Supporting Information Reaction Based Color-Convertible Fluorescent Probe for Ferroptosis Identification Leilei Shi, Qinghua Guan, Xihui Gao, Xin Jin, Li Xu, Jian Shen,*, Chenwei Wu, Xinyuan Zhu, and Chuan Zhang*, School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Nanjing Normal University, Nanjing 210046, China *Corresponding authors Emai: chuanzhang@sjtu.edu.cn; shenjianbio@hotmail.com Contents: 1. Materials and instruments...s2 2. Synthesis of compounds...s4 3. Supplementary figures....s10 4. Table S1....S22 S-1
Materials and instruments. Benzene-1,2-diamine, ethyl-2-oxopropanoate, 3-bromoprop-1-ene and methyl-2-bromoacetate were purchased from Adamas. 5-(Methylthio)thiophene-2-carbaldehyde was purchased from J&K chemical. All reagents were of analytical grade, and used without purfication. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchase from sigma. Dulbecco s Modified Eagle Medium (DMEM) was purchased from Gibco. Fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco. H 2 O 2 (30% aqueous solution) was purchased from Sigma-Alderich. Hemeoxygenase-1 and hemeoxygenase-1 monoclonal antibody were purchased from Abcam. Anti HO-1 sirna (5 -AAGCCACACAGCACUAUGUAAdTdT-3 (sense) and 5 -UUACAUAGUGCUGUGUGGCUUdTdT-3 (anti-sense)) was purchased from Shanghai Sangon Biotech. All reagents were of analytical grade, and used as received. 1 H and 13 C nuclear magnetic resonance spectroscopy ( 1 H and 13 C NMR) were recorded using a Mercury plus 400 MHz spectrometer (Varian, USA) with dimethyl sulfoxide-d6 (DMSO-d 6 ) and chloroform-d (CDCl 3 ) as solvents. Liquid chromatography-mass spectrometry (LC-MS) was performed on a Water ACQUITY UPLC system equipped with a binary solvent delivery manager and a sample manager, coupled with a Waters Q-TOF Premier Mass Spectrometer equipped with an electrospray interface (Waters Corporation, Milford, MA). Ultraviolet-visible (UV-Vis) absorption of the sample solutions was determined by Thermo Electron-EV300 UV-Vis spectrophotometer. Steady-state and time-resolved fluorescence measurements were performed with a PTI-QM/TM/IM steady-state & time-resolved fluorescence spectrofluorometer (USA/CAN) photo technology international (PTI) laserstrobe fluorescence. Three replicate measurements were performed at the same conditions for each sample, with some repetitions on selected. Quartz cells (1*1 cm) were used for all spectral measurements. The absolute fluorescence quantum yields were determined by integrating sphere. S-2
1. Synthesis of QS-1, QS-2, QS-3 and QS-4 Scheme S1. Design and synthesis of AIEE-active molecule (QS-1). Scheme S2. Design and synthesis of AIEE-active molecule (QS-2). Scheme S3. Design and synthesis of AIEE-active molecule (QS-3). Scheme S4. Design and synthesis of AIEE-active molecule (QS-4). S-3
