Supporting information for Dual-site Controlled and Lysosome-targeted ICT-PET-FRET Fluorescent Probe for Monitoring ph Changes in Living Cells Baoli Dong, Xuezhen Song, Chao Wang, Xiuqi Kong, Yonghe Tang and Weiying Lin* Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Biological Science, University of Jinan, Jinan, Shandong 250022, P. R. China. Fax: (+) 86-531-82769031, E-mail: weiyinglin2013@163.com S1
Table of contents page 1. synthesis..s3-s5 2. pka value calculation...s5 3. Figure S1.....S5 4. Figure S2-S3....S6 5. Figure S4-S5.....S7 6. Figure S6-S7......S8 7. Figure S8-S9.....S9 8. Figure S10...S10 9. 1 H and 13 C NMR spectra.....s11-s17 S2
Synthesis of compound 2. A mixture of 7-hydroxycoumarin-3-carboxylic acid (1.030 g, 5 mmol), t-butyl piperazine-1-carboxylate (931.25 mg, 5 mmol), EDCl (1.342 g, 7.5mmol), HOBt (1.6788 g, 12.5 mmol) and DIEA (0.5 ml) in DMF(2.5 ml) was stirred for 12 h at room temperature. Then 5 ml water was added into the mixture and extracted with CH 2 Cl 2, washed three times with water, dried over Na 2 SO 4 and evaporated under reduced pressure to provide crude compound 1. Then, compound 1 was dissolved into 15mL CH 2 Cl 2, and 3mL CF 3 COOH was dropwise added to the solution under vigorous stirring condition. After stirred at room temperature for 2 h, the reaction mixture was concentrated under vacuum, and then the crude product was purified by silica column chromatography (CH 2 Cl 2 : MeOH = 15:1) to afford compound 2 (959 mg,70% ). 1 H NMR (DMSO, 400 MHz):δ 8.15 (s, 1H), 7.61-7.63 (d, J=8.8 Hz, 1H), 6.85-6.88 (dd, 1H), 6.78-6.79 (d, 1H),4.14 (s,1h), 3.79 (s, 2H), 3.59 (s, 2H), 3.40 (s, 1H), 3.10-3.17 (m, 4H). 13 C NMR (DMSO, 100 MHz): δ 164.2, 163.1, 158.6, 156.2, 144.7, 131.0, 119.1, 114.1, 111.1, 102.5, 49.0, 43.9, 43.4, 43.0. HRMS (ESI): m/z calculated for C 14 H 14 N 2 O 4 275.1026 [M+H] +, found: 275.1032. Synthesis of CN-1. Compound 2 (274 mg, 1 mmol), propionic acid (74 mg, 1 mmol), HOBT (337.5 mg, 2.5 mmol), EDCl (270 mg, 1.5 mmol) and DIEA (0.3 ml) were mixed in DMF (3.0 ml) and stirred overnight for 12h. Then 5 ml water was added into the mixture and extracted with CH 2 Cl 2, washed three times with water, dried over Na 2 SO 4 and evaporated under reduced pressure. Purification by column chromatography (CH 2 Cl 2 : MeOH = 10:1) afford CN-1 (100 mg, 0.30 mmol) as a white solid. Yelid: 30%. 1 H NMR (DMSO, 400 MHz): δ 10.85 (s,1h), 8.10-8.12 (d, J= 5.6 Hz, 1H), 7.59-7.61 (d, J= 8.8 Hz,1H), 6.83-6.86 (dd,1h), 6.77-6.78 (d, J= 1.6 Hz,2H), 3.34-3.61 (m, 8H), 2.31-2.37 (m, 2H), 0.98-0.99 (m, 3H). 13 C NMR (DMSO, 100 MHz): δ 172.0, 164.1, 162.8, 158.5, 156.1, 144.0, 143.8, 131.0, 120.0, 114.2, 111.2, 102.5, 55.4, 46.7, 44.9, 26.0, 9.7. HRMS (ESI): m/z calculated for C 17 H 28 N 2 O 5 331.1288 [M+H] +, found: 331.1289; 353.1108 [M+Na] +, found: 353.1104. Synthesis of compound 3. A mixture of 4-bromo-1,8-naphthalic anhydride( 1.10 g, 4 mmol) and 3-aminopropanoic acid (712 mg, 8 mmol) in 10 ml ethanol was refluxed for 3 h. After cooled to room temperature, the precipitated solid was filtered and washed with ethanol and dried under a vacuum to give an gray solid of compound 3 (1.253 g, 90%). 1 H NMR (DMSO, 400 MHz): δ 8.45-8.51 (m, 2H), 8.25-8.27 (d, J= 7.6 Hz, 1H), 8.15-8.17 (d, J= 7.6 Hz, 1H), 7.94 (t, 1H), 4.20-4.24 (t, 2H), 2.57-2.61 (t, 2H). 13 C NMR (DMSO, 100 MHz): δ 173.0, 163.1, 163.0, 132.9, 131.9, 131.7, 131.2, 130.0, 129.6, 129.1, 128.4, 123.0, 122.1, 36.4, 32.6. HRMS (ESI): m/z calculated for C 15 H 10 BrN 2 O 4 347.9866 [M+H] +, found: 347.9882. S3
