Highly Sensitive Fluorescence Molecular Switch for the Ratio Monitoring of Trace Change of Mitochondrial Membrane Potential

Transcription

1 Highly Sensitive Fluorescence Molecular Switch for the Ratio Monitoring of Trace Change of Mitochondrial Membrane Potential Caixia Wang, Ge Wang, Xiang Li, Kui Wang, Jing Fan, Kai Jiang,, Yuming Guo, Hua Zhang *, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education; Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals; School of Chemistry and Chemical Engineering, Henan Normal University; Xinxiang Medical University; Key Laboratory for Yellow River and Huai River Water Environment and Pollution Control, Ministry of Education; School of Environment, Henan Normal University; Xinxiang, 453, P.R. China. Corresponding Author Tel/Fax: Contents 1. Synthetic procedures of probe and intermediates 2. Spectral properties of probe to sodium-dodecyl sulphate 3. Quantum calculations 4. Specific selectivity of probe for sodium-dodecyl sulphate 5. The photostability of probe and MitoTracker Red CMXRos 6. Cell viability of probe 7. The spectrum extraction of different concentrition with catechol in living cancer cells 8. ph effect on the emission spectra of the TPP-CY/TPP-SP 9. Incubation and staining of living cells with probe Attached Figure: 1 H and 13 C NMR spectra of probe and intermediates. Supporting Information-Video 1: The dynamic imaging of HepG2 cells that were treated with the membrane-potential uncoupler 3-chlorophenylhydrazone (CCCP,.5mM) with AVT (*. avi). S-1

2 1. Synthetic procedures of probe and intermediates Figure S1. Synthetic procedures of probe and intermediates. (1) 2,3,3-Trimethylindolenine, iodoethane, reflux, 85%; (2) HNO 3, CH 3 COOH, room temperature, 96%; (3) Compound a, 5-nitrosalicylaldehyde, reflux, 74%; (4) Compound SP, triphenylphosphine, 4%; Synthesis of Compound SP Compound a was prepared by the literature methods Figure S1. To a solution of (2.4 mmol) in anhydrous ethanol (1 ml) was stirred at 8 C for 4 h, and the Compound a in 1 ml was dropwise added into, simultaneously. The mixture was cooled to room temperature, and precipitate solids, washed with little anhydrous ethanol. Yield: 74 %; mp: 111 C; 1 H NMR (6 MHz, DMSO) δ 8.67 (s, 1H), 8.1 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 16.4 Hz, 1H), (m, 3H), 7.32 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.3 Hz, 1H), 7.7 (d, J = 9. Hz, 1H), 1.41 (s, 6H), 1.23 (s, 4H). 13 C NMR (1 MHz, DMSO) δ: 183.9, 154., 147.1, 131.5, 128., 126.4, 125.9, 124.3, , 12.6, 52.4, 4., 39.6, 39.4, 37.4, ESI-MS: m/z calcd for C 2 H 19 BrN 2 O 3 : , found: Synthesis of the probe Compound TPP (1. mmol), Compound SP (3.5 mmol) and K 2 CO 3 (1.2 mol) in THF (1 ml) were refluxed under nitrogen for 8 h. After the mixture was cooled to room temperature, concentrated hydrochloric acid (45 ml) was added dropwise under nitrogen. The solvent was evaporated and the crude product was purified by column chromatography using CH 2 Cl 2 / petroleum ether (3:5-1:9) as the eluent to give Compound probe. Yield: 53 %; mp: 167 C; 1 H NMR (6 MHz, DMSO) δ: (m, 18H), (m, 3H), (m, 1H), 5.18 (d, J = Hz, 1H), (m, 2H), 3.93 (d, J = 5.4 Hz, 2H), 3.51 (s, 1H), 2.27 (t, J = 6.8 Hz, 4H), (m, 2H). 13 C NMR (1 MHz, DMSO) δ: 183.9, 154.1, 147.1, 135.8, 135.3, 134.9, 131.4, 13.8, 13.5, 13., 128.2, 125.9, 124.3, , 121.7, 117.8, 117.6, 117.2, 52.4, 4., 39.4, 38.7, ESI-MS: m/z calcd for C 38 H 34 N 2 O 3 P + : , found: S-2

