Supporting Information Development of a Highly Sensitive Fluorescence Probe for Hydrogen Peroxide Masahiro Abo,, Yasuteru Urano, Kenjiro Hanaoka,, Takuya Terai,, Toru Komatsu, and Tetsuo Nagano,, * Graduate School of Pharmaceutical Sciences and Graduate School of Medicine, the University of Tokyo Hongo, Bunkyo-ku, Tokyo,113-33, Japan.. CREST, JST, 4-8-1 Sanbancho-blg, 5 Sanbancho, Chiyoda-ku, Tokyo, 12-75, Japan * To whom correspondence should be addressed. E-mail: tlong@mol.f.u-tokyo.ac.jp Abbreviations LUMO, lowest unoccupied molecular orbital; fl, fluorescence quantum efficiency; HPLC, high-performance liquid chromatography; CH 3 CN, acetonitrile; DMSO, dimethylsulfoxide; DMF, N,N-dimethylformamide; MeOH, methanol; H 2 O 2, hydrogen peroxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; AcOEt,ethyl acetate; NaOCl, sodium hypochlorite; OCl, hypochlorite; NO, nitric oxide; O 2, superoxide anion; OH, hydroxyl radical; ONOO, peroxynitrite; KO 2, potassium superoxide; NOC13, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene; EP-1, 3-(1,4-dihydro-1,4-epidioxy-1-naphthyl)propionic acid; Fe(ClO 4 ) 2, iron (II) perchlorate; Na 2 SO 4, sodium sulfonate; NaHCO 3, sodium bicarbonate; K 2 CO 3, potassium carbonate; ROS, reactive oxygen species; SOD, superoxide dismutase. S1
4 45 5 55 6 M 1 M 2 M + SOD 4 3 2 1 45 5 55 6 65 M 1 M 2 M + SOD Figure S1. and fluorescence spectral changes of NBzF in the presence of superoxide. M 1 M 2 M 5 4 3 2 1 M 1 M 2 M 4 45 5 55 6 45 5 55 6 65 Figure S2. and fluorescence spectral changes of NBzF in the presence of hydrogen peroxide. 4 45 5 55 6 M 1 M 2 M 8 M 6 1 M 4 2 M 2 45 5 55 6 65 Figure S3. and fluorescence spectral changes of NBzF in the presence of hydroxyl radical. S2
4 45 5 55 6 M 1 M 2 M 1 8 M 6 1 M 4 2 M 2 45 5 55 6 65 Figure S4. and fluorescence spectral changes of NBzF in the presence of peroxynitrite. 4 45 5 55 6 M 1 M 2 M 1 8 M 6 1 M 4 2 M 2 45 5 55 6 65 Figure S5. and fluorescence spectral changes of NBzF in the presence of hypochlorite. 4 45 5 55 6 M 1 M 2 M 1 8 M 6 1 M 4 2 M 2 45 5 55 6 65 Figure S6. and fluorescence spectral changes of NBzF in the presence of tert-butyl hydroperoxide. S3
4 45 5 55 6 M 1 M 2 M 8 M 6 1 M 4 2 M 2 45 5 55 6 65 Figure S7. and fluorescence spectral changes of NBzF in the presence of nitric oxide. M 1 M 2 M 4 45 5 55 6 1 8 M 6 1 M 4 2 M 2 45 5 55 6 65 Figure S8. and fluorescence spectral changes of NBzF in the presence of singlet oxygen. S4
fluorescein NBzF NBzF ONOO - 4 8 12 16 4 8 12 16 1 M H 2 O 2 ONOO - 4 8 12 16 4 8 12 16 1 M H 2 O 2 + 5-CF 1 M TBHP 4 8 12 16 4 8 12 16 5-CF 5-CF 2 M KO 2 4 8 12 16 4 8 12 16 Figure S9. HPLC chromatograms of NBzF (1 M) after reaction with hydrogen peroxide (H 2 O 2 ), peroxinitrite (ONOO - ), tert-butyl hydroperoxide (TBHP), or potassium superoxide (KO 2 ). Chromatograms of unreacted dye and 5-carboxyfluorescein (5-CF) are also shown. Elution was done with a linear gradient (eluent, min, 8 % CH 3 CN/% TFA aq. ~ 2 min, 8 % CH 3 CN/ % TFA aq.; flow rate = 1. ml/min). Detection wavelength was 49 nm. Fluorescein was used as a standard (peak: 1 min). S5