Syntheis of compound 1: 3-methylquinoxalin-2(1H)-one (1a) and 7-methoxy-3-methylquinoxalin-2(1H)-one (1b) o-phenylenediamine (0.1 mol, 10.8 g) was suspended in anhydrous ethanol (150 ml). The mixture was then cooled in an ice bath. A solution of ethyl pyruvate (0.12 mol, 13.92 g) in anhydrous ethanol (10 ml) was added dropwise over a period of 20 min under stirring. The resulting solution was allowed to react at room temperature for 12 h. The resulting precipitate was filtered and washed with ethanol, and dried in vacuum to give the product 1a (13.6 g, yield: 85%) as a white solid which was pure without any purification. ESI-MS m/z: 161.6 [M+H] +. For 7-methoxy-3-methylquinoxalin-2(1H)-one (1b), the synthetic procedure of 1b is the same as 1a. 1 H-NMR (400 MHz, CDCl 3 ): δ = 12.18 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 6.83-6.86 (m, 1H), 6.70-6.72 (m, 1H), 3.78 (s, 3H), 2.32 (s, 3H) ppm; ESI-MS m/z: 190.2 [M+H] +. Synthesis of compound 2: 1-allyl-3-methylquinoxalin-2 (1H)-one (2a) and 1-allyl-7-methoxy-3-methylquinoxalin-2 (1H)-one (2b) Compound 1a (20 mmol, 3.2 g) and K 2 CO 3 (24 mmol, 3.31 g) were suspended in acetone (100 ml). Then a solution of 3-bromoprop-1-ene (24 mmol, 2.88 g) was added dropwise into the mixture. The reaction mixture was stirred on oil bath at 62 o C for 12 h. After completion of reaction, the solvent was removed via rotary evaporators and the residue was partitioned between distilled water (20 ml) and ethyl acetate (EA) (40 ml). The organic layer was separated and dried over anhydrous MgSO 4, filtered, and concentrated via rotary evaporators to obtain crude product, which was further purified via silica gel chromatography (hexane: EA = 20:1, v:v) to afford 2.21 g of 2a as a white solid, yield 55%. 1 H-NMR (400 MHz, CDCl 3 ): δ = 7.83 (dd, J 1 = 8.0 Hz, J 2 = 2.0 Hz, 1H), 7.46-7.50 (m, 1H), 7.32-7.33 (m, 1H), 7.28-7.29 (m, 1H), 5.88-5.98 (m, 1H), 5.24-5.27 (m, 1H), 5.13-5.18 (m, 1H), 4.90 (d, J = 8.0 Hz, 2H), 2.61 (s, 3H) ppm; HRMS: m/z (ESI) calcd for C 12 H 12 N 2 O (M+H) +, 201.0950, Found 201.0984. For 1-allyl-7-methoxy-3-methylquinoxalin-2 (1H)-one (2b), the synthetic procedure is the same as 2a. 1 H-NMR (400 MHz, CDCl 3 ): δ = 7.71 (d, J = 8.0 Hz, 1H), 6.90 (d, J = S-4
8.0 Hz, 1H), 6.70 (s, 1H), 5.99-5.85 (m, 1H), 5.28 (d, J = 12 Hz, 1H), 5.18 (d, J = 12 Hz, 1H), 4.85-4.88 (m, 2H), 3.88 (s, 3H), 2.55 (s, 3H) ppm; HRMS: m/z (ESI) calcd for C 13 H 12 N 2 O 2 (M+H) +, 231.1055, Found 231.1118. Synthesis of compound 3: methyl-2-(3-methyl-2-oxoquinoxalin-1(2h)-yl)acetate (3a), methyl-2-(7-methoxyl-3-methyl-2-oxoquinoxalin-1(2h)-yl)acetate (3b) Compound 1a (20 mmol, 3.2 g) and K 2 CO 3 (24 mmol, 3.31 g) were suspended in acetone (100 ml). Then a solution of methyl-2-bromoacetate (24 mmol, 3.67 g) was added dropwise into the mixture. The reaction mixture was stirred on oil bath at 62 o C for 12 h. After completion of reaction, the solvent was removed via rotary evaporators and the residue was partitioned between distilled water (20 ml) and ethyl acetate (EA) (40 ml). The organic layer was separated and dried over anhydrous MgSO 4, filtered, and concentrated via rotary evaporators to obtain crude product, which was further purified via silica gel chromatography (hexane: EA = 10:1, v:v) to afford 3.0 g of 3a as a white solid, yield 54%. 