Synthesis of compound 4. Compound 3 (1.038 g, 3mmol), 4-(2-aminoethyl)morpholine (1.560 g,12 mmol ), Pd(OAc) 2 (100.8 mg,0.45 mmol), BINAP (186.6 mg, 0.3 mmol) and Cs 2 CO 3 (1.755 g, 5.4 mmol) in DMSO (15 ml) were stirred at 80 C under N 2 atmosphere for 8 h. After cooled to room temperature, the reaction mixture was purified by column chromatography (CH 2 Cl 2 :MeOH = 5:1) to afford compound 4 (714.6 mg, 60%) as an orange powder. 1 H NMR (DMSO, 400 MHz): δ 8.66-8.68 (d, J=8.4 Hz,1H), 8.41-8.43 (d, J=7.2 Hz, 1H), 8.24-8.26 (d, J=8.4 Hz, 1H), 7.68-7.74 (d, 2H), 6.79-6.81 (d, J=8.8 Hz, 1H), 4.20-4.22 (t, 2H), 3.59-3.60 (m, 4H), 3.51-3.52 (m, 4H), 2.64-2.67 (t, 2H), 2.51-2.53 (m, 4H). 13 C NMR (DMSO, 100 MHz): δ 164.1, 163.2, 151.0, 134.6, 131.0, 129.8, 129.0, 124.7, 122.2, 120.5, 108.1, 104.2, 66.7, 66.5, 56.6, 55.6, 53.8, 53.4, 36.7, 35.9, 34.6. HRMS (ESI): m/z calculated for C 21 H 23 N 3 O 5 398.1710 [M+H] +, found: 398.1729. Synthesis of CN-pH. Compound 2 (274mg, 1mmol), compound 4 (397 mg, 1 mmol), EDCl (270 mg,1.5mmol), HOBt (337.5 mg, 2.5 mmol) and DIEA (0.2 ml) in DMF (2.5 ml) were stirred for 12 h at room temperature. Then 5 ml water was added into the mixture and extracted with CH 2 Cl 2, washed three times with water, dried over Na 2 SO 4 and evaporated under reduced pressure. Purification by column chromatography (CH 2 Cl 2 : MeOH=10:1) afford CN-pH (359.15 mg, 55%) as a yellow solid. 1 H NMR (DMSO, 400 MHz): δ 8.66-8.68 (d, J=8.4 Hz, 1H), 8.41-8.43 (d, J=6.8 Hz, 1H), 8.24-8.26 (d, J=8.4 Hz,1H), 8.10 (s, 1H), 7.69-7.73 (m, 2H), 7.56-7.58 (d, J=8.4 Hz, 1H), 6.79-6.81 (d, J=8.4 Hz, 2H), 6.72 (s, 1H), 4.19-4.23 (t, 2H), 3.59-3.61 (t,6h), 3.51-3.52 (m, 6H), 3.12-3.17 (q, 2H), 2.69-2.73 (m,4h), 2.41-2.43 (t,2h), 2.27-2.31 (m,2h). 13 C NMR (DMSO, 100 MHz): δ 170.3, 169.4, 164.2, 164.1, 163.3, 163.2, 158.6, 156.4, 151.1, 144.1, 134.7, 134.6, 131.2, 130.8, 129.9, 129.0, 128.9, 124.8, 122.4, 122.3, 120.6, 114.6, 110.7, 108.1, 104.4, 102.6, 66.7, 57.9, 56.6, 53.8, 36.6, 36.4, 36.2, 34.3, 31.7. HRMS (ESI): m/z calculated for C 35 H 35 N 5 O 8 654.2558 [M+H] +, found: 654.2665; 676.2378 [M+Na] +, 676.2456. Synthesis of compound 5. 4-Bromo-1,8-naphthalic anhydride( 1.10 g, 4 mmol) and propan-1-amine (1.18 g, 20 mmol) in 10 ml ethanol were refluxed for 3 h. After the mixture was cooled to room temperature, filtered, washed with cold ethanol and then dried to afford a gray solid of compound 5 (1.37 g, 90%). 1 H NMR (CDCl 3, 400 MHz): δ 8.67-8.69 (d, J=7.2 Hz, 1H), 8.58-8.60 (d, J=8.0 Hz, 1H), 8.43-8.45 (d, J=7.6 Hz,1H), 8.05-8.07 (d, J=8.0 Hz, 2H), 7.87-7.89 (t, 1H), 4.14-4.18 (m, 2H), 1.74-1,83 (m, 2H), 1.02-1.06 (t, 3H). 13 C NMR (CDCl 3, 100 MHz): δ 163.7, 163.6, 133.2, 132.0, 131.2, 131.1, 130.6, 130.2, 129.0, 128.1, 123.2, 122.3, 42.1, 21.4, 11.5. HRMS (ESI): m/z calculated for C 15 H 12 BrNO 2 318.0124 [M+H] +, found: 318.0132. S4
