Selective detection of endogenous H2S in living

Electronic Supplementary Information for 

Selective detection of endogenous H2S in living cells and mouse hippocampus using a ratiometric fluorescent probe Ling Zhang1,2‡, Wen-qi Meng2‡, Liang Lu1, Yun-Sheng Xue2, Cheng Li2, Fang Zou2, Yi Liu2* & Jing Zhao1,3* 1

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Institute of Chemistry and

BioMedical Sciences, Nanjing University, Nanjing, 210093, China. Fax:+8602584687371. E-mail: [email protected],[email protected]. 2

School of Pharmacy, Xuzhou Medical College, Xuzhou, 221002, China. E-mail: [email protected].

3

Guangdong Key Lab of Nano-Micro Material Research, School of Chemical Biology and Biotechnology,

Shenzhen Graduate School of Peking University, Shenzhen, 518055, China.

Table of contents General information Synthesis and Characterisation of compounds Evidence of mechanism detection Quantum yields Preparation of the test solution Determination of the detection limit MTT Assay Determination of sulphide in mouse hippocampus with SFP-2 probe Sucrose preference test Tail suspension test Forced swimming test Theoretical and Computational Methods References Figure S1. Fluorescence spectra spectra of compound NAH-NH2 Figure S2. The detection limits of RHP-2 Figure S3. Time profile of RHP-2 toward sulphide Figure S4.1st-order kinetics kobs and 2nd-order K2 of RHP-2 Figure S5. Effects of pH on RHP-2 in PBS buffer Figure S6. Photosatbility of RHP-2 Figure S7. Selectivity of RHP-2 to various thiols Figure S8. Selectivity of RHP-2 to various amino acids Figure S9. Cell viability of MCF-7 cells in the presense of RHP-2 Figure S10. Cell viability of MCF-7 cells in the presense of NAP-NH2 Figure S11. The corresponding bright images of cells Figure S12. Confocal fluorescence imaging of exogenous sulphide in living MCF-7 cells using RHP-2 Figure S13. The average fluorescence emission intensity ratios (R) of cells Figure S14. Confocal fluorescence images of H2S in living MCF-7 cells using RHP-2 Figure S15. Determination of sulphide concentrations in mouse hippocampus with RHP-2 probe Figure S16. Determination of sulphideconcentrations in mouse hippocampus with SFP-2 probe Table S1 Measurement of sulphide concentrations in mouse hippocampus Figure S17. Sucrose consumption of mice Figure S18. Immobility time of mice in the tail suspension test Figure S19. Immobility time of mice in the forced swimming test Figure S20. Full-length blots of Fig. 6B in main text . CBS protein levels in mouse hippocampus. Figure S21. Full-length blots of Fig. 6C in main text . CBS mRNA levels in mouse hippocampus. Figure S22. Optimized structures of ground state for RHP-2 and NAP-NH2 Figure S23. Plots of the HOMO and LUMO of the ground state for RHP-2 and NAP-NH2 Figure S24. Electron density difference computed for the ground and the first excited states of RHP-2 and NAP-NH2 Table S2. Calculated electronic transitions energies for RHP-2 and NAP-NH2 NMR and HRMS spectrum of compounds      

General Information Thin layer chromatography was performed on silica gel 60 F254 plates (250 μm) and column chromatography was conducted over silica gel (300-400 mesh). Visualisation of the developed chromatogram was accomplished by a UV lamp. Nuclear magnetic resonance (NMR) spectra were acquired on Bruker DRX-500/400 operated at 125/100 MHz for 1H NMR and 13C NMR, respectively, residual protio solvent signals serving as internal criteria for calibration purposes. Data for 1H NMR are reported as follows: chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), integration, coupling constant (Hz). High-Resolution Mass was performed by Mass Spectrometry. All fluorescence measurements were recorded on a Hitachi F4600 Fluorescence Spectrophotometer. The pH measurements were performed on a Mettler-Toledo Delta 320 pH meter. All fluorescence imaging experiments were conducted on a FV1000 confocal laser scanning microscope (Olympus, Japan). Chemicals and media Unless noted otherwise, reagents and solvents were obtained from commercial suppliers and employed without further purification: Na2S·9H2O (≥ 99.99%), NaHS, TRI reagent and High Capacity RNA-to-cDNA kit (Sigma-Aldrich, St. Louis, MO, USA); DMEM media, fetal bovine serum (FBS), penicillin (100 μg/mL) and streptomycin (100 μg/mL) (Life Technologies, CA, USA); Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA); MCF-7 cells (the Committee on type Culture Collection of Chinese Academy of Sciences); CBS (Santa Cruz Biotechnology, Santa Cruz, CA, USA); β-actin (ZSGB-BIO, Nanjing, China); alkaline phosphatase-conjugated antibodies (1:1000, ZSGB-BIO, Jiangsu, China); BCIP/NBT alkaline phosphatase colour development Kits (Beyotime Institute of Biotechnology, Jiangsu, China). Synthesis and Characterisation of compounds

O

O

O

O

N

O

O

N

CH3CH2CH2CH2NH2

NaN3

H2S

AcOH, reflux, 6 h

DMF, 40 oC, 4 h

30 oC, 3 h

Br

N3

Br 2

1

O

HO O

N

O

N

O

O NO2

trisphosgene/DMAP reflux, 6 h

rt, overnight O2N

HN

NH2 NAP-NH2

O O

RHP-2

 

