SUPPORTING INFORMATION. Highly Sensitive and Selective Bioluminescence Based Ozone Probes and Their Applications to Detect Ambient Ozone

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1 Electronic upplementary Material (EI) for ChemComm. This journal is The Royal ociety of Chemistry 15 UPPRTIG IFRMATI Highly ensitive and elective Bioluminescence Based zone Probes and Their Applications to Detect Ambient zone Younseok am, Beom eok Kim and Injae hin* Center for Biofunctional Molecules, Department of Chemistry, Yonsei University, eoul 37, Korea Corresponding author 1

2 upplementary Experimental Methods General methods Chemicals were purchased from igma-aldrich (t. Louis, M) and Alfa Aesar (Ward Hill, MA). Analytical thin-layer chromatography was performed using silica gel 6 F54 glass plates (Merck, Darmstadt). Compound spots on TLC were visualized by UV light (54 nm) and/or by staining with 1 wt% phosphomolybdic acid in ethanol. Flash column chromatography was conducted on silica gel 6 (3-4 Mesh, Merck). MR spectra were recorded with a Bruke Avance II-4 or DPX-5 instrument. Coupling constants were reported in Hertz (Hz). Analytical reverse-phased HPLC analysis was performed on Denali C-18 column (5 x 4.6 mm) at a flow rate of 1. ml/min with a Waters HPLC system. Electrospray ionization (EI) mass spectra were recorded in a positive ion mode on a Waters 31 LC/M ystem. A B H H D-cysteine C MeH, H 65% C 4-Bromo-1-butene K C 3,DMF 6% 1. Phosgene, DMF. 3-Buten-1-ol DMAP, THF 7% 1 H CH C C D-cysteine MeH, H 78% H CH cheme 1. ynthesis of bioluminescent ozone probes (A) 1 and (B). -Cyano-6-(3-butenyloxy)benzothiazole. A stirring mixture of -cyano-6- hydroxybenzothiazole 1 (5 mg,.14 mmol), K C 3 (58.9 mg,.43 mmol) and 4-bromo-1- butene (43. L,.43 mmol) in ml DMF was heated at 7 o C for 4 h. The reaction mixture was poured into water (5 ml) and extracted with EtAc (3 ml x 3). The combined organic layers were washed with water and brine, dried over anhydrous a 4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (hexane : EtAc = 4:1) to afford a product as a white solid in 6% yield: 1 H MR (4 MHz, CDCl 3 ) 8.8 (d, 1 H, J = 9.5 Hz), 7.35 (d, 1 H, J =. Hz), 7.4 (dd, 1 H, J = 1.3,. Hz), (m, 1 H), (m, 1 H), 5.15 (dd, 1 H, J = 1.3,. Hz), 4.1 (t, H, J = 7. Hz), (m, H); 13 C MR (1 MHz, CDCl 3 ) 159.9, 147., 137.5, 133.9, 133.4, 15.9, 118,9, 117,7, 113.3, 13.8, 68., 33.5; EI-M calcd for C 1 H 11 [M + H] , found 31.7.

