Supporting Information for

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1 Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 214 Supporting Information for A Fluorescent Probe for Hydrazine and Its in Vivo Applications Liangliang Xiao, a Jia Tu, a Shiguo Sun, *a Zhichao Pei, a Yuxin Pei, a Yi Pang b and Yongqian Xu,* a a College of Science, Northwest A&F University, Yangling, Shaanxi, P. R. China, 7121, xuyq@nwsuaf.edu.cn b Department of Chemistry & Maurice Morton Institute of Polymer Science, The University of Akron, Akron, H, Synthesis material and instruments All chemicals and reagents were used directly as obtained commercially unless otherwise stated. All solvents were of reagent grade and water used was ultra filter deionized. Probe 4 was synthesized according to our previous work. S1 Absorption and emission spectra were collected by using a Shimadzu 175 UV-visible spectrometer and a RF-531 fluorescence spectrometer (Japan), respectively. NMR spectra were collected on a Bruker 5 avance III spectrometer. Chemical shifts (δ) were reported as ppm with TMS as the internal standard. Mass spectrometric (MS) data were obtained with HP11LC/MSD MS and an LC/Q-TF-MS instruments. The quantum yield of the sample was measured using quinine sulfate as the standard (Φ =.53,.1 M H 2 S 4 ). S2 Sample Preparation and Titration Stock solutions of metal ions, anions, amino acids and amine complexes were prepared in deionized water. The concentration are fixed to M. Stock solution of fluorescent probes ( M) were prepared in CH 3 CN and then further diluted to M for titration experiments. Every time an appropriate volume of each analyte was added to the test solution. Excitation was provided at 39 nm. Cell culture and fluorescence image Hela cells were seeded on 35 mm glass-bottomed dishes (NEST) and incubated in RPMI-164 in an incubator (37 C, 5% C 2 and 2% 2 ) for 24 h. The cells were rinsed slightly 3 times with fresh RPMI-164 and incubated in RPMI-164 medium spiked with or without sensor (5 μm) for 3 min, respectively. After washing with fresh RPMI-164, the cells treated with sensor were further incubated in fresh RPMI-

2 164 containing of 5 μm hydrazine for.5 h. Cells were then analyzed by Laser Scanning Confocal Microscope (A1R). Synthesis routine H Et Et Et H CH 3 CH 2 I CH 3 CN Et HBr,HCH Br Et Br 1.KAc 2.NaH 3.PCC/DCM Et o-aminophenol/ MeH 2.DDQ/DCM Et N BBr 3 dry DCM H N Et H 3 1 H H N + CH 3 CH 2 I K 2 C 3 dry DMF N H 1 Et 2 1,4-Bis(ethyoxyl)benzene (5) A mixture of 1,4-hydroquinone (4.4 g, 4 mmol), iodoethane (1. ml, 12 mmol), and K 2 C 3 (22. g, 16 mmol) in acetonitrile (1 ml) was heated to reflux for two days. After cooling to room temperature, the precipitates were filtered, then evaporation of the solvent under reduced pressure. The solid residues were collected and purified on a silica gel column by using an eluent (petroleum ether), the product 5 was obtained as a white solid (4.9 g, 75.% yield). 2,5-Bis(bromomethyl)-1,4-bis(ethyoxyl)benzene (6) HBr (1 ml, 3 wt % in acetic acid) was added to a suspension of 5 (4. g, 23 mmol) and paraformaldehyde (1.4 g, 46 mmol) in acetic acid (8 ml). The mixture