Synthesis of 1 (4-bromo-N-butyl-1,8-naphthalimide). Compound 1 was synthesized according to the method reported by Zhu et al.1 A solution of 4-bromo-1,8-naphthalic anhydride (1.50 g, 5.4 mmol) and n-butylamine (3.55 g, 48.6 mmol) in acetic acid (30 mL) was heated to reflux under N2 for 6 h. The solution was poured into a beaker containing distilled water, forming a precipitate, which was filtered and rinsed with water. The crude product (yellow powder) was collected and recrystallized with ethanol to afford compound 1 (white crystals). Yield: 1.4 g, 78.2%. m.p. 101-102°C . 1H NMR (500 MHz, CDCl3): δ 8.61 (d, J = 6.5 Hz, 1 H, ArH), 8.50 (d, J

= 8.5 Hz, 1 H, ArH), 8.36 (d, J = 8.0 Hz, 1 H, ArH), 7.99 (d, J = 7.5 Hz, 1 H, ArH), 7.80 (t, J = 7.0 Hz, J = 8.5 Hz, 1 H, ArH), 4.16 (t, J = 7.5 Hz, 2 H, NCH2-), 1.69-1.74 (m, 2 H, NCH2CH2-), 1.41-1.48 (m, 2 H, -CH2CH3), 0.98 (t, J = 7.5 Hz, J = 7.0 Hz, 3 H, -CH3). 13C NMR (125 MHz, CDCl3): δ 163.5, 133.1, 131.9, 131.1, 131.0, 130.5, 130.1, 128.9, 127.9, 123.2, 122.3, 40.4, 30.2, 20.4, 13.8; HRMS (m/z): [M+H]+ calcd. for C16H15BrNO2, 332.0286; found, 332.0239. Synthesis of 2 (4-azido-N-butyl-1,8-naphthalimide). Compound 2 was synthesized according to the previous method.2 A solution of 1 (0.5 g, 1.5 mmol) and sodium azide (0.97 g, 15.0 mmol) in DMF (10 mL) were stirred for 4 h at 40°C. The solution was poured into a beaker containing water, forming a precipitate, which was filtered and rinsed with water. The crude product, a yellow powder, was collected. Yield: 0.3 g, 68.2%. m.p. 126.2-127.6°C. TLC (silica, hexane: EtOAc, 10:1 v/v): Rf = 0.5; 1H NMR (500 MHz, CDCl3): δ 8.64 (d, J = 7.5 Hz, 1 H, ArH), 8.58 (d, J = 8.0 Hz, 1 H, ArH), 8.44 (d, J = 8.5 Hz, 1 H, ArH), 7.74 (t, J = 8.5 Hz, J = 7.5 Hz, 1 H, ArH), 7.47 (d, J = 7.5 Hz, 1 H, ArH), 4.18 (t, J = 7.0 Hz, J = 8.0 Hz, 2 H, NCH2-), 1.69-1.75 (m, 2 H, NCH2CH2-), 1.42-1.49 (m, 2 H, -CH2CH3), 0.99 (t, J = 7.5 Hz, J = 7.0 Hz, 3 H, -CH3); 13C NMR (125 MHz, CDCl3): δ 163.9, 163.5, 143.3, 132.1, 131.6, 129.2, 128.6, 126.8, 124.4, 122.8, 119.1, 114.6, 40.3, 30.2, 20.4, 13.8; HRMS (m/z): [M+H]+ calcd. for C16H15N4O2, 295.1195; found, 295.1193. Synthesis of NAP-NH2 (4-amino-N-butyl-1,8-naphthalimide). Compound 2 (0.15g, 0.51 mmol) was dissolved in acetonitrile (25 mL). With the bubbling of H2S, the mixture was stirred for 3 h at 30 °C. The mixture for reaction was dried by evaporation under reduced pressure and an orange powder was obtained. Yield: 0.9 g, 66.2%. m.p. 174.8-176.2°C. TLC (silica, hexane:EtOAc, 2:1 v/v): Rf = 0.2; 1H NMR (400 MHz, DMSO-d6): δ 8.59 (d, J = 8.4 Hz, 1 H, ArH), 8.40 (d, J = 7.2 Hz, 1 H, ArH), 8.17 (d, J = 8.4 Hz, 1 H, ArH), 7.62 (t, J = 8.0 Hz, J = 7.6 Hz,1 H, ArH), 7.40 (s, 2 H, ArH), 6.83 (d, J = 8.4 Hz, 1 H, ArH), 3.99 (t, J = 7.2 Hz, 2 H, NCH2-), 1.52-1.60 (m, 2 H, NCH2CH2-), 1.28-1.34 (m, 2 H, -CH2CH3), 0.90 (t, J = 7.6 Hz, J = 7.2 Hz, 3 H, -CH3); 13C NMR (100 MHz, DMSO-d6): δ 164.1, 163.2, 152.9, 134.2, 131.3, 129.9, 129.6, 124.3, 122.1, 119.7, 108.5, 107.9, 39.3, 30.2, 20.2, 14.1; HRMS (m/z): [M+H]+ calcd. for C16H17N2O2, 269.1290; found, 269.1282. Evidence of mechanism detection RHP-2 (48 mg, 0.11 mmol) was dissolved in CH3CN (15 mL), followed by the addition of the solution of Na2S• 9H2O (80 mg, 0.33 mmol) in PBS buffer (16.5 mL, 20 mM, pH = 7.4). The resultant mixture was stirred for 3 h at room temperature. Subsequently, EtOAc (3 x 10 mL) was added into the solution for extraction. The fluorescent product was thereafter purified by column chromatography, and the spectra obtained by 1H NMR and 13C NMR were consistent with those of compound NAP-NH2, hence the confirmation of the fluorescent product as compound NAP-NH2. Quantum Yields Quantum yields were determined using fluorescein as a standard according to a published method.3 For RHP-2 and fluorescein, the absorbance spectra were measured within an absorbance range of 0.01 to 0.1. The quantum yield was calculated according to the equation: Φsample = Φstandard (Gradsample/Gradstandard)(η2sample/η2standard); where Φ is the quantum yield, Φfluorescein = 0.79 in 0.1 M NaOH, Grad is the slope of the plot of absorbance versus integrated emission intensity, and η is the refractive index of the solvent. Preparation of the test solution RHP-2 stock solution preparation: RHP-2 (48 mg, 0.11 mmol) was dissolved into CH3CN (5 mL) to get 2.0 mM stock solution. Cys (L-Cysteine) stock solution preparation: Cys (24.2 mg, 0.2 mmol) was dissolved into DI H2O (10 mL) to