3 ynthesis of 1. D-Cysteine hydrochloride monohydrate (71.6 mg,.41 mmol) was dissolved in water ( ml) degassed with argon and ph of the solution was adjusted to 8. with.5 M K C 3. -Cyano-6-(3-butenyloxy)benzothiazole (39.1 mg,.17 mmol) dissolved in MeH (4 ml) degassed with argon was added to the D-cysteine solution. The mixture was stirred at room temperature with light shielding for 1 h under argon atmosphere. The reaction mixture was adjusted to about ph 4 with addition of several drops of concentrated HCl. The organic solvent was removed under reduced pressure. The remaining aqueous solution was extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over anhydrous a 4, and concentrated in vacuo. The residue was purified by flash column chromatography (CH Cl : EtAc : MeH = 3:3:1) to provide a product as a yellow solid in 65% yield: 1 H MR (4 MHz, CD 3 D) 7.94 (d, 1 H, J = 8.8 Hz), 7.53 (d, 1 H, J =.9 Hz), 7.15 (dd, 1 H, J = 9.,.6 Hz), (m, 1 H), 5.9 (t, 1 H, J = 9. Hz), 5. (dq, 1 H, J = 17.1, 1.6 Hz), 5.11 (dt, 1 H, J=1.3,. Hz), 4.1 (t, H, J=6. Hz), 3.76 (d, H, J = 9.5 Hz),.58 (qt, H, J = 6.6, 1.5 Hz); 13 C MR (1 MHz, DM-d 6 ) 17.4, 161.3, 158.8, 157.9, 147., 137., 134.7, 14.5, 117.1, 117., 15.3, 81.7, 67.4, 35.7, 3.9; EI-M calcd for C 15 H 15 3 [M + H] , found Butenyloxycarbonylamino--cyanobenzothiazole. To a stirred solution of 6-amino-- cyanobenzothiazole (98 mg,.6 mmol) in dry THF (1 ml) was added phosgene (.7 ml, % w/v in toluene, 5.5 mmol). The solution was heated at reflux for 4.5 h. After cooling to room temperature, the remaining phosgene was removed by purging with nitrogen into a KH trap for 1 min. The solvent was removed under reduced pressure to give a white solid. The solid was dissolved into anhydrous toluene (1 ml) and then the solvent was removed again under reduced pressure. After re-dissolving the resulting residue in dry THF (5 ml) under atmosphere, 3-buten-1-ol (96 μl, 1. mmol) and DMAP (6. mg,.5 mmol) were added to the solution. After stirring overnight at room temperature, the volatile material was removed under reduced pressure. The residue was re-dissolved in EtAc and the organic layer was washed with brine, dried over anhydrous a 4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (hexane : EtAc = 5:1) to give a product as a pale yellow solid in 7% yield: 1 H MR (4 MHz, CDCl 3 ) 8.41 (s, 1 H), 8.8 (d, 1 H, J = 9.5 Hz), 7.38 (dd, 1 H, J = 9., 1.8 Hz), 7.17 (s, 1 H), (m, 1 H), (m, H), 4.8 (t, H, J = 6.6 Hz), (m, H); 13 C MR (1 MHz, CDCl 3 ) 154.1, 147.6, 14., 136.9, 134.3, 134.1, 14.5, 119.7, 116.3, 11.8, 19.1, 64.1, 33.1; EI-M calcd for C 13 H 1 3 [M + H] , found ynthesis of. D-Cysteine hydrochloride monohydrate (77. mg,.44 mmol) was dissolved in water ( ml) degassed with argon and ph of the solution was adjusted to 8. with.5 M K C 3. 6-Butenyloxycarbonylamino--cyanobenzothiazole (5 mg,.18 mmol) dissolved in MeH (4 ml) degassed with argon was added to the D-cysteine solution. The mixture was stirred at room temperature with light shielding for 1 h under argon atmosphere. The reaction mixture was adjusted to about ph 4 with addition of several drops of concentrated HCl. The organic solvent was removed under reduced pressure. The remaining aqueous solution was 3

