, 2H), 6.73 (m, 4H), 3.95 (d, 1H, J = 1.2 Hz), 3.63 (d, 1H, J = 1.2 Hz), 3.00 (m, 1H), 2.63 (m, 1H), 2.40 (m, 1H), 1.68 (m, 2H), 1.30 (m, 1H), 1.20 (m, 1H). 13 C NMR (100 MHz, DMSO-d 6 ) 175.2, 165.2, 158.2, 157.9, 156.4, 150.8, 147.1, 136.6, 132.8, 132.3, 131.6, 131.0, 130.0, 128.5, 123.9, 122.7, 115.0, 103.0, 92.7, 89.3, 46.2, 27.8, 27.2. HRMS (ESI+) Calcd for [M+H], 589.16109; Found, 589.15914 ( 1.95 mmu). For synthesis of compound 4, compound 3 (9.2 mg, 15.6 µmol) and palladium(ii) chloride (13.8 S 3
mg, 78.0 µmol) were mixed in 1 ml of DMSO and the mixture was heated for 3.5 h at 60 70 C under an argon atmosphere. The mixture was purified by preparative HPLC, affording a product (3.20 mg, 5.16 µmol, 33%). 1 H NMR (300 MHz, DMSO-d 6 ) 8.46 (d, 2H, J = 8.7 Hz), 8.29 (m, 3H), 8.18 (d, 1H, J = 1.5 Hz), 6.98 (d, 2H, J = 8.7 Hz), 6.56 (m, 4H), 3.93 (m, 1H), 1.66 (m, 2H), 1.20 (m, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) 191.3, 191.2, 175.2, 165.1, 158.4, 156.4, 150.9, 150.2, 137.3, 136.9, 136.8, 132.8, 131.6, 131.5, 131.3, 130.7, 128.7, 124.2, 121.2, 114.9, 103.1, 46.3, 27.8, 27.2. HRMS (ESI+) Calcd for [M+H], 619.13527; Found, 619.13149 ( 3.78 mmu). For synthesis of NBzF-BG, compound 4 (1.50 mg, 2.42 µmol), NHS (1.42 mg, 12.3 µmol) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDCI HCl; 2.54 mg, 13.2 µmol) were mixed in 0.25 ml of DMF, and the mixture was stirred for 3 h at 25 C. After further addition of NHS (1.36 mg) and EDCI HCl (2.05 mg), the mixture was stirred for further 30 min at 25 C. O 6 -[4-(aminomethyl)benzyl]guanine (1.60 mg, 5.93 µmol) was then added to the mixture, which was stirred for 1.5 h at 25 C. DMF was evaporated and the mixture was purified by preparative HPLC, affording a pure product (1.6 mg, 1.83 mmol, 76%). 1 H NMR (300 MHz, DMSO-d 6 ) 8.46 (d, 2H, J = 9.0 Hz), 8.30 (m, 4H), 8.19 (d, 1H, J = 1.5 Hz), 8.08 (s, 1H), 7.77 (d, 2H, J = 8.1 Hz), 7.45 (d, 2H, J = 7.8 Hz), 7.22 (d, 2H, J = 7.2 Hz), 6.99 (d, 2H, J = 9.6 Hz), 6.58 (m, 4H), 5.48 (s, 2H), 4.22 (d, 2H, J = 5.1 Hz), 4.07 (m, 1H), 1.61 (m, 2H), 1.24 (m, 2H). 13 C NMR (100 MHz, DMSO-d 6 ) 191.3, 191.2, 173.5, 165.2, 158.7, 158.4, 158.0, 156.3, 153.2, 150.9, 149.2, 140.1, 137.3, 136.9, 136.8, 133.9, 132.8, 131.6, 131.1, 130.8, 128.9, 128.6, 127.0, 124.2, 117.4, 114.7, 114.5, 103.3, 68.2, 46.5, 41.6, 41.1, 28.5, 27.8, 25.3. HRMS (ESI-) Calcd for [M-H], 871.24761; Found, 871.24383 ( 3.78 mmu). NMR spectra of NBzF-BG and its precursors were recorded on a JEOL JNM-LA300 instrument or a JEOL JNM-LA 400. Mass spectra of NBzF-BG and its precursors were measured with a JEOL JMS-T 100LC AccuTOF. S 4
Figure S2. Fluorescence of NBzF-BG at various ph. NBzF-BG (20 M) was incubated with or without 1 mm H 2 O 2 for 20 min at 25 C in 10 mm sodium phosphate (ph 7.4) containing 20 % DMSO. The reaction mixture (25 L) was diluted with 475 L of 100 mm sodium phosphate at indicated ph and fluorescence spectra were measured with excitation light at 500 nm. (A) Fluorescence spectra. (B) Fluorescence at 520 nm. Error bars, SD (n = 3). S 5
References 1) Jiao, G. S.; Han, J. W.; Burgess, K. J. Org. Chem. 2003, 68, 8264-8267. 2) Abo, M.; Urano, Y; Hanaoka, K.; Terai, T.; Komatsu, T.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 10629-10637. S 6