get 20.0 mM stock solution, which was then diluted to 1.0 mM and 100 µM solution for general use. Hcy (Homocysteine) stock solution preparation: Hcy (27.0 mg, 0.2 mmol) was dissolved into DI H2O (10 mL) to get 20.0 mM stock solution, which was then diluted to 1.0 mM and 100 µM solution for general use. GSH (Glutathione) stock solution preparation: GSH (61.5 mg, 0.2 mmol) was dissolved into DI H2O (10 mL) to get 20.0 mM stock solution, which was then diluted to 1.0 mM and 100 µM solution for general use. Na2S stock solution preparation:4 5 mg EDTA was dissolved in 10 mL DI H2O in a 25 mL Schlenk tube. The solution was purged vigorously with nitrogen for 15 min. Then 48 mg sodium sulfide (Na2S·9H2O) was dissolved in the solution under nitrogen. The resulting solution was 20 mM Na2S, which was then diluted to 1.0 mM-100 µM stock solution for general use. Stock solutions of other biological analytes, including Ala, Glu, Trp, Met, Tyr, Leu, Val, Ser, Pro, Arg, Gly, Phe, His, Gln, Asn, Ile, Thr, KCl, CaCl2, NaCl, MgCl2, ZnSO4, FeCl3, NaH2PO4, H2O2, ·OCl-, O2-, ·OH, tBuOOH, NO2-, NO, Na2S2O3, Na2S2O5, Na2SO4, Na2S2O4, Na2SO3, KSCN, NADH, and Glucose, were prepared in DI H2O. Superoxide radicals (O2-) were generated according to the previous reported method.5 ·OH was generated by Fenton reaction between FeII(EDTA) and H2O2 quantitively.6 NO is generated in form of 3-(Aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5, 50 μmol/ml). Absorption analyses Absorption spectra were recorded at room temperature on a Shimadzu PharmaSpec UV-2401PC UV-Visible spectrophotometer. The RHP-2 probe (CH3CN) was added to a quartz cuvette. With the probe diluted to 10 µM with 20 mM PBS buffer, Na2S was added. The resulting solution was incubated for 40 min prior to measurements (n = 3), with the mean ± SD expressed.

  Fig. S1 Fluorescence spectra of compound NAP-NH2 in PBS buffer (20 mM, pH = 7.4, 5 % CH3CN). Excitation: 415 nm, emission: 430 nm to 650 nm.

Determination of the detection limit The detection limit was calculated based on the method reported in the previous literature.7 The fluorescence emission spectrum of RHP-2 without Na2S was measured by 10 times and the standard deviation of blank measurement was obtained. Then the solution was treated with Na2S of concentration from 0 to 100 μM. A linear regression curve was then achieved according to the intensity ratio F532/F467 in the range of Na2S from 0 to 4 μM. The detection limit was calculated with the following equation: Detection limit = 3σ/k. Where σ is the standard deviation of blank

measurements, k is the slope between the fluorescence intensity ratios versus Na2S concentrations. The detection limit was 270 nM and 280 nM in PBS buffer and bovine serum, respectively.

Fig. S2 The emission intensity ratios (F532/F467) of RHP-2 (5 μM) to various concentrations of Na2S (0-4 μM) in PBS buffer (20 mM, pH 7.4, 5% CH3CN) and bovine serum (5% CH3CN) at 37 °C for 40 min. Data are presented as the mean ± SD (n = 3).

Fig. S3 (A) Fluorescence spectra of RHP-2 (5 μM) with Na2S (100 μM) in PBS buffer (20 mM, pH 7.4, 5% CH3CN) at 37 °C for 0, 5, 10, 15, 20, 25, 30, 40 and 60 min. (B) Time profile of RHP-2 (5 μM) toward Na2S (100 μM) in PBS buffer (20 mM, pH 7.4, 5% CH3CN) at 37 oC for 0, 5, 10, 15, 20, 25, 30, 40, 60 and 90 min. Data are presented as the mean ± SD (n = 3).

 

 

Fig. S4 (A) Pseudo first-order kinetic plots of the reaction of RHP-2 (5 μM) with Na2S (20 equiv.) in PBS buffer (20 mM, pH 7.4, 5% CH3CN). kobs = 1.0 × 10-3 s-1. (B) Plots of kobs vs Na2S concentration. Second-order rate constant, k2 = 5.0 M-1 s-1.