1 H-NMR (400 MHz, CDCl 3 ): δ = 7.64 (d, J = 8.0 Hz, 1H), 7.14-7.17 (m, 1H), 6.93 (J 1 = 8.0 Hz, J 2 = 2.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H) ppm; HRMS: m/z (ESI) calcd for C 12 H 12 N 2 O 3 (M+H) +, 233.2353, Found 233.6002. For 3b, the synthetic procedure is the same as 3a. 1 H-NMR (400 MHz, CDCl 3 ): δ = 7.66 (d, J = 8.0 Hz, 1H), 6.96 (dd, J 1 = 8.0 Hz, J 2 = 2.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 5.06 (s, 2H), 3.83 (s, 3H), 3.68 (s, 3H), 2.36 (s, 3H) ppm; HRMS: m/z (ESI) calcd for C 13 H 14 N 2 O 4 (M+H) +, 263.0954, Found 263.4613. Synthesis of The Final Product: E-1-allyl-3-(2-(5-methylthio)thiophene-2-yl)vinyl)quinoxalin-2(1H)-one (QS-1), E-1-allyl-7-methoxy3-(2-(5-methylthio)thiophene-2-yl)vinyl)quinoxalin-2 (1H)-one (QS-2), E-methyl-2-(3-(2-(5-methylthio)thiophene-2-yl)vinyl)-2-oxoquinoxalin-1 (2H)-yl)acetate (QS-3), and E-methyl-2-(7-methoxy-3-(2-(5-methylthio)thiophene-2-yl)vinyl)-2-oxoquinoxalin -1 (2H)-yl)acetate (QS-4) 5-(Methylthio)thiophene-2-carbaldehyde (3 mmol, 570 mg) and catalytic concentrated sulfuric acid were added into a solution of compound 3a (2.5 mmol, 500 S-5
mg) in acetic acid (10 ml). The resulting solution was heated to 50 o C and reacted for 8 h. The reaction mixture was concentrated by rotary evaporators and then water (20 ml) and ethyl acetate (50 ml) were added, the organic layer was collected, and the water phase was basified by K 2 CO 3, then extracted by ethyl acetate, the combined organic phase was dried via anhydrous MgSO 4, and purified by silica chromatography (PE: EA = from 20 :1 to 10:1) to afford 322 mg of QS-1 as a orange solid, yield 38%. 1 H-NMR (400 MHz, CDCl 3 ): δ = 8.18-8.22 (m, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.41-7.45 (m, 1H), 7.33-7.37 (m, 1H), 7.32-7.33 (m, 1H), 7.24-7.26 (m, 1H), 7.11-7.12 (m, 1H), 6.92-6.93 (m, 1H), 5.88-6.01 (m, 1H), 5.25-5.28 (m, 1H), 5.15-5.19 (m, 1H), 4.92-4.93 (m, 2H), 2.55 (s, 3H) ppm; 13 C-NMR (100 MHz, CDCl 3 ): δ = 154.6, 151.9, 143.6, 141.3, 133.6, 132.0, 131.3, 130.8, 130.0, 129.8, 129.7, 123.8, 121.3, 121.3, 118.1, 114.3, 44.6, 20.8 ppm; HRMS: m/z (ESI) calcd for C 35 H 27 N 3 OS (M+H) +, 341.4624, Found 341.0748; QS-2, orange solid, yield 31%, 1 H-NMR (400 MHz, CDCl 3 ): δ = 8.10-8.14 (m, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.31-7.35 (m, 1H), 7.07-7.08 (m, 1H), 6.89-6.93 (m, 1H), 6.68-6.69 (m, 1H), 5.89-5.98 (m, 1H), 5.27-5.29 (m, 1H), 5.17-5.22 (m, 1H), 4.88-4.90 (m, 2H), 3.88 (s, 3H), 2.54 (s, 3H) ppm; 13 C-NMR (100 MHz, CDCl 3 ): δ = 155.0, 150.1, 147.5, 143.0, 142.8, 139.5, 136.5, 135.0, 130.9, 129.2, 127.5, 126.6, 126.5, 126.1, 125.9, 125.3, 125.0, 124.8, 123.2,121.9, 120.9, 120.1, 119.6, 119.4, 119.3, 118.9, 118.7, 118.5, 116.9, 113.5, 109.6, 40.1 ppm; HRMS: m/z (ESI) calcd for C 39 H 29 N 