Supporting Information

Near-Infrared Fluorescent Chemodosimeter for Real-Time in vivo Evaluating H2S-Releasing

Efficiency of Prodrug

1. Materials and Apparatus:

2. Synthesis

3. Response of NRh-N3 to H2S in vitro

4. Response of NRh-N3 to other reductants in vitro:

5. Response of NRh-N3 to ADT-OH in vitro

6. MTT assay

7. Confocal Imaging

8. Cell culture

9. MTT assay

10. Fluorescent imaging in living mice

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020

Materials and Apparatus: Hyperpure water was used to prepare all aqueous solutions. Most

materials were purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD. ADT-OH was

purchased from Bidepharm. Tert-Butyl nitrite was purchased from J&K. The confocal

fluorescence images of the cells were determined with confocal laser scanning microscope (CLSM,

LSM800, Zeiss, Germany) and the fluorescence images of the mice were determined with CCD

from princeton instrument. All NMR data was collected using a Bruker 400 MHz. Mass

spectroscopy data was collected on Waters Q-TOF MicroTM. High Performance Liquid

Chromatography data was collected on Thermo Scientific dionex ultimate 3000.

Synthesis:

Scheme S1. Synthesis itinerary of NRh-N3

Synthesis of compound 1:

1,1,2-Trimethylbenz[e]indole (2.09 g 10.0 mmol) and iodoethane (1.56 g 10.0 mmol) were placed

in a flask containing dry toluene (15.0 ml), and the mixture was stirred at 130°C with solvent

reflux under a nitrogen atmosphere for 8 hours. The solution was removed under vacuum filtration

and washed with ether to produce compoud 1 as a blue solid (2.92 g, yield 80%). 1H NMR (400

MHz, MeOD) δ 8.34 (t, J = 12.6 Hz, 1H), 8.27 (d, J = 8.9 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.05

(d, J = 8.9 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 4.89 (s, 3H), 4.70 (q, J = 7.3

Hz, 2H), 1.87 (s, 6H), 1.66 (t, J = 7.3 Hz, 3H). MS (ESI+): calcd for C17H20N+, 238.2 [M]+; found,

238.1589 [M]+.

Synthesis of NIR-Cl:

Compound 1 (2.92 g, 8.0 mmol) and N-((2-Chloro-3((phenylimino)methyl)cyclohex-2en-1-

ylidene)methyl)aniline hydrochloride (1.44 g, 4.0 mmol) was added into a flask containing dry

acetic anhydride and potassium acetate under a nitrogen atmosphere. The mixture was stirred at

60°C for 3 hours. The solution was hydrolysed by sodium bicarbonate and removed under vacuum

filtration to produce NIR-Cl as a red solid (2.35 g, yield 80%). 1H NMR (400 MHz, MeOD) δ 8.90

(d, J = 15.3 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 8.9 Hz, 1H), 8.11 (t, J = 7.3 Hz, 1H),

7.86 (d, J = 8.9 Hz, 1H), 7.78 (ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.68 – 7.62 (m, 1H), 7.53 (d, J = 8.3

Hz, 1H), 7.30 (s, 1H), 7.25 – 7.19 (m, 1H), 7.13 – 7.02 (m, 1H), 6.68 (d, J = 15.2 Hz, 1H), 4.60 (q,

J = 7.3 Hz, 2H), 2.80 (dt, J = 11.9, 5.9 Hz, 4H), 2.12 (d, J = 5.1 Hz, 6H), 1.99 (dd, J = 10.5, 4.4

Hz, 2H), 1.59 (d, J = 7.3 Hz, 2H). MS (ESI+): calcd for C42H44ClN2+, 611.3 [M]+; found,

611.3186 [M]+.

Synthesis of NRh-NH2:

NIR-Cl (1.47 g, 2.0 mmol) and 3-Nitrophenol (0.42 g, 3.0 mmol) were placed in a flask containing

dry N,N-dimethylformamide and triethylamine under a nitrogen atmosphere at 85°C for 3 hours.