Fig. S5 (A) Fluorescence spectra of RHP-2 (5 μM) with Na2S (100 μM) in different pH buffer (20 mM, pH 4.2, 4.6, 5.0, 5.4, 5.8, 6.2, 6.6, 7.0, 7.4, 7.8, 8.2, 8.6, and 9.0, 5% CH3CN) at 37 oC for 40 min. (B) The emission intensity ratios (F532/F467) of RHP-2 (5 μM) to Na2S (100 μM) in different pH buffer at 37 oC for 40 min. Data are presented as the mean ± SD (n = 3).

Fig. S6 Photosatbility of RHP-2 under visible light and UV light. The last column was the same solution with the addition of 100 μM Na2S. Data are presented as the mean ± SD (n = 3).

Fig. S7 Fluorescence spectra of RHP-2 (5 μM) with Na2S and various thiols in 20 mM PBS (pH 7.4, 5% CH3CN) after 40 min of incubation. 1.Na2S (0 μM); 2.Na2S (10 μM); 3.Na2S (100 μM); 4.Cys (100 μM); 5.Cys (1 mM); 6.Hcy (100 μM); 7.Hcy (1 mM); 8.GSH (1 mM); 9.GSH (10 mM); 10.Na2S (10 μM) + Cys (100 μM); 11.Na2S (100 μM) + Cys (100 μM); 12.Na2S (10 μM) + Cys (1 mM); 13.Na2S (100 μM) + Cys (1 mM); 14.Na2S (10 μM) + Hcy (100 μM); 15.Na2S (100 μM) + Hcy (100 μM); 16.Na2S (10 μM) + Hcy (1 mM); 17.Na2S (100 μM) + Hcy (1 mM); 18.Na2S (10 μM) + GSH (1 mM); 19.Na2S (100 μM) + GSH (1 mM); 20.Na2S (10 μM) + GSH (10 mM); and 21.Na2S (100 μM) + GSH (10 mM).

Fig. S8 Fluorescence responses of RHP-2 (5 μM) towards Na2S (100 μM) and other amino acids (1 mM). (1) Blank, (2) Na2S, (3) Ala, (4) Glu, (5) Trp, (6) Met, (7) Tyr, (8) Leu, (9) Val, (10) Ser, (11) Pro, (12) Arg, (13) Gly, (14) Phe, (15) His, (16) Gln, (17) Asn, (18) Ile, (19) Thr. Data are presented as the mean ± SD (n = 3).

MTT assay Cell growth inhibitory effects of RHP-2 and compound NAP-NH2 were measured using a colorimetric MTT assay kit (Sigma-Aldrich). MCF-7 cells were seeded in 96-well plates at a density of 50,000 cells/well and then maintained at 37 °C in a 5 % CO2 incubator. The cells were incubated with different concentrations of RHP-2 and NAP-NH2 for 24 h, respectively. Cells in culture medium without RHP-2 or NAP-NH2 were used as control. After the incubation time, 20 μL of MTT dye (3-[4, 5-dimethylthiazol-2-yl]- 2, 5-diphenyl tetrazolium bromide, 5 mg/ml in phosphate buffered saline), was added to each well, and the plates were incubated for 4 h at 37 ºC. Then, the remaining MTT solution was removed, and 150 μL of DMSO was added to each well to dissolve the formazan crystals. The plate was shaken for 10 min and the absorbance was measured at 570 nm on a microplate reader (ELX808IU, Bio-tek Instruments Inc, USA). Each sample was performed in triplicate, and the entire experiment was repeated three times. Calculation of IC50 values was done according to Huber and Koella. IC50 of RHP-2 and compound NAP-NH2 was calculated to be of 101.2 ± 1.3 μM and 82.6 ± 1.1 μM, respectively. The cell viability of RHP-2 and compound NAP-NH2 (5 μM) at 0, 6, 12, 18 and 24 h further demonstrated that the RHP-2 and NAP-NH2 were of low toxicity to cultured MCF-7 cells.

Fig. S9 (A) The inhibitory effect of RHP-2 on cell growth in MCF-7 cells treated for 24 h. (B) Cell viability of RHP-2 (5 μM) at different times in MCF-7 cell. Data are presented as the mean ± SD (n = 3).

Fig. S10 (A) The inhibitory effect of compound NAP-NH2 on cell growth in MCF-7 cells treated for 24 h. (B) Cell viability of compound NAP-NH2 (5 μM) at different times in MCF-7 cell. Data are presented as the mean ± SD (n = 3).

Fig. S11 The corresponding bright images of Fig. 4, panels 3A, 3B and 3C.

Fig. S12 Confocal fluorescence imaging of exogenous sulphide in living MCF-7 cells using RHP-2. Cells were incubated with RHP-2 (5 μM) for 30 min (5A, 5B, 5C, and 5D). Cells in panels 5A, 5B, 5C, and 5D were then treated with Na2S (50 μM) for 40 min (6A, 6B, 6C, and 6D). Cells were pretreated with 1 mM ZnCl2 for 30 min and then incubated with RHP-2 (5 μM) for 30 min (7A, 7B, 7C, and 7D). Cells in panels 7A, 7B, 7C, and 7D were thereafter treated with Na2S (300 μM) for 30 min (8A, 8B, 8C, and 8D). Blue channel images were obtained from 445 nm to 495 nm (5A, 6A, 7A and 8A). Green channel images were obtained from 520 nm to 580 nm (5B, 6B, 7B and 8B). Ratiometric images (F520–580 nm/F445–495 nm) were generated by Olympus software (5C, 6C, 7C and 8C) and the corresponding bright images were shown in 5D, 6D, 7D and 8D. Scale bars=10 μm.