3 2. Spectral properties of probe to sodium-dodecyl sulphate (SDS) 3 a d I g SDS(mM) s b SDS(mM) e SDS(mM) Absorbance h I 563 I c I 563 = C SDS R 2 = C SDS /mm f I 563 = C SDS R 2 = CSDS/mM TPP-SP+SDS (6. mm) TPP-CY TPP-SP Time / s Figure S2. Probe (3.3 µm) recongnize the different concentrations of SDS. (a) Probe (C = 3.3 µm, ε = 14375) appears a low fluorescence (Φfree state =.3) of black line, and its fluorescence quantum yield Φ =.11 with 8.82 mm SDS of red line. (b) Fluorescence intensity of probe response to SDS (-4.2 mm). (c) The linear fluorescent response of probe to SDS (-4.2 mm) in secondary water, I 663 = C SDS (R 2 =.99). (d) Emission spectra of 3.3 µm probe mixtures with different concentrations of SDS ( mm); (e) Emission spectra of 3.3 µm probe mixtures with different concentrations of SDS ( mm); (f) The linear fluorescent response of probe to SDS (-5.1 mm) in secondary water. I 563 = C SDS (R 2 =.99). (g) Reponse time of probe to SDS (4.2 mm). (h) the absorbance spectre of probe to SDS (6. mm). S-3

4 3. Quantum calculations The Gaussian 9 suite was used to obtain the data of quantum chemical calculations. The density functional theory (DFT) with Becke s three-parameter hybrid exchange function used to perform the geometry optimizations of probe and SP. C(9)-O(12): length:1.435; angles: C(9)-O(15):Length: 1.429; angles: Figure S3. Geometry optimizations of the probe and SP. 4. Specific selectivity of probe for sodium-dodecyl sulphate (SDS) I 563 /I a SDS/Zeph 5 b ZEPH SDS Zeta Potential/mv -14 c Zeta Potential = C SDS R 2 = CSDS/mM Figure S4. (a) The influence of common coexisting substances for probe under 54nm. 1, Glycyl-DL-phenylalanine (.1 mm); 2, Lysine (.1 mm); 3, DL-Threonine (.1 mm); 4, Glutamine (Gln,.1 mm); 5, Cystine (.1 mm); 6, Arginine (.1 mm); 7, Glycyl-DL- tyrosine (.1 mm); 8, DL-methionine (.1 mm); 9, D-cysteine (.1 mm); 1, Glycine (.1 mm); 11, DL-leucine (.1 mm); 12, Serine (Ser,.1 mm); 13, Homocysteine (.1 mm); 14, Dithiothreitol (DTT,.1 mm); 15, Glutamic acid (.1 mm); 16, 6-Hydroxypurine (.1 mm); 17, L-aspartic acid (.1 mm); 18, SDS (.1 mm). (b) The fluorescence intensity of probe respond to cationic surfactant (Benzalkonium Chloride, Zeph) and anion surfactant (SDS). (c) The linear fluorescent response of zeta potential to SDS (R 2 = -.99). S-4

5 5. The photostability of probe and MitoTracker Red CMXRos Time/s Figure S5. The green line is probe and the red line is MitoTracker Red CMXRos. The fluorecence intencity were detected by LS Cell viability of probe HepG2, MCF-7, CHO, 293T, L929 and HeLa cell were prepared for cell viability studies in 96 well plates (1 1 5 cells per well that were incubated in 1 µl). The cells were incubated for an additional 24 h in different concentrations of probe. Subsequently, 2 µl of 5 mg/ml 3 - (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co. USA) was added into each well, followed by further incubation for 4 h at 37 C. After DMEM was removed, DMSO (15 µl /well) was added to dissolve the reddish blue crystals of formazan. Optical density was determined on a Microplate Reader (Spectra Max M5, Molecular Devices) at 57 nm with subtraction of the absorbance of the cell free blank volume at 63 nm. The results from the six individual experiments were averaged. The relative cell viability (%) was calculated using the following equation: cells viability (%) = (OD dye ODK dye ) / (OD contral -ODK contral ) 1 Survival Rate / % 1..5 MCF-7 CHO HePG2 HeLa 293T L929. 1X 1X Figure S6. Cell viability. Data were obtained from replicate experiments (n = 5). S-5