SnCl2 (3.80 g, 20.0 mmol) dissolved in concentrated HCl (4.0 mL) was added the above solution

under nitrogen atmosphere. The mixture was stirred at 85°C for 8 hours. Then the solution was

removed under vacuum filtration at 85°C. The crude product was purified by silica column

chromatography using CH2Cl2/CH3OH (50:1) as the eluent to produce the NRh-NH2 compound as

a black solid (0.72 g, yield 50%). 1H NMR (400 MHz, MeOD) δ 8.77 (d, J = 14.4 Hz, 1H), 8.30 (d,

J = 8.6 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.69 (dd, J = 12.7, 6.4 Hz, 2H),

7.54 (dd, J = 14.6, 7.2 Hz, 2H), 7.40 – 7.33 (m, 1H), 6.78 (dd, J = 5.6, 1.8 Hz, 2H), 6.33 (d, J =

14.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 2.78 (dt, J = 16.7, 5.9 Hz, 4H), 2.11 (d, J = 14.3 Hz, 6H),

2.00 – 1.93 (m, 2H), 1.52 (t, J = 7.2 Hz, 3H). MS (ESI+): calcd for C31H31N2O+, 447.2 [M]+; found,

447.3 [M]+.

Synthesis of NRh-N3：

NRh-NH2 (0.47 g,1.0 mmol) and tert-Butyl nitrite (0.52 g,5.0 mmol), trimethylsilyl azide (0.58 g,

5.0 mmol) were placed in a flask containing dry N,N-dimethylformamide under a nitrogen

atmosphere for 5 hours. The solution was removed under vacuum filtration at 85°C. The crude

product was purified by silica column chromatography using CH2Cl2/CH3OH (50:1) as the eluent

to produce the NRh-N3 compound as a black solid (0.37 g, yield 79%). 1H NMR (400 MHz,

MeOD) δ 8.90 (d, J = 15.3 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H), 8.18 (d, J = 8.9 Hz, 1H), 8.11 (t, J =

7.3 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.78 (ddd, J = 8.4, 6.9, 1.2 Hz, 1H), 7.68 – 7.62 (m, 1H),

7.53 (d, J = 8.3 Hz, 1H), 7.30 (s, 1H), 7.25 – 7.19 (m, 1H), 7.13 – 7.02 (m, 1H), 6.68 (d, J = 15.2

Hz, 1H), 4.60 (q, J = 7.3 Hz, 2H), 2.80 (dt, J = 11.9, 5.9 Hz, 4H), 2.12 (d, J = 5.1 Hz, 6H), 1.99

(dd, J = 10.5, 4.4 Hz, 2H), 1.59 (d, J = 7.3 Hz, 2H). 13C NMR (101 MHz, DMSO) δ 159.08,

153.58, 144.86, 142.92, 139.03, 137.07, 133.00, 131.36, 131.13, 129.67, 128.66, 127.52, 126.85,

123.15, 119.40, 116.88, 114.65, 113.00, 106.89, 105.82, 52.88, 31.58, 30.49, 30.29, 29.50, 27.34,

24.05, 20.30, 13.59. MS (ESI+): calcd for C31H29N4O+, 473.2336 [M]+; found, 473.2353 [M]+.

Figure S1. 1H NMR spectra of compound 1 in MeOD

Figure S2. Mass spectra of compound 1.

Figure S3. 1H NMR spectra of NIR-Cl in MeOD.

Figure S4. Mass spectra of NIR-Cl.

Figure S5. 1H NMR spectra of NRh-NH2 in MeOD.

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

%

0

100SAMPLE-1018-01 3919 (73.031) TOF MS ES+

5.77e3447.3

448.3

NRh-NH2

Figure S6. Mass spectra of NRh-NH2.

Figure S7. 1H NMR spectra of NRh-N3 in MeOD.

Figure S8. 13C NMR spectra of NRh-N3 in DMSO.

Figure S9. Mass spectra of NRh-N3.