Fig. S13 The average fluorescence emission intensity ratios (R=F520–580 nm/F445–495 nm) generated by Olympus software according to the corresponding ratiometric images (i.e., Fig. S12, panels 5C, 6C, 7C and 8C) of cells. The R values were obtained from triplicate experiments (n = 3).

 

Fig. S14 Confocal fluorescence images of H2S in living MCF-7 cells using RHP-2. Cells were pretreated with 1 mM PPG for 30 min, then incubated with RHP-2 (5 μM) for 30 min (3A, 3B, 3C and 3D). Cells in panels 3A, 3B, 3C and 3D were thereafter treated with SNP (400 μM) for 30 min (4A, 4B. 4C and 4D). Blue channel images were obtained from 445 to 495 nm (3A and 4A). Green channel images were obtained from 520 to 580 nm (3B and 4B). Ratiometric images (F520–580 nm/F445–495 nm)

generated by Olympus software (3C and 4C) andthe corresponding bright images were shown in 3D and 4D.

Scale bars = 10 μm.

Fig. S15 Determination of sulphide concentrations in mouse hippocampus using RHP-2 probe (see the sulphide detection protocols). Sulphide concentrations in hippocampus homogenates (the test solution, 2 %, v/v) were 1.0 μM, 0.90 μM, 1.1 μM, 0.80 μM, 0.70 μM, 0.85 μM, 0.75 μM and 0.84 μM, respectively. Total protein concentrations in hippocampus homogenates (1 %, w/v) were 0.28 g/L, 0.27 g/L, 0.31 g/L, 0.25 g/L, 0.22 g/L, 0.26 g/L, 0.24 g/L and 0.24 g/L, respectively. The sulphide concentrations in mouse hippocampus were expressed as μmol g -1protein. The sulphide concentrations in mouse hippocampus were 1.78 μmol g-1 protein, 1.67 μmol g-1 protein, 1.77 μmol g-1 protein, 1.60 μmol g-1 protein, 1.59 μmol g-1 protein, 1.63 μmol g-1 protein, 1.59 μmol g-1 protein and 1.75 μmol g-1 protein, respectively. The average sulphide concentration was 1.67 ± 0.08 μmol g-1 protein (Data are presented as the mean ± SD, n = 8).

Determination of sulphide in mouse hippocampus with SFP-2 probe For the measurement of sulphide, the mice were sacrificed, and the hippocampus was immediately isolated and homogenised with 9 volumes (w/v) of ice-cold 100 mM PBS buffer (pH 7.4); the homogenate was centrifuged at 10,000 ×g for 10 min at 4°C. All of the procedures were performed in an ice bath, and the homogenate supernatants were immediately transferred for sulphide determination. All fluorescence measurements were recorded on a Hitachi F4600 Fluorescence Spectrophotometer. Protein concentrations of the mouse hippocampus were determined with a Pierce BCA Protein Assay Kit. The determination of sulphide concentration in hippocampus homogenates spiked with Na2S were used as an internal standard (X, X+0.4, X+0.8, X+1.2 and X+1.6 μM). Twenty microlitres of 10 % homogenate supernatant (final concentration 2 %, w/v) was added into Eppendorf tubes containing 69 µL of PBS buffer (100 mM, pH 7.4) and DI H2O (10, 9.6, 9.2, 8.8 and 8.4 µL, respectively). Thereafter, 0, 0.4, 0.8, 1.2, 1.6 µL

of Na2S stock solution (100 μM) were spiked into the samples as an internal standard, followed by the addition of 1 µL of 1.0 mM SFP-2 probe (final concentration 10 µM). Emission spectra (λex = 450 nm, emission at 512 nm) were determined at the end of a 40-min incubation of the mixture at 37°C. The zero point was obtained by the addition of 1 µL of 100 mM (final concentration 1 mM) ZnCl2 to trap H2S in the samples. The sulphide concentration in each sample was calculated using a calibration curve of Na2S, and the results were expressed as μmol g-1 protein. All data are expressed as the mean ± SD (n = 3).

Fig. S16 Determination of sulphide concentrations in mouse hippocampus using SFP-2 probe (see the sulphide detection protocols). Sulphide concentrations in hippocampus homogenates (the test solution, 2%, v/v) were 0.89 μM, 1.18 μM, 1.1 μM, 1.0 μM, 0.9 μM, 1.2 μM, 0.9 μM and 1.24 μM, respectively. Total protein concentrations in hippocampus homogenates (1%, w/v) were 0.25 g/L, 0.32 g/L, 0.32 g/L, 0.29 g/L, 0.25 g/L, 0.32 g/L, 0.26 g/L and 0.33 g/L, respectively. The sulphide concentrations in mouse hippocampus were expressed as μmol g-1 protein. The sulphide concentrations in mouse hippocampus were 1.79 μmol g-1 protein, 1.84 μmol g-1 protein, 1.78μmol g-1 protein, 1.73μmol g-1 protein, 1.81 μmol g-1 protein, 1.88 μmol g-1 protein, 1.75 μmol g-1 protein and 1.87 μmol g-1 protein, respectively. The average sulphide concentration was 1.81 ± 0.05 μmol g-1 protein (Data are presented as the mean ± SD, n = 8).