4 extracted with ethyl acetate three times. The combined organic layers were washed with brine, dried over anhydrous a 4, and concentrated in vacuo. The residue was purified by flash column chromatography (CH Cl : EtAc : MeH = 3:3:1) to provide a product as a yellow solid in 78% yield: 1 H MR (4 MHz, CD 3 D) 8.6 (s, 1 H), 7.95 (d, 1 H, J = 9.5 Hz), 7.5 (dd, 1 H, J = 9., 1.8 Hz), (m, 1 H), 5.9 (t, 1 H, J = 9. Hz), (m, H), (m, H), 3.76 (d, H, J = 9.5 Hz), (m, H); 13 C MR (1 MHz, DM-d 6 ) 173., 161.7, 159.5, 153.7, 148., 138.6, 136.4, 134.7, 14.1, 118.8, 117.4, 11., 81.9, 63.7, 35.9, 33.; EI-M calcd for C 16 H [M + H] , found Production of ozone and determination of its concentration. zone produced by using an ozone generator (Fischer, Meckenheim, Germany) was collected in potassium phosphate buffer (5 mm, ph 7.). The concentration of ozone solutions was determined by iodometric titration and UV spectroscopy methods. Measurements of ozone concentrations by iodometric titration. zone concentrations by iodometric titration were determined according to the method described previously. 3 Briefly, to a solution of ozone (5 ml) in potassium phosphate buffer (5 mm, ph 7.) was added 5 ml of. M KI solution. The ph of solutions was adjusted to ca.. by adding drops of 1 M H 4. The solutions were titrated with mm a 3 solutions until the yellow solutions became coloreless. Measurements of ozone concentrations by UV spectroscopy. 4 zone concentrations were determined by using a JAC Corporation UV/VI spectrophotometer (ε 54 nm ( 3 ) = 315 M -1 cm -1 ). HPLC analysis of products obtained from reaction of ozone and probes. A solution ( mm) of probe 1 or in DM (5 μl) was diluted with 1 ml potassium phosphate buffer (5 mm, ph 7). Each probe (a final concentration: 1 μm) in potassium phosphate buffer (5 mm, ph 7.) was reacted with ozone (a final concentration: 15 μm) in potassium phosphate buffer (5 mm, ph 7.) for 1 h at 37 C. The mixture was treated with mm pyrrolidine for 5 min. The mixture treated or untreated with pyrrolidine was analyzed by analytical RP-HPLC on a C-18 column at a flow rate of 1 ml/min with a linear gradient of 5-1% CH 3 C (.1% TFA) in water (.1% TFA) over 45 min. The products were characterized by EI-M. Fluorescent detection of a mixture of probe 1 or with ozone. A solution of each probe (4 μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with various concentrations of ozone ( 1 μm) in potassium phosphate buffer at room temperature for 3 min and then further treated with mm pyrrolidine for 5 min. Fluorescent spectra were recorded on a Perkin Elmer L 55 fluorometer. The samples were excited at 33 nm for probe 1 and 35 nm for probe. 4

5 Luciferase activity test. To a reaction mixture (1 μl) of 1 or with ozone in potassium phosphate buffer was added a solution (1 μl) of luciferase (1 μg/ml) in assay buffer (1 mm ATP, 6 mm DTT.6 mm CoA and 16 mm Mg + in 5 mm Tris buffer at ph 8.) at 5 o C. Black, opaque 384-well plates were used for bioluminescent measurements. Bioluminescence intensity was measured by using a Victor X5 Multilabel Plate Reader. The experiment was performed in triplicate. Bioluminescent detection of a mixture of probe 1 or with ozone. A solution of each probe (1 mm) in potassium phosphate buffer (5 mm, ph 7.) containing 5% DM was treated with various concentrations of ozone ( 5 μm) in potassium phosphate buffer at room temperature for 3 min and then further treated with mm pyrrolidine. After 5 min, luciferase (1 μg/ml) in assay buffer was added to the mixture. After incubation for 1 min at 5 o C, bioluminescence spectra were recorded on a Perkin Elmer L 55 fluorimeter (a bioluminescence measurement mode) at room temperature. Bioluminescence images were obtained by using a Xenogen IVI pectrum In Vivo Imaging ystem. Time-dependent changes in bioluminescence intensity. A solution of each probe ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with ozone (1 μm) in potassium phosphate buffer at 5 o C. After incubation for the indicated time, luciferase (1 μg/ml) in assay buffer was added to the mixture and then bioluminescence intensity was measured by using a bioluminescent microplate reader. Response of probes to R. A solution of each probe ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with M 3, 1 mm 1, 1 mm H, 1 mm. - H, 1 mm. or 1 mm Cl - at 5 C. After incubation for 1 h at 37 C, luciferase (1 μg/ml) in assay buffer was added to the mixture, and bioluminescence intensity was measured by using a bioluminescent microplate reader. zone: A solution of probe 1 or ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with ozone ( μm) in potassium phosphate buffer for 1 h at 37 C. Luciferase activity was measured as described above. Hydrogen peroxide (H ): A solution of probe 1 or ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with 1 mm hydrogen peroxide in water for 1 h at 37 C. Luciferase activity was measured as described above. Hydroxyl radical (. H): A solution of probe 1 or ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with 1 mm hydroxyl radical generated from a mixture of Fe 4 7H and H in water for 1 h at 37 C. Luciferase activity was measured as described above. 5