3 OS 2 (M+H) +, 371.4884, Found 371.0869. QS-3, red solid, 36%, 1 H-NMR (400 MHz, CDCl 3 ): δ = 8.16-8.20 (m, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.43-7.47 (m, 1H), 7.31-7.36 (m, 2H), 7.11-7.12 (m, 1H), 7.03-7.05 (m, 1H), 6.91-6.92 (m, 1H), 5.05 (s, 2H), 3.77 (s, 3H), 2.54 (s, 3H) ppm; 13 C-NMR (100 MHz, CDCl 3 ): δ = 167.6, 154.8, 151.7, 143.4, 141.3, 132.1, 131.5, 130.7, 130.2, 130.0, 129.7, 124.2, 120.8, 113.2, 52.7, 43.8, 20.8 ppm; HRMS: m/z (ESI) calcd for C 40 H 31 N 3 O 2 S 2 (M+H) +, 372.0602, Found 372.0911. QS-4, red solid, 21%, 1 H-NMR (400 MHz, CDCl 3 ): δ = 8.22-8.25 (m, 1H), 7.34-7.38 (m, 2H), 7.11-7.14 (m, 2H), 6.93-6.99 (m, 2H), 5.05 (s, 2H), 3.89 (s, 3H), 3.78 (s, 3H), 2.56 (s, 3H) ppm; 13 C-NMR (100 MHz, CDCl 3 ): δ = 168.1, 156.4, 154.5, 152.3, 143.3, 134.4, 131.5, 130.5, 121.5, 119.2, 114.1, 111.0, 55.9, 53.1, 43.7, 20.9 ppm; HRMS: S-6
m/z (ESI) calcd for C 40 H 31 N 3 O 2 S 2 (M+H) +, 403.4872, Found 403.0771. S-7
Supplementary Figures Figure S1. Ultraviolet-Visible (UV-Vis) absorption spectra of quinoxalinone derivatives. (a) UV-Vis absorption spectra of QS-1 in the mixed solvents with different DMSO/H 2 O ratios. (b) UV-Vis absorption spectra of QS-2 in the mixed solvents with different DMSO/H 2 O ratios. (c) UV-Vis absorption spectra of QS-3 in the mixed solvents with different DMSO/H 2 O ratios. (d) UV-Vis absorption spectra of QS-4 in the mixed solvents with different DMSO/H 2 O ratios. Figure S2. DLS (a), TEM image (b), and stability study (c) of nanodot self-assembled by QS-4 in PBS buffer, the scale bar is 100 nm. S-8
Figure S3. Fluorescence spectra of QS-1 in the mixed solvents with different DMSO/H 2 O ratios. Figure S4. Fluorescence spectra of QS-2 in the mixed solvents with different DMSO/H 2 O ratios. Figure S5. Fluorescence spectra of QS-3 in the mixed solvents with different DMSO/H 2 O ratios. S-9
Figure S6. 2,7-Dichlorofluorescin diacetate (DCFH-DA) assay for identification of the ROS levels. (a) Confocal imaging study of HeLa cells when they were treated ferroptosis inducer erastin. (b) Quantification of fluorescence intensity. The scale bar is 50 µm. Figure S7. The relative expression level of hemeoxygenase-1 (HO-1) in HeLa cells being treated with 1 µm and 2 µm erastin for 24 h, respectively. Cells without anytreatment were used as negative control. (a) Western blot analysis of HO-1 HeLa cells. (b) Quantification of the western blot band densities. Figure S8. Expressions of HO-1 in HeLa cells treated by PTX, CDDP, and ferroptosis inducer erastin, as determined by ELISA analysis. S-10
Figure S9. 2,7-Dichlorofluorescin diacetate (DCFH-DA) assay for detection the level of ROS when HeLa cells were treated by PTX, CDDP and ferroptosis inducer erastin. (a) Flow cytometry histograms illustrating different cellular ROS concentration in HeLa cells. (b) Fluorescence intensities of HeLa cells treated with PTX, CDDP and erastin. Figure S10. Representative UV-Vis absorption spectra of QS-4 (20 µm) under different solutions. Figure S11. Cell viabilities of HeLa cells incubated with different concentration of QS-4. S-11