3 was heated to 6 C, and then stirred for 3 h. As the reaction proceeded, the suspension changed to clear solution first and then became a thick suspension again. After cooling to room temperature, the suspension was poured into water (4 ml). The precipitates were filtered and washed with water (3 1 ml). Then dried under vacuum and the crude product 6 was obtained as an off-white solid (6.7 g, 78.% yield). 2,5-Bis(ethyoxyl)benzene-1,4-dialdehyde (7) A solution of 2 (2.5 g, 7.7 mmol), potassium acetate (2.5 g, 23.1 mmol), and tetra(n-butyl)ammonium bromide (.3 g) in a mixture of acetonitrile (5 ml) and chloroform (25 ml) was heated to reflux for 8 h. The mixture was washed with 1 ml water, and organic solution was removed on a rotary evaporator. The solid residues were dissolved in a 1:l mixture of THF and MeH, then a solution of NaH (1. g, 25 mmol) and H 2 (2 ml) was added dropwise to this solution. This mixture was heated to reflux for 8 h. After cooling to room temperature, the solvent was removed, the solid residues were washed with water to remove the salt, then dried under vacuum. The dried crude product was dissolved in 2 ml CH 2 Cl 2. The solution was added dropwise to a stirred solution of freshly prepared pyridium chlorochromate (6.3 g, 29.3 mmol) in CH 2 Cl 2 (2 ml) at room temperature. After addition was completed, stirring was continued for an additional 3 h. The reaction mixture was then directly transferred onto the top of a short silica gel column. The yellow and highly fluorescent compound was then washed off the column with CH 2 Cl 2 /petroleum ether (1:2), affording product 7 (1.1 g, 61.5% yield). 1 H NMR (5 MHz, CDCl 3 ) δ = 1.56 (d, J=1.2 Hz, 2H), 7.46 (s, 2H), 4.21 (q, J=7. Hz, 4H), 1.51 (t, J=7. Hz, 6H). 13 C NMR (125 MHz, CDCl 3 ) δ = , 155.8, , , 64.85, ,5-bis(ethyoxyl)-4-(benzoxazolyl) benzaldehyde (3) A solution of o-aminophenol (.24 g, 2.25 mmol) in methyl alcohol (2 ml) was added dropwise to a stirred solution of compound 7 in methyl alcohol (1 ml) at 7 C. After addition was completed, stirring was continued for an additional 5 h. Then the solvent was removed using rotary evaporator. After that the solution of DDQ (.5 g, 2.25 mmol) in 15 ml CH 2 Cl 2 was added dropwise to the above residue. The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was treated with 1 ml saturated Na 2 C 3, then washed with water, and dried over

4 Na 2 S 4. After evaporation of the solvent, the solid residues were collected and purified on a silica gel column by using an eluant (petroleum ether: CH 2 Cl 2 =1:1), the product 3 was obtained as a yellow solid (.15 g, 21.42% yield). 1 H NMR (5 MHz, CDCl 3 ) δ = 1.56 (d, J=1.2 Hz, 1H), (m, 2H), (m, 1H), 7.55 (s, 1H), (m, 2H), (m, 4H), 1.55 (t, J=7. Hz, 6H). 13 C NMR (125 MHz, CDCl 3 ) δ = , 161.3, , , 15.86, , 127.8, , 124.7, , 12.37, , , 11.78, 65.69, 65.7, 14.77, Synthesis of probe 1 A solution of compound 3 (.2 g, 1.6 mmol) in 1 ml of dry CH 2 Cl 2 was cooled to -3 C with ice/ethyl alcohol. BBr 3 (.3 ml) in 1 ml CH 2 Cl 2 was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred at -3 C for 1 h and then at room temperature for 24 h. Following the addition of 4. ml distilled H 2, the reaction mixture was stirred for additional 2 h and poured into a mixture of water (2 ml) and CH 2 Cl 2 (2 ml). The organic layer was separated, and the aqueous layer was extracted twice with CH 2 Cl 2 (3 ml). The combined organic layer was washed with brine and dried over anhydrous MgS 4. After removal of the solvent on a rotary evaporator, the solid residues were collected and purified on a silica gel column by using an eluent (petroleum ether/ch 2 Cl 2, 1:1), the product probe 1 was obtained as a yellow solid (.1 g, 62.5% yield). 1 H NMR (5 MHz, DMS-d 6 ) δ = 1.59 (s, 1H), 1.47 (s, 1H), 1.31 (s, 1H), 7.87 (d, J = 7.7 Hz, 2H), 7.63 (s, 1H), (m, 2H), 7.26 (s, 1H). 13 C NMR (125 MHz, DMS-d 6 ) δ = 19.41, , , 15.27, , 14.1, , , , 12.14, , , , m/z (TF-LD): Calcd. [M+H] + For C 14 H 9 N 4 : , found: Synthesis of probe 2 A solution of probe 1 (.2 g) in anhydrous DMF (5 ml) was treated with K 2 C 3 (.12 g) at 25 C, and the mixture was stirred for 3 min. Ethyl iodide (.1 g) was added, and the reaction mixture was further stirred for 2 h before being quenched by the addition of H 2 (1 ml). The aqueous layer was extracted with CH 2 Cl 2 (2 1 ml). The combined organic layer was dried over Na 2 S 4 and concentrated under vacuum. The product was isolated by flash chromatography on silica gel by using an eluent (petroleum ether/ch 2 Cl 2, 1:1), the product probe 2 was obtained as a yellow solid (.15 g, 65.% yields). 1 H NMR (5 MHz, CDCl 3 ) δ ppm 1.48 (s, 1H), 7.71