Chronic unpredictable mild stress (CUMS) induction The CUMS procedures were performed as described with slight modifications.8 Stressors were administered once daily for 5 consecutive weeks, i.e., 24-h water deprivation, 24-h food deprivation, 1-min tail pinched with a clothes-pin (1 cm distal from the tail tip), 5-min cold swimming (at 4°C), damp sawdust with 200 ml of water per cage (sufficient to reach the moisture of the sawdust bedding) and 24-h cage tilting, 4-h immobilisation and overnight illumination. The same stressor was not applied successively so that mice could not anticipate the onset of stress. Immediately after the closure of each stress session, the animals were returned to their home cages and maintained in standard conditions until the subsequent session. Normal control animals were housed (n = 4 each) without disturbance except for necessary procedures such as weighting or cage cleansing, with ad libitum access to food and water except for a 24-h period of fasting prior to the sucrose consumption test. Sucrose preference test Sucrose preference test was carried out as previously described with minor modifications,9 and was conducted before stress and 5 weeks after stress. Briefly, 72 h before the test, mice were trained to adapt 1% sucrose solution (w/v): two bottles of 1% sucrose solution were placed in each cage, and 24 h later 1% sucrose in one bottle was replaced with tap water for 24 h. After the adaptation, mice were deprived of water and food for 24 h. Sucrose preference test was conducted at 9:00 a.m., in which mice were housed in individual cages and were permitted ad libitum access to two bottles containing 100 mL of sucrose solution (1% w/v) and 100 mL of water, respectively. After 1 h, the volumes of sucrose solution and water consumed were recorded and the sucrose preference was calculated by the following formula: Sucrose preference = sucrose consumption / (water consumption + sucrose consumption) × 100 % Tail suspension test

The test was performed as described.10 Mice were individually suspended 50 cm above the surface of a floor, using an adhesive tape placed 1 cm away from the tail tip. In a 6-min test session, the duration for which animal remains immobile was recorded during the final 5 min. Mice were considered immobile if there were no body movements. Forced swimming test The forced swimming test was performed as previously described except for slight modifications.11 Briefly, mice were forced to swim individually in a transparent glass vessel (25 cm in height, 10 cm in diameter) with water filled to 10 cm at 24-26 °C, which was to be refreshed prior to each trial. Animals were trained for 15 min for swimming 24 h before commencement of test. The total duration of immobility (seconds) was measured during the final 4 min of a single 6 min test session. Mice were considered immobile when they made no attempts to escape except for the movements necessary to keep their heads above the water. This immobile posture reflects a state of behavioural despair or helplessness.

Fig. S17 Sucrose consumption of mice. The sucrose consumption test was carried before stress (0 week) and 5 weeks after stress. Data are presented as the mean ± SEM (n = 8). #p< 0.001 vs control, ##p< 0.001 vs model.

Fig. S18 Immobility time of mice in the tail suspension test. Data are presented as the mean ± SEM (n = 8). #p< 0.001 vs control, ##p< 0.001 vs model.

Fig. S19 Immobility time of mice in the forced swimming test. Data are presented as the mean ± SEM (n = 8). #p< 0.001 vs control, ##p< 0.001 vs model.

Fig. S20 Full-length blots of Fig. 6B in main text. CBS protein levels in mouse hippocampus. The samples derive from the same experiment and that blots were processed in parallel.

Fig. S21 Full-length blots of Fig. 6C in main text. CBS mRNA levels in mouse hippocampus.

Table S1. Measurement of sulphide concentrations in the mouse hippocampus Control H2S a (μM)

Model H2S c

Protein

b

(g/L)

(μmol g-1 protein)

H2S a (μM)

NaHS H2S c

Protein

b

(g/L)

(μmol g-1 protein)

H 2S c

H2S a

Protein

(μM)

(g/L)

b

(μmol g-1 protein)

1

1.00

0.28

1.78

0.59

0.32

0.92

0.93

0.30

1.54

2

0.90

0.27

1.67

0.49

0.27

0.91

0.82

0.27

1.52

3

1.10

0.31

1.77

0.53

0.31

0.85

0.94

0.30

1.56

4

0.80

0.25

1.60

0.48

0.29

0.83

0.82

0.28

1.45

5

0.70

0.22

1.59

0.59

0.31

0.95

0.87

0.29

1.49

6

0.85

0.26

1.63

0.46

0.28

0.83

0.92

0.29

1.58

7

0.75

0.24

1.59

0.47

0.27

0.86

1.09

0.33

1.65

8

0.84

0.24

1.75

0.49

0.31

0.79

0.87

0.31

1.41

Mean ± SD

1.67 ± 0.08

0.87 ± 0.05#

a

Sulphide concentrations in the hippocampus homogenates (the test solution, 2 %).

b

Sulphide protein concentrations in the hippocampus homogenate (dilution to 1 % homogenate).

c

Sulphide concentrations in the mouse hippocampus.

1.53 ± 0.08##

#

p< 0.001 vs control, ##p< 0.001 vs model.

Data are presented as the mean ± SD (n=8).

Theoretical and Computational Methods In order to understand the sensing mechanism of the RHP-2, we carried out theoretical calculations by using the density functional theory (DFT) and time-dependent functional theory (TDDFT) method. Molecular excitation energies, oscillator strengths (f) and electron transitions were listed in Table S2. Frontier molecular orbitals and corresponding energies of RHP-2 and compound NAP-NH2 are shown in Fig S23. Both the HOMO and LUMO of NAP-NH2 are mainly localized on naphthalimide moiety. As for RHP-2, the HOMO has no significant change with respect to NAP-NH2 and mainly localized on naphthalimide moiety, while the LUMO is essentially outspreaded on 4-nitrophenyl group. Obviously, the electron promotion from the HOMO to LUMO is accompanied by charge transfer from the donor moiety (naphthalimide moiety) to the acceptor group (4-nitrophenyl group), so that HOMO–LUMO transition of RHP-2 can be classified as an intramolecular charge transfer (ICT). In support of the assertion of charge transfer nature, Figure S24 depicts the differential charge densities (Δρ) between the ground and excited state. The electron density difference map gives a dynamic visualization of