6 uperoxide ( -. ): A solution of probe 1 or ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with 1 mm superoxide generated from K in DM for 1 h at 37 C. Luciferase activity was measured as described above. inglet oxygen ( 1 ): A solution of probe 1 or ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with 1 mm singlet oxygen generated from acl and H for 1 h at 37 C. Luciferase activity was measured as described above. Hyperchlorite ion (Cl - ): A solution of probe 1 or ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM were treated with 1 mm hyperchlorite ion generated from acl and ah for 1 h at 37 C. Luciferase activity was measured as described above. Myeloperoxidase (MP): A solution of probe 1 or ( μm) in potassium phosphate buffer(5 mm, ph 7.) containing.5% DM were treated with 4 nm myeloperoxidase and M H in 15 mm acl in water for 1 h at 37 C. Luciferase activity was measured as described above. Bioluminescent detection of a mixture of probe 1, amino acid and 6-FP after UV light irradiation. A solution of probe 1 ( μm), 6-FP (1 μm) and mm of each amino acid (Cys, Met, Trp, His, Lys, Ala) was irradiated for 5 min at 3. mw/cm by using a UVA irradiation apparatus (XX-15BLB; UVP). The mixture was treated with mm pyrrolidine. After 5 min, luciferase (1 μg/ml) in assay buffer was added to the mixture. After incubation for 5 min, bioluminescence intensity was measured by using a bioluminescent microplate reader. zone (1 μm) was used as a positive control. For detection using indigo carmine, a solution of indigo carmine (6 μm), 6-FP (1 μm) and each amino acid (Cys, Met, Trp, His, Lys, Ala;.5- mm) was irradiated for 5 min at 3. mw/cm by using a UVA irradiation apparatus. Decomposition of indigo carmine was monitored by measuring its absorbance at 61 nm. Measurements of ozone concentrations in ambient samples. pen, wide mouth glass bottles containing potassium phosphate buffer (5 mm, ph 7.) were placed for 8 h at four locations with different atmospheric conditions. During this period, the sample bottles were protected from direct sunlight. A blank used as a negative control indicates the potassium phosphate buffer (5 mm, ph 7.) without exposing to ozone. The collected samples were treated with 1 M 1 for 3 min and then with mm pyrrolidine for 5 min. The luciferase activity of each sample was measured as described above. The experiments were carried out three times. 6

7 upplementary References 1. D. C. McCutcheon, M. A. Paley, R. C. teinhardt, J. A. Prescher. J. Am. Chem. oc., 1, 134, H. Takakura, R. Kojima, Y. Urano, T. Terai, K. Hanaoka, T. agano, Chem. Asian. J., 11, 6, H. Gallard, U. von Gunten, Water Res.,, 36, A. L. Garner, C. M. t Croix, B. R. Pitt, G. D. Leikauf,. Ando, K. Koide, at Chem., 9, 1,

8 A B D-luciferin Aminoluciferin + pyrrolidine + pyrrolidine a 6 min 6 min c -pyrrolidine - pyrrolidine b 6 min 6 min 1 min min Time(min) Time(min) H CH H H H b CH H CH CH H a H H c Fig. 1. HPLC profiles of products of reaction of probe (A) 1 and (B) with ozone. A solution of each probe (1 M) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with 15 M ozone in potassium phosphate buffer in the absence or presence of pyrrolidine ( mm) for 1 h at 37 C. 8

9 A HPLC profiles M data D-luciferin H CH a [M + 1] + H CH a H [M + 1] + a 6 min 1 min B Time(min) HPLC profiles M data Aminoluciferin [M + 1] + CH H H c c 6 min b [M + 1] + an aldehyde form (c) H H H [M + 1] + b CH min Time(min) Fig.. HPLC profiles and M data of products of reaction of probe (A) 1 and (B) with ozone. M data of b in (B) indicate that a hydrate form b is partially converted to its aldehyde form a during acquisition of mass data. 9

10 A 1 B 1 Luminescence Intensity ( x 1 5 ) [zone] (μm) Luminescence intensity ( x 1 5 ) [Pyrrolidine] (mm) Fig. 3. Effect of ozone and pyrrolidine on the luciferase activity. Luciferase (1 μg/ml) in assay buffer was added to a solution of D-luciferin ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing (A) 5 μm ozone and (B) 1 mm pyrrolidine. Bioluminescence intensity was measured by using a bioluminescent microplate reader (error bar: mean ± s.d., n = 3). 1