Synthesis of CN-2. Compound 5 (634 mg, 2 mmol), 4-(2-aminoethyl)morpholine (1.3 mg,10 mmol), Pd(OAc) 2 (67.2 mg,0.3 mmol), BINAP (124.4 mg, 0.2 mmol) and Cs 2 CO 3 (910 mg, 2.8 mmol) in DMSO (15 ml) were stirred at 80 C for 8 h under N 2 atmosphere. After cooled to room temperature, the reaction mixture was purified by column chromatography (CH 2 Cl 2 : MeOH=15:1) to afford CN-2 (588 mg, 80%) as a white powder. 1 H NMR (CDCl 3, 400 MHz): δ 8.58-8.60 (d, J=7.2 Hz, 1H), 8.44-8.46 (d, J=8.4 Hz, 1H), 8.16 (s,1h),7.63-7.67 (t,1h), 6.65-6.68 (d, J=8.4 Hz, 1H), 4.12-4.16 (m, 2H), 3.83 (s, 4H), 3.46-3.47 (m, 2H), 2.90 (s, 2H), 2.64 (s, 4H), 1.74-1.80 (m, 2H), 1.00-1.04 (t, 3H). 13 C NMR (CDCl 3, 100 MHz): δ 164.7, 164.2, 149.3, 134.4, 132.5, 131.1, 129.7, 128.0, 126.1, 124.9, 123.1, 120.4, 110.5, 104.4, 66.9, 55.9, 53.0, 41.7, 38.8, 21.5, 11.6. HRMS (ESI): m/z calculated for C 21 H 25 N 3 O 3 368.1969 [M+H] +, found: 368.2044. pka value calculation. The pka values of CN-1 and CN-2 were calculated using the Henderson-Hasselbalch equation: log [(I-I min )/(I max -I)] = ph - pka for CN-1, log[(i max -I)/(I-I min )] = ph - pka for CN-2. Where I is the observed fluorescence intensity, I max and I min are the corresponding maximum and minimum fluorescence intensity, respectively. Figure S1. Absorption spectra of 5 µm CN-1 (a) and CN-2 (b) at various ph in B-F buffer solution (5% MeOH). S5
(a) 900 (b) 120 Fluorescence Intensity 600 300 0 ph = 10.0 ph = 3.0 400 450 500 550 600 Wavelength/nm Fluorescence Intensity 100 80 60 40 20 0 ph = 3.0 ph = 10.0 450 500 550 600 650 700 Wavelength/nm Figure S2. Fluorescence spectra of 5 µm CN-1 (a, λ ex = 380 nm) and CN-2 (b, λ ex = 410 nm) at various ph in B-F buffer solution (5% MeOH). Figure S3. Highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy values of 4-aminonaphthalimide fluorophore and 4-ethylmorpholine calculated by Gaussian 09 software using DFT theory with 6-31G basis sets. S6
900 Fluorescence Intensity 750 600 450 300 150 0 ph = 10.0 ph = 3.0 400 450 500 550 600 650 Wavelength / nm Figure S4. Fluorescence spectra of the mixture of 5 µm CN-1 and 5 µm CN-2 at various ph in B-F buffers (5% MeOH). λ ex = 380 nm 900 Fluorescence Intensity 750 600 450 300 150 0 454 nm 530 nm 3 4 5 6 7 8 9 10 ph Figure S5. Fluorescence intensity at 454 nm and 530 nm of the mixture of 5 µm CN-1 and 5 µm CN-2 at various ph in B-F buffers (5% MeOH). λ ex = 380 nm S7
Distribution / % 100 80 60 40 20 form a form b form c 0 3 4 5 6 7 8 9 10 ph Figure S6. The distribution of form a, c and c at various ph calculated by MarvinSketch software (version 14.12.15.0) using macro mode and dynamic acid/base prefix at 298 K. I 530 /I 454 8 6 4 2 0 blank CaCl 2 MgCl 2 ZnCl 2 CuCl 2 FeSO 4 KF Fe 2 (SO 4 ) 3 KI KNO 2 NaS 2 Na 2 SO 3 VC Cys Hcys GSH NO H 2 O 2 Figure S7. Ratios (I 530 /I 454 ) of fluorescence intensity of CN-pH (5 µm) coexisting with diverse species in B-R buffer solution (5 % MeOH) at ph 4.5 ( ), ph 5.5 ( ) and ph 7.4 ( ). GSH, 1 mm; the other species, 100 µm. λ ex = 380 nm. S8
25 20 I 530 /I 454 15 10 5 0 1 2 3 4 5 6 7 8 9 10 Time/min Figure S8. Time-dependent ratio intensities (I 530 /I 454 ) of CN-pH in B-F buffer solutions (5% MeOH) at ph 4.5 ( ), 5.5 ( ) and 7.4 ( ). λ ex = 380 nm. 28 21 I 530 /I 454 14 7 0 1 2 3 4 5 6 Figure S9. ph reversibility study of CN-pH (5 µm) in B-R buffer solution (5% MeOH) between ph 5.5 and 7.4. λ ex = 380 nm. S9
Figure S10. Survial of HeLa cells in the presence of CN-pH at various concentrations measured using MTS assay. The cells were incubated with CN-pH for 24 h. S10
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