electronic rearrangement for a transition, with green regions (positively valued) denoting an accumulation of density and red regions (negatively valued) representing a depletion of density upon excitation.12 The density depletion zones (red) of RHP-2 are mostly located on naphthalimide moiety. This finding is consistent with a weak donor character of the naphthalimide moiety. On the other hand, the regions of density increment (green) look more localized on 4-nitrophenyl group. These results further demonstrated that RHP-2 possess obvious ICT character, which result in blue-shifted absorption and fluorescence emission. With respect to compound NAP-NH2, the electrons in LUMO exhibits large overlap with those in HOMO, which would consequently result in localized state (LE) and induce strong fluorescence emission. These calculations were consistent with the experimental results and rationalize the ICT process.

 

              

RHP-2

NAP-NH2

Fig. S22 Optimized structure of ground states for RHP-2 and compound NAP-NH2

  HOMO (-6.5210 eV)

LUMO (-2.5348 eV)

  HOMO (-6.0187 eV)

  LUMO (-2.0256 eV)

Fig. S23 Plots of the HOMO and LUMO of the ground state for RHP-2 (up) and compound NAP-NH2 (down).

  (a)

(b)

Fig. S24 Electron density difference computed for the ground and the first excited states (Δρ (r) =ρexcited (r) – ρ ground (r)) of RHP-2 (a) and compound NAP-NH2 (b). Green regions denote a positive density difference (accumulation of density upon electronic excitation), and red regions represent a negative density difference (depletion of density upon excitation). Both densities are plotted using the same isosurface contour value of 0.001 au.

Table S2. Calculated electronic transitions energies for RHP-2 and compound NAP-NH2 at TD-DFT/PBE1PBE/6-31+G(d,p) level Compound

Transitons

λexp.(nm)

λcalc. (nm)

f

Transitions (CI expansion coefficients)

RHP-2

S0–S1

370

361

0.1519

HOMO → LUMO (0.59119)

NAP-NH2

S0–S1

428

373

0.2140

HOMO → LUMO (0.69601)

Computational method The ground geometries and electron transition of the title compounds have been investigated by using Gaussian 09 program.13 The ground state (S0) geometries were fully optimized without constraint at the PBE1PBE/6-31G(d,p) level, and all the local minima were confirmed by the vibrational frequencies analysis. On the basis of the optimized structures of the S0-states, the TDDFT calculations with PBE1PBE/6-31+G(d,p) method were performed to compute the absorption energies of title compounds. All calculation was performed in gas phase. The electron density difference map of the title compounds have been generated using Multiwfn 14 and Gauss View embedded in Gaussian 09 program. References 1.

Song L, Yang Y, Zhang Q, Tian H, Zhu W. Synthesis and photochromism of naphthopyrans bearing naphthalimide chromophore: predominant thermal reversibility in color-fading and fluorescence switch. J Phys Chem B. 2011, 115, 14648-14658.

2.

CN. Pat., 101 302 197, 2008.

3.

Williams ATR, Winfield SA and Miller JN. Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer. Analyst, 1983, 108, 1067-1071.

4.

Qian, Y., Zhang, L., Ding, S., Deng, X., He, C., Zhu, H.L., Zhao, J. A Fluorescent Probe for rapid detection of hydrogen Sulfide in blood plasma and brain tissues in mice. Chem Sci. 2012, 3, 2920-2923.

5.

Arudi R, Allen A and Bielski B. Some observations on the chemistry of KO2-DMSO solutions. FEBS Lett., 1981, 135, 265-267.

6.

Halliwell B and Gutteridge J. Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys. 1986, 246, 501-514.

7.

Joshi BP, Park J, Lee WIand Lee K. Ratiometric and turn-on monitoring for heavy and transition metal ions in aqueous solution with a fluorescent peptide sensor. Talanta. 2009, 78, 903-909.

8.

Harro J, Haidkind R, Harro M, Modiri AR, Gillberg PG, Pahkla R, Matto V, Oreland L. Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioral and biochemical effects of DSP-4 treatment. Eur Neuropsychopharmacol. 1999, 10, 5-16.

9.

Luo DD, An SC, Zhang X. Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress. Brain Res Bull. 2008, 77, 8-12.

10.

Steru L, Chermat R, Thierry B, et al. The tail suspension test: a newmethod for screening antidepressants in mice. Psychopharmacology. 1985, 85, 367-370.

11.

Porsolt RD, Bertin A, Blavet N, Deneil M, Jalfre M. Immobility induced by forced swimming in rats: effects of agents which modify central catecholamine and serotonin activity. Eur J Pharmacol, 1979, 57, 201-210.

12.

Xue YS, An L, Zheng YG, Zhang L, Gong XD, Qian Y and Liu Y, Comput Theor Chem. 2012, 981, 90-99.

13.

M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A.02, Gaussian, Inc., Pittsburgh PA, 2009.

14.

Lu T, Chen F. Multiwfn: a multifunctional wavefunction analyzer. J Comput Chem. 2012, 33, 580-592.