5 (dd, J=7.5, 1.5 Hz, 1H), 7.61 (t, J=7.3 Hz, 1H), (m, 2H), (m, 2H), (m, 2H), (m, 3H). 13 C NMR (125 MHz, CDCl 3 ) δ ppm , , , , , 139.9, , , , , 116.3, , 11.79, 11.35, 65., m/z (TF-LD): Calcd. [M+H]+ For C 16 H 13 N 4 : , found: FL Intensity (a.u.) CH 3 H THF Wavelength (nm) Fig. S1 The fluorescence spectra of probe 2 (2 μm) in CH 3 H and THF, λ ex =39 nm. The fluorescent bands at 47 and 58 nm are attributed to enol and keto emission, which is consistent with ESIPT characteristic a) 35 3 b) Probe Probe -N 2 eq N 2 Absorption eq N 2 1 eq N 2 FL Intensity (a.u.) eq N Wavelength (nm) Wavelength (nm) Fig. S2 UV-Vis absorption (a) and fluorescence spectra (b) of probe 1 (2 μm) upon addition of N 2 in CH 3 CN (λ ex =39 nm). Inset: The fluorescence color change before and after addition of hydrazine excited by hand-held UV lamp. Each spectrum was recorded after 5 min of reaction.

6 F 56 /F NH 2 H NH 2 NH Time (min) Fig. S3 Time-dependent fluorescence intensity of probe 1 (2. μm) upon addition of NH 2 NH 2 (.2 mm) and NH 2 H (.2 mm) in CH 3 CN/HEPES Buffer (1:2, v/v, ph 7.4), λ ex =39 nm F Fig. S4 The fluorescence intensity change of probe 1 (2 µm) at 56 nm response to the different value of ph. ph

7 ppm 1. eq N 2.8 eq N 2.6 eq N 2.4 eq N 2.2 eq N ppm eq N 2 Fig. S5 The 1 H NMR spectra titration of probe 1 in DMS-d 6 /D 2 (1:1) upon addition of N 2. Inset: the enlarged spectra in the region of x1 6 +ESI Scan (.187 min) Frag=15.V SUNWEI-6.d Subtract 1.2 * ([C14 H11 N3 3]+H) Counts vs. Mass-to-Charge (m/z) Fig. S6 HRMS spectrum of the reaction product of probe 1 with hydrazine.

8 Fig. S7 HM LUM energy levels and interfacial plots of the orbitals for probe 1 and the corresponding product. Fig. S8 The optimizational molecular structures for probe 1 (a) and the corresponding product (b).

9 Table S1 The calculated data for probe 1 TDDFT //B3LYP/6-31G* Compoud Electronic Transition Excitation Energy f S S ev nm. S S ev nm.3446 S S ev nm.3958 Probe 1 S S ev 34.9 nm.311 S S ev nm.858 S S ev nm.1

10 Table S2 The calculated data for the corresponding product TDDFT //B3LYP/6-31G* Compoud Electronic Transition Excitation Energy f S S ev nm.785 S S ev nm.5142 S S ev nm.185 product S S ev nm.44 S S ev nm.27 S S ev nm.184

11 36 3 a) F 56 /F probe 1 probe Time (min) F 56 /F b) probe 1 probe 2 C) Time (min) = NH 2NH 2 = probe = SDS micelles Fig. S9 Time-dependent fluorescence intensity of probe (2. μm) was treated with hydrazine (.2 mm) (a) in CH 3 CN/HEPES Buffer (1:2, v/v, ph 7.4) and (b) in SDS solution (1 mm), λ ex =39 nm. c) Schematic diagram of micelle-induced enrichment of hydrazine on micelle surface, conductive to reaction with probe.