Figure S12. Mean fluorescence intensity of the HeLa cells incubated with QS-4 nanodot (20 µm) at different times (15 min, 30 min, 1 h, 2 h, and 4 h) quantified by flow cytometry. Figure S13. HPLC-MS based identification of the oxidized product QSO-4 in the cell lysate after incubating the QS-4 with ferroptotic cells. S-12
Green Red Overlay Erastin CDDP PTX Figure S14. Confocal imaging of HeLa cells treated with Erastin (1 µm), CDDP (10 µm) and PTX (1 µm) for 24 h then further incubated with QS-4 (20 µm) for another 1 h. The scale bar is 50 µm. Figure S15. Confocal imaging of HeLa cells treated with fluorescent nanodots. Top: HeLa cells were cultured in normal medium without any treatment. Middle: HeLa cells were incubated with erastin (1 µm) for 24 h, then cells were treated with QS-4 nanodots (20 µm) for 1 h. Bottom: HeLa cells were pretreated with anti HO-1 sirna, then cells were incubated with erastin and treated with QS-4 nanodots (20 µm) for 1 h. The scale bars are 25 µm. S-13
Figure S16. Flow cytometry analysis of HeLa cells stained by QS-4. (a) HeLa cells without any treatment. (b) HeLa cells were treated with erastin (1 µm) for 24 h and then incubated with QS-4 (20 µm) for 30 min. (c) HeLa cells were treated with QS-4 (20 µm) alone for 1 h. Figure S17. Confocal imaging of HeLa cells treated with different concentrations of erastin (from 50 nm to 2 µm) and then incubated with QS-4 (20 µm) for another 1 h. S-14
Figure S18. (a) Confocal imaging of tumor sections. The blue channel indicated nucleus staining by DAPI. The green channel signal was collected in the region of 510-550 nm with laser excitation at 488 nm. The red channel signal was collected in the region of 595-650 nm with laser excitation at 488 nm. (b) Fluorescence intensity ratios of different fluorescence channels for both control and ferroptosis samples. The scale bar is 25 µm. Data are presented as average ±standard error (n = 3), and the statistical significance level is *p<0.05. S-15
Figure S19. 1 H-NMR spectrum of QS-1 (400 MHz, CDCl 3 ). Figure S20. 13 C-NMR spectrum of QS-1 (100 MHz, CDCl 3 ). S-16
Figure S21. 1 H-NMR spectrum of QS-2 (400 MHz, CDCl 3 ). Figure S22. 13 C-NMR spectrum of QS-2 (100 MHz, CDCl 3 ). S-17
Figure S23. 1 H-NMR spectrum of QS-3 (400 MHz, CDCl 3 ). Figure S24. 13 C-NMR spectrum of QS-3 (100 MHz, CDCl 3 ). S-18
Figure S25. 1 H-NMR spectrum of QS-4 (400 MHz, CDCl 3 ). Figure S26. 13 C-NMR spectrum of QS-4 (100 MHz, CDCl 3 ). S-19
Table S1. Quantum yields and Stokes shifts of QS-1, QS-2, QS-3, and QS-4. Samples QS-1 QS-2 QS-3 QS-4 Quantum Yield (%) 6.06 7.46 6.25 8.47 Stokes Shift (nm) 140 nm 143 nm 133 nm 134 nm Maxium excitation wavelength (nm) Maxium emission wavelength (nm) 441 nm 447 nm 453 nm 461 nm 581 nm 590 nm 586 nm 595 nm Extinction coefficient 1.46 10 4 2.15 10 4 1.57 10 4 2.21 10 4 S-20