Response of NRh-N3 to H2S in vitro:

4.70 mg NRh-N3 was soluted to 1.0 mL DMSO (10.0 mM NRh-N3 solution), 8.30 mg 67%NaHS

was soluted in 10.0 mL water (10.0 mM NaHS solution). 3.0 μL NRh-N3 solution was added into

cuvette after the addition of 3.0 mL PBS buffer (pH 7.4) and initial absorbance spectrum were

obtained. Then 3.0 μL NaHS solution was added many a time and each time reacted at room

temperature for 10 min, the metabolic absorbance spectra were obtained.. 3.0 μL NRh-N3 solution

was added into all sides cuvette after the addition of 3.0 mL PBS buffer (pH 7.4) and initial

fluorescence spectrum were obtained. The same operation as the previous step, we got the

metabolic fluorescence spectra. We signed five bottles(1-5). We added the 10.0 μM NRh-N3 into

(1-4)four bottles and 10.0 μM NRh-NH2 into the remaining bottle(5). Then we added various

concentration NaHS solutions into (1-4) bottles and injected into High Performance Liquid

Chromatography.

Figure S10. Plot of fluorescence intensity at 740 against the concentration of NaHS. And the the

limited of detection (LOD) was approximately 26.0 nM.

Figure S11. Image showing colour change after addition of sufficient NaHS into NRh-N3.

Figure S12. Image showing colour change after addition of various NaHS into NRh-N3 before

adding into High Performance Liquid Chromatography.

m/z50 100 150 200 250 300 350 400 450 500 550 600 650

%

0

100SAMPLE-0424-01 1924 (35.831) TOF MS ES+

1.29e3447.3

445.3

432.3448.3

473.3

N3

Figure S13. Mass spectra of solution in bottle 3.

Response of NRh-N3 to other reductants in vitro:

We prepared 10μM NRh-N3, And reacted with 1.0 mM H2O2, 200.0 μM NaNO2, 200.0 μM

NaNO3, 1.0 mM Vc, 1.0 mM Va, NaHS, 200.0 μM NaS2O3, 200.0 μM NaSO4, 1.0 mM Hcy, 1.0

mM GSH, 1.0 mM Cys and 200.0 μM NaHS solutions for 15 min at room temperature. We got the

fluorescence spectra. And we selected the fluorescence intensity of 730 nm and analyzed with

origin8 software.

Figure S14. relative pixel intensity of NRh-N3 (10.0 μM) toward oxygen, nitrogen species,

reactive sulfur.

.

Response of NRh-N3 to ADT-OH in vitro:

We prepared 10.0 μM NRh-N3, And reacted with 0-300.0 μM ADT-OH under simulated

physiological conditions (PBS buffers contains 50% cell lysates) for 30 min at room temperature.

We got the fluorescence spectra. And we selected the fluorescence intensity of 730 nm and

analyzed with origin8 software.

MTT assay:

Figure S15. MTT assay for estimating cell viability (%) of U87 cells treated with various

concentrations of NRh-N3 (0-100.0 μM) after 48 h incubation.

Confocal Imaging :

Fluorescent images were observed with confocal laser scanning microscope (CLSM, LSM800,

Zeiss, Germany) with objective lens (×63),And processed using the ZEN imaging software. The

excitation wavelength was 635 nm. Cell imaging was carried out after washing cells with fresh

complete medium (DMEM+10% FBS, 2×1.0 mL) and preserve in 4% PFA.

Cell culture:

We use human glioma cells U87 cells for biological experiments and grew them in DMEM with

10% FBS. Cultures were maintained at 37 °C under a humidified atmosphere containing 5%

CO2.The cells were subcultured by scraping and seeding on 20 mm glass bottom cell culture dish.

Fluorescent imaging in living mice：

BALB/c nude mice (20.0 g-25.0 g) were obtained from Nangjing University. Mice were group-

housed on a 12:12 light-dark cycle at 25 °C with free access to food and water. BALB/c nude

mice were selected and divided into four groups. First group was given subcutaneous injection

with NRh-N3 as a control group. Second groups were given subcutaneous injection with NaHS

after NRh-N3 as a treatment group. Then the rest mice were administrated with NRh-N3 by tail

vein injection. And last group intraperitoneally injected ADT-OH was chosen as treatment group.