12 FL Intensity (a.u.) a) 1 eq N FL Intensity (a.u.) b) 2 eq N Wavelength (nm) Wavelength (nm) 25 c) FL Intensity (a.u.) eq N Wavelength (nm) Fig. S1 Fluorescence emission spectra of (a) probe 2 (2 μm), (b) probe 3 (2 μm) and (c) probe 4 (2 μμ) upon addition of N 2 in CH 3 CN/HEPES Buffer (1:2, v/v, ph 7.4), λ ex =39 nm. Each spectrum was recorded after 15 min of reaction. FL Intensity (a.u.) a) 5 min FL Intensity (a.u.) b) 5 min Wavelength (nm) Wavelength (nm) Fig. S11 The fluorescence spectra response of probe 1 (2μM) (a) and probe 2 (2μM) (b) with time duration in the presence of 1 μm hydrazine in cationic CTAB (5 mm) micelles solution, λ ex =39 nm. Probe 1 and 2 showed no obvious fluorescence spectra change with increasing time upon to 1 equivalents of hydrazine in cationic cetyltrimethyl ammonium bromide (CTAB) micelles (5 mm) solution. These phenomena are unlike that in anionic SDS micelles solution (Fig. S8b). It is presumed that cationic CTAB micelles-encapsulated probe cannot access to positively charged hydrazine (pk a =14.9) because of electrostatic repulsion.

13 Fig. S12 Fluorescent color changes of probe 1 (2 μm)-coated filter paper after exposure to an excess quantity of various vapors, including hydrazine, ammonia, methylamine, n-butylamine, formaldehyde, dimethylamine, H 2 2, HCl, and C 2 for 1 min, respectively. The fluorescent color changes were observed using a hand-held UV lamp with an excitation at 365 nm.

14 The 1 H NMR spectrum of compound 7 PRTN CDCl3 {D:\214-1} ZHL ppm Current Data Parameters NAME XYQ-17-XL-SQJ EXPN 1 PRCN 1 F2 - Acquisition Parameters Date_ Time INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zg3 TD SLVENT CDCl3 NS 8 DS 2 SWH Hz FIDRES Hz AQ sec RG 128 DW 48.4 usec DE 6.5 usec TE K D1 1. sec TD 1 ======== CHANNEL f1 ======== NUC1 1H P1 9. usec PL1 db PL1W -1.#IND W SF MHz F2 - Processing parameters SI SF 5.13 MHz WDW EM SSB LB.3 Hz GB PC The 13 C NMR spectrum of compound 7 C13CPD CDCl3 {D:\214-1} ZHL F2 - Processing parameters SI SF MHz WDW EM SSB ppm LB 1. Hz GB PC Current Data Parameters NAME XYQ-17-XL-SQJ EXPN 11 PRCN 1 F2 - Acquisition Parameters Date_ Time 16.5 INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zgpg3 TD SLVENT CDCl3 NS 47 DS 4 SWH Hz FIDRES Hz AQ sec RG 23 DW 16.8 usec DE 6.5 usec TE 299. K D1.62 sec D11.3 sec TD 1 ======== CHANNEL f1 ======== NUC1 13C P1 9.5 usec PL1-1. db PL1W -1.#IND W SF MHz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 8. usec PL2 db PL db PL db PL2W -1.#IND W PL12W -1.#IND W PL13W -1.#IND W SF MHz

15 The 1 H NMR spectrum of probe 3 PRTN CDCl3 {D:\214-1} ZHL ppm Current Data Parameters NAME XYQ-17-XL-DQDH EXPN 1 PRCN 1 F2 - Acquisition Parameters Date_ Time INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zg3 TD SLVENT CDCl3 NS 8 DS 2 SWH Hz FIDRES Hz AQ sec RG 128 DW 48.4 usec DE 6.5 usec TE K D1 1. sec TD 1 ======== CHANNEL f1 ======== NUC1 1H P1 9. usec PL1 db PL1W -1.#IND W SF MHz F2 - Processing parameters SI SF 5.13 MHz WDW EM SSB LB.3 Hz GB PC The 13 C NMR spectrum of probe 3 C13CPD CDCl3 {D:\214-1} ZHL ppm Current Data Parameters NAME XYQ-17-XL-DQDH EXPN 11 PRCN 1 F2 - Acquisition Parameters Date_ Time INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zgpg3 TD SLVENT CDCl3 NS 111 DS 4 SWH Hz FIDRES Hz AQ sec RG 23 DW 16.8 usec DE 6.5 usec TE K D1.62 sec D11.3 sec TD 1 ======== CHANNEL f1 ======== NUC1 13C P1 9.5 usec PL1-1. db PL1W -1.#IND W SF MHz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 8. usec PL2 db PL db PL db PL2W -1.#IND W PL12W -1.#IND W PL13W -1.#IND W SF MHz F2 - Processing parameters SI SF MHz WDW EM SSB LB 1. Hz GB PC 1.4