(A) (B) (C) (D) (E) (F) 25 Averaged F.I. of ROIs 2 15 1 5 PMA- PMA+ APO EBS L-NAME Figure S1. Confocal fluorescence images of RAW 264.7 macrophages stained with FDA after treatment with PMA and inhibitors. (A) Without PMA stimulation. (B) PMA stimulation only. (C) PMA stimulation in the presence of 5 mm apocynin. (D) PMA stimulation in the presence of ebselen. (E) PMA stimulation in the presence of 5 mm L-NAME. Esterase activity was not critically affected by PMA or inhibitors. S6
1 2 Fluorescence intensity at 52 nm (a.u.) 7 6 5 4 3 2 1 blank H 2 O 2 ONOO - TBHP O - - 2 OH OCl NO 1 O 2 3 4 Fluorescence intensity at 52 nm (a.u.) 7 6 5 4 3 2 1 blank H 2 O 2 ONOO - TBHP O - - 2 OH OCl NO 1 O 2 Fluorescence intensity at 52 nm (a.u.) 7 6 5 4 3 2 1 blank H 2 O 2 ONOO - TBHP O - - 2 OH OCl NO 1 O 2 Fluorescence intensity at 52 nm (a.u.) 6 5 4 3 2 1 blank H 2 O 2 ONOO - TBHP O - - 2 OH OCl NO 1 O 2 Figure S11. Fluorescence response of 1-4 (1 M) to various ROS. The reaction and fluorescence measurements were performed in M sodium phosphate buffer at ph 7.4 with excitation at 49 nm. H 2 O 2 : 1 M H 2 O 2 at 37 C for 6 min; ONOO-: 1 M ONOONa; TBHP: 1 M t-buooh at 37 C for 6 min; O - 2 : 1 M KO 2, OH: 1 M H 2 O 2 and 1 M Fe(OCl 4 ) 2 (Fenton reaction); - OCl: 1 M NaOCl at 25 C for 5 min; NO: 1 M NOC 13 at 37 C for 3 min; 1 O 2 : 1 M EP-1 at 37 C for 3 min. S7
NBzF in DMSO-d 6 NBzF in DMSO-d 6 with 2 % PBS Reaction mixture (3 min) NBzF 5-CF 4-NO 2 BA Reaction mixture (3 min) 5-CF in DMSO-d 6 with 2 % PBS 4-NO 2 BA in DMSO-d 6 with 2 % PBS Figure S12. Monitoring of the reaction between NBzF and hydrogen peroxide by means of NMR spectroscopy. The reaction was performed with 2 mm NBzF and 2 mm hydrogen peroxide at 25 C in DMSO-d 6 containing 2 % PBS at ph 7.4. NBzF is converted to 5-carboxyfluorescein (5-CF) and 4-nitrobenzoic acid (4-NO 2 BA) within 3 minutes. The spectrum of NBzF in DMSO-d 6 with 2 % PBS indicates that some equilibration of NBzF occurs under neutral aqueous conditions. S8
Figure S13. Kinetic analysis of the reaction between NBzF (1 M) and hydrogen peroxide in M sodium phosphate buffer at ph 7.4. The indicated concentration of hydrogen peroxide was added at 6 sec and the reaction mixture was incubated at 25 or 37 C with excitation at 495 nm. The fluorescence at 52 nm was measured. The results for dichlorodihydrofluorescein (DCFH, 1 M) under the same conditions are also presented. References (9) Capasso, M.; Bhamrah, M. K.; Henley, T.; Boyd, R. S.; Langlais, C.; Cain, K.; Dinsdale, D.; Pulford, K.; Khan, M.; Musset, B.; Cherny, V. V.; Morgan, D.; Gascoyne, R. D.; Vigorito, E.; DeCoursey, T. E.; MacLennan, I. C.; Dyer, M. J. Nat Immunol 21, 11, 265-272. S9