6 7. The spectrum extraction of different concentrition with catechol in living cancer cells. 1 a 25 b Figure S7. The spectrum extraction of living cells that were stained by catechol with the same time (36 h), but different concentration (a:.1 mm and b: 2. mm) in the apoptotic model. 8. ph effect on the emission spectra of the TPP-CY/TPP-SP ph Figure S8. The fluorescence intensities in PBS buffer as a function of ph ( ). Solution of ph 5.:.2 mol/l KH 2 PO 3, ph was adjusted to 5. by.2 mol/l NaOH; Solution of ph 6.5: KH 2 PO 3.68 g, ph was adjusted to 5. by.1 mol/l NaOH; Solution of ph 7.: KH 2 PO 3.68 g, ph was adjusted to 5. by.1 mol/l NaOH; Solution of ph 7.8: (1) Na 2 HPO g was diluted by H 2 O (.5 L), ph was adjusted to 5. by.1 mol/l NaOH; (2) NaH 2 PO g was diluted by H 2 O (.1 L), then, 91.5ml of solution; (3) and L of solution; (4) Solution of ph 8.8:.2 mol/l KH 2 PO 3, ph was adjusted to 8.8 by.1 mol/l NaOH; Solution of ph 1.3:.1 mol/l KH 2 PO 3, ph was adjusted to 1.3 by.1 mol/l NaOH; Solution of ph 11.7:.1mol/L KH 2 PO 3, ph was adjusted to 11.7 by.1 mol/l NaOH. S-6

7 9. Incubation and staining of living cells with probe Figure S9. The cells were stained by different concentration (.5,.1,.23 and 3.3 µm) of probe at 5. min. S-7

8 Attached Figure: 1 H and 13 C NMR spectra of probe and intermediates S-8

9 S-9

10 Length and angles of probe: C(1)-C(2) C(1)-C(6) C(1)-H(76) C(2)-C(3) C(2)-H(75) C(3)-C(4) C(3)-H(78) C(4)-C(5) C(4)-N(7) C(5)-C(6) C(5)-C(8) C(6)-H(77) N(7)-C(9) N(7)-C(25) C(8)-C(9) C(8)-C(1) C(8)-C(11) C(9)-O(12) C(9)-C(16) C(1)-H(48) C(1)-H(49) C(1)-H(5) C(11)-H(45) C(11)-H(46) C(11)-H(47) O(12)-C(13) O(12)-Lp(79).6.6 O(12)-Lp(8) C(13)-C(14) C(13)-C(17) C(14)-C(15) C(14)-C(2) C(15)-C(16) C(15)-H(7) C(16)-H(72) C(17)-C(18) C(17)-H(71) C(18)-C(19) C(18)-H(68) C(19)-C(2) C(19)-N(21) C(2)-H(69) N(21)-O(22) N(21)-O(23) P(24)-C(26) P(24)-C(27) P(24)-C(28) P(24)-C(29) C(25)-C(26) C(25)-H(73) C(25)-H(74) C(26)-H(66) C(26)-H(67) S-1

11 C(27)-C(35) C(27)-C(39) C(28)-C(3) C(28)-C(34) C(29)-C(4) C(29)-C(44) C(3)-C(31) C(3)-H(65) C(31)-C(32) C(31)-H(63) C(32)-C(33) C(32)-H(62) C(33)-C(34) C(33)-H(61) C(34)-H(64) C(35)-C(36) C(35)-H(6) C(36)-C(37) C(36)-H(58) C(37)-C(38) C(37)-H(57) C(38)-C(39) C(38)-H(56) C(39)-H(59) C(4)-C(41) C(4)-H(55) C(41)-C(42) C(41)-H(53) C(42)-C(43) C(42)-H(52) C(43)-C(44) C(43)-H(51) C(44)-H(54) C(1)-C(2) C(1)-C(6) C(1)-H(76) C(2)-C(3) C(2)-H(75) C(3)-C(4) C(3)-H(78) C(4)-C(5) C(4)-N(7) C(5)-C(6) C(5)-C(8) C(6)-H(77) N(7)-C(9) N(7)-C(25) C(8)-C(9) C(8)-C(1) C(8)-C(11) C(9)-O(12) C(9)-C(16) C(1)-H(48) C(1)-H(49) C(1)-H(5) S-11