16 The 1 H NMR spectrum of probe 1 PRTN DMS {D:\214-1} ZHL Current Data Parameters NAME SSG-117-XL-NH EXPN 1 PRCN 1 F2 - Acquisition Parameters Date_ Time 1.8 INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zg3 TD SLVENT DMS NS 9 DS 2 SWH Hz FIDRES Hz AQ sec RG 114 DW 48.4 usec DE 6.5 usec TE K D1 1. sec TD 1 ======== CHANNEL f1 ======== NUC1 1H P1 9. usec PL1 db PL1W -1.#IND W SF MHz F2 - Processing parameters SI SF 5.13 MHz WDW EM ppm SSB LB.3 Hz GB PC 1. The 13 C NMR spectrum of probe 1 C13CPD DMS {D:\214-1} ZHL Current Data Parameters NAME SSG-117-XL-NH EXPN 11 PRCN 1 F2 - Acquisition Parameters Date_ Time 1.12 INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zgpg3 TD SLVENT DMS NS 64 DS 4 SWH Hz FIDRES Hz AQ sec RG 23 DW 16.8 usec DE 6.5 usec TE K D1 2. sec D11.3 sec TD 1 ======== CHANNEL f1 ======== NUC1 13C P1 9.5 usec PL1-1. db PL1W -1.#IND W SF MHz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 8. usec PL2 db PL db PL db PL2W -1.#IND W PL12W -1.#IND W PL13W -1.#IND W SF MHz F2 - Processing parameters SI SF MHz WDW EM SSB LB 1. Hz ppm GB PC 1.4

17 The HRMS spectrum of probe 1 x1 5 +ESI Scan (.195 min) Frag=15.V SUNWEI-5.d Subtract ([C14 H9 N 4]+H) Counts vs. Mass-to-Charge (m/z) The 1 H NMR spectrum of probe Current Data Parameters NAME SSG XL-SEt EXPN 1 PRCN 1 F2 - Acquisition Parameters Date_ Time INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zg3 TD SLVENT CDCl3 NS 16 DS 2 SWH Hz FIDRES Hz AQ sec RG 9.5 DW 48.4 usec DE 6.5 usec TE K D1 1. sec TD 1 ======== CHANNEL f1 ======== NUC1 1H P1 9. usec PL1 db PL1W -1.#IND W SF MHz F2 - Processing parameters SI SF 5.13 MHz WDW EM SSB ppm LB GB.3 Hz PC 1.

18 The 13 C NMR spectrum of probe ppm Current Data Parameters NAME SSG XL-SEt EXPN 11 PRCN 1 F2 - Acquisition Parameters Date_ Time INSTRUM spect PRBHD 5 mm PABB BB- PULPRG zgpg3 TD SLVENT CDCl3 NS 222 DS 2 SWH Hz FIDRES Hz AQ sec RG 23 DW 16.8 usec DE 6.5 usec TE K D1 2. sec D11.3 sec TD 1 ======== CHANNEL f1 ======== NUC1 13C P1 9.5 usec PL1-1. db PL1W -1.#IND W SF MHz ======== CHANNEL f2 ======== CPDPRG2 waltz16 NUC2 1H PCPD2 8. usec PL2 db PL db PL db PL2W -1.#IND W PL12W -1.#IND W PL13W -1.#IND W SF MHz F2 - Processing parameters SI SF MHz WDW EM SSB LB 1. Hz GB PC 1.4 The HRMS spectrum of probe 2 XXL #4-5 RT:.7-.1 AV: 2 SB: NL: 9.5E5 T: FTMS + p ESI Full ms [12.-6.] Relative Abundance m/z

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