XYZ Coordinates for compound NAP-NH2 and RHP-2 Compound NAP-NH2 Atomic

Atomic

Number

Type

Coordinates (Angstroms) X

Y

Z

-----------------------------------------------------------6

0

1.034093

2.599893

0.053697

6

0

2.410469

2.635745

0.324016

6

0

3.142197

1.467597

0.357483

6

0

2.532004

0.214368

0.126967

6

0

1.141614

0.185700

-0.174505

6

0

0.410389

1.395179

-0.197326

6

0

3.249226

-1.028539

0.170796

6

0

2.577797

-2.209650

-0.131961

6

0

1.218889

-2.208862

-0.446421

6

0

0.489482

-1.034137

-0.460379

6

0

-0.945610

-1.074252

-0.769580

7

0

-1.626383

0.150871

-0.748973

6

0

-1.039384

1.386730

-0.497964

6

0

-3.059434

0.148461

-1.053021

6

0

-3.968703

0.258762

0.168741

6

0

-3.871430

-0.886483

1.184254

6

0

-2.786299

-0.710151

2.242897

8

0

-1.535940

-2.112828

-1.025992

8

0

-1.703864

2.411050

-0.526665

7

0

4.590422

-1.042683

0.462271

1

0

0.435783

3.504987

0.024140

1

0

2.901838

3.587577

0.499030

1

0

4.212174

1.527747

0.535336

1

0

3.123189

-3.149366

-0.107773

1

0

0.700850

-3.135867

-0.671686

1

0

-3.251758

-0.782585

-1.588458

1

0

-3.242928

0.995464

-1.718381

1

0

-4.991150

0.303207

-0.227613

1

0

-3.791171

1.222245

0.661088

1

0

-3.719448

-1.833229

0.652092

1

0

-4.836729

-0.974072

1.698127

1

0

-2.811931

-1.529128

2.968959

1

0

-2.927291

0.226637

2.793757

1

0

-1.781704

-0.689204

1.811879

1

0

4.992304

-1.946497

0.653915

1

0

4.946314

-0.313174

1.057605

RHP-2 Atomic

Atomic

Number

Type

Coordinates (Angstroms) X

Y

Z

----------------------------------------------------------6

0

3.467312

-2.199674

2.048644

6

0

2.190709

-2.745928

2.271398

6

0

1.139141

-2.432609

1.433110

6

0

1.307880

-1.556905

0.328680

6

0

2.612095

-1.014281

0.102790

6

0

3.675812

-1.348716

0.979542

6

0

0.253084

-1.184781

-0.578306

6

0

0.520992

-0.341201

-1.649518

6

0

1.813130

0.167675

-1.847918

6

0

2.852387

-0.148675

-0.992580

6

0

4.195320

0.423173

-1.236376

7

0

5.208754

0.072503

-0.325168

6

0

5.036998

-0.789662

0.763020

6

0

6.565543

0.616106

-0.545695

6

0

6.906791

1.807822

0.358290

6

0

6.025406

3.046946

0.159615

6

0

6.458680

4.222102

1.041347

8

0

4.429000

1.168596

-2.179235

8

0

5.976676

-1.064710

1.499267

7

0

-1.028247

-1.695703

-0.332669

6

0

-2.174169

-1.525991

-1.073369

8

0

-3.183413

-2.184907

-0.440911

8

0

-2.292361

-0.909347

-2.114333

6

0

-4.490353

-2.092514

-1.064712

6

0

-5.274799

-0.914993

-0.535677

6

0

-6.126959

-1.073875

0.565331

6

0

-6.846631

0.004146

1.072430

6

0

-6.699906

1.245807

0.459133

6

0

-5.861913

1.437817

-0.637177

6

0

-5.149507

0.348992

-1.131341

7

0

-7.459580

2.393490

0.986584

8

0

-8.189227

2.196398

1.957613

8

0

-7.317363

3.478343

0.424623

1

0

4.304425

-2.432790

2.697270

1

0

2.031130

-3.420048

3.106691

1

0

0.177071

-2.891356

1.641173

1

0

-0.277496

-0.077536

1

0

2.015297

0.829310

-2.683160

1

0

6.611525

0.901309

-1.596612

1

0

7.265745

-0.199600

-0.357342

1

0

7.954016

2.070720

0.154829

1

0

6.870343

1.481336

1.403974

1

0

4.981213

2.794092

0.382085

1

0

6.043564

3.344501

-0.895727

1

0

5.812325

5.092789

0.891081

1

0

7.486719

4.529261

0.817298

1

0

6.419188

3.957688

2.104322

1

0

-1.165350

-2.237800

0.505838

1

0

-4.978983

-3.034461

-0.809737

1

0

-4.357639

-2.026966

-2.145733

1

0

-6.231123

-2.050746

1.028860

1

0

-7.512079

-0.097359

1.920170

1

0

-5.782970

2.421686

-1.081870

1

0

-4.482153

0.474280

-1.977268

-2.327520

O

N

O

Br

 

 

 

 

Fig. S25 HR-MS identification of compound 1 (calculated for C16H15BrNO2 (M+H)+, 332.0286; found, 332.0239).

O

N

O

N3

 

 

 

 

Fig. S26 HR-MS identification of compound 2 (calculated for C16H15N4O2 (M+H)+ 295.1195; found 295.1193) .

O

N

O

NH2

 

 

Fig. S27 HR-MS identification of compound NAP-NH2 (calculated for C16H17N2O2 (M+H)+ 269.1290; found 269.1282).

           

O

O2 N

N

O

HN

O O

 

 

 

  Fig. S28 HR-MS identification of RHP-2 probe (calculated for C24H20N3O6 (M-H)+ 446.1352; found 446.1359) .

Fig. S29 1H NMR spectra of the isolated fluorescent product of RHP-2 + Na2S (b) in DMSO-d6.

Fig. S30 13C NMR spectra of the isolated fluorescent product of RHP-2 + Na2S (b) in DMSO-d6.