11 A 1 B 1 Luminescence Intensity (L.U. x 1 5 ) Luminescence Intensity (L.U. x 1 4 ) Fig. 4. Changes in bioluminescence intensity after treatment of probe (A) 1 and (B) with ozone. A solution of each probe ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with 1 μm ozone. The mixture was treated with mm pyrrolidine for 5 min and then luciferase (1 μg/ml) in assay buffer was added to the mixture. Bioluminescence intensity was measured by using a bioluminescence microplate reader (error bar: mean ± s.d., n = 3). 11

12 A Luminescence Intensity ( x 1 5 ) pyrrolidine - pyrrolidine Time (min) B Luminescence Intensity ( x 1 4 ) pyrrolidine - pyrrolidine Time (min) Fig. 5. Time-dependent bioluminescent detection of ozone using probe (A) 1 and (B). A solution of each probe ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with ozone (1 μm) in the absence or presence of mm pyrrolidine at 37 C. After incubation for the indicated time, luciferase (1 μg/ml) in assay buffer was added to the mixture and then time-dependent bioluminescence intensity was measured by using a bioluminescence microplate reader. 1

13 A 18 Fluorescence Intensity (a.u.) M 3 M 3 B Fluorescence Intensity (a.u.) Wavelength (nm) 4 1 M M Wavelength (nm) Fig. 6. Fluorescence spectra after treatment of probe (A) 1 and (B) with ozone. A solution of each probe (4 μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with various concentrations of ozone and then the mixture was treated with mm pyrrolidine for 5 min (1; ex = 33 nm, ; ex = 35 nm). 13

14 A Fluorescence Intensity (a.u.) pyrrolidine - pyrrolidine B Fluorescence Intensity (a.u.) pyrrolidine - pyrrolidine Time (min) Time (min) Fig. 7. Time-dependent changes in fluorescence intensity after treatment of probe (A) 1 and (B) with ozone. A solution of each probe (4 μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with μm ozone in the absence or presence of mm pyrrolidine. Time-dependent fluorescent intensity was measure by using a fluorescence microplate reader at 37 C (1; ex = 33 nm, em = 53 nm, ; ex = 35 nm, em = 5 nm). 14

15 A Luminescence Intensity (x1 5 ) B Luminescence Intensity (x1 4 ) zone concentration (nm) zone concentration (nm) Fig. 8. zone concentration dependent bioluminescence intensity of (A) 1 and (B). A solution of each probe ( μm) in potassium phosphate buffer (5 mm, ph 7.) containing.5% DM was treated with ozone (-4 nm) in potassium phosphate buffer at 5 C. The mixture was treated with mm pyrrolidine for 5 min and then luciferase (1 μg/ml) in assay buffer was added to the mixture. Bioluminescence intensity was measured by using a bioluminescence microplate reader (error bar: mean ± s.d., n = 3). 15

16 A Absorbance (A 61 ) mM 1mM mm B Luminescence Intensity (L.U x 1 5 ) C y s M e t T r p H i s L y s A l a Fig. 9. (A) A solution of each amino acid, 6-FP (1 M) and indigo carmine (6 M) was irradiated for 5 min by using a UV lamp. Loss of indigo carmine was monitored by measuring its absorbance at 61 nm (error bar: mean ± s.d., n = 3). (B) Changes in bioluminescence intensity. A solution of probe 1 ( M), 6-FP (1 M) and each amino acid was irradiated for 5 min by using a UV lamp. zone (1 M) was used as a positive control and no treatment () is a negative control. The mixture was treated with mm pyrrolidine for 5 min and then luciferase (1 μg/ml) in assay buffer was added to the mixture. Bioluminescence intensity was measured by using a bioluminescence microplate reader (error bar: mean ± s.d., n = 3). 16

17 4 Luminescence Intensity (x1 4 ) 3 1 Blank A B C D E Fig. 1. zone detection in environmental samples using probe 1. pen, wide mouth glass bottles containing 5 mm potassium phosphate buffer (ph 7.) were placed at four different locations for 8 h: A, indoor cience building of Yonsei University; B, the road nearby threeway intersection of the Yeonhui-ro; C, the main gate of Yonsei University; D, the back gate of the Ewha Women's University. Blank contains 5 mm potassium phosphate buffer (ph 7.) without ozone (error bar: mean ± s.d., n = 3). The ozone concentration of each sample was calculated by using a calibration curve shown in Figure 8A. 17

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