12 C(11)-H(45) C(11)-H(46) C(11)-H(47) O(12)-C(13) O(12)-Lp(79).6.6 O(12)-Lp(8) C(13)-C(14) C(13)-C(17) C(14)-C(15) C(14)-C(2) C(15)-C(16) C(15)-H(7) C(16)-H(72) C(17)-C(18) C(17)-H(71) C(18)-C(19) C(18)-H(68) C(19)-C(2) C(19)-N(21) C(2)-H(69) N(21)-O(22) N(21)-O(23) P(24)-C(26) P(24)-C(27) P(24)-C(28) P(24)-C(29) C(25)-C(26) C(25)-H(73) C(25)-H(74) C(26)-H(66) C(26)-H(67) C(27)-C(35) C(27)-C(39) C(28)-C(3) C(28)-C(34) C(29)-C(4) C(29)-C(44) C(3)-C(31) C(3)-H(65) C(31)-C(32) C(31)-H(63) C(32)-C(33) C(32)-H(62) C(33)-C(34) C(33)-H(61) C(34)-H(64) C(35)-C(36) C(35)-H(6) C(36)-C(37) C(36)-H(58) C(37)-C(38) C(37)-H(57) C(38)-C(39) C(38)-H(56) C(39)-H(59) S-12

13 C(4)-C(41) C(4)-H(55) C(41)-C(42) C(41)-H(53) C(42)-C(43) C(42)-H(52) C(43)-C(44) C(43)-H(51) C(44)-H(54) C(2)-C(1)-C(6) C(2)-C(1)-H(76) C(6)-C(1)-H(76) C(1)-C(2)-C(3) C(1)-C(2)-H(75) C(3)-C(2)-H(75) C(2)-C(3)-C(4) C(2)-C(3)-H(78) C(4)-C(3)-H(78) C(3)-C(4)-C(5) C(3)-C(4)-N(7) C(5)-C(4)-N(7) C(4)-C(5)-C(6) C(4)-C(5)-C(8) C(6)-C(5)-C(8) C(1)-C(6)-C(5) C(1)-C(6)-H(77) C(5)-C(6)-H(77) C(4)-N(7)-C(9) C(4)-N(7)-C(25) C(9)-N(7)-C(25) C(5)-C(8)-C(9) C(5)-C(8)-C(1) C(5)-C(8)-C(11) C(9)-C(8)-C(1) C(9)-C(8)-C(11) C(1)-C(8)-C(11) N(7)-C(9)-C(8) N(7)-C(9)-O(12) N(7)-C(9)-C(16) C(8)-C(9)-O(12) C(8)-C(9)-C(16) O(12)-C(9)-C(16) C(8)-C(1)-H(48) C(8)-C(1)-H(49) C(8)-C(1)-H(5) H(48)-C(1)-H(49) H(48)-C(1)-H(5) H(49)-C(1)-H(5) C(8)-C(11)-H(45) C(8)-C(11)-H(46) C(8)-C(11)-H(47) H(45)-C(11)-H(46) H(45)-C(11)-H(47) H(46)-C(11)-H(47) C(9)-O(12)-C(13) S-13

14 C(9)-O(12)-Lp(79) C(9)-O(12)-Lp(8) C(13)-O(12)-Lp(79) C(13)-O(12)-Lp(8) Lp(79)-O(12)-Lp(8) O(12)-C(13)-C(14) O(12)-C(13)-C(17) C(14)-C(13)-C(17) C(13)-C(14)-C(15) C(13)-C(14)-C(2) C(15)-C(14)-C(2) C(14)-C(15)-C(16) C(14)-C(15)-H(7) C(16)-C(15)-H(7) C(9)-C(16)-C(15) C(9)-C(16)-H(72) C(15)-C(16)-H(72) C(13)-C(17)-C(18) C(13)-C(17)-H(71) C(18)-C(17)-H(71) C(17)-C(18)-C(19) C(17)-C(18)-H(68) C(19)-C(18)-H(68) C(18)-C(19)-C(2) C(18)-C(19)-N(21) C(2)-C(19)-N(21) C(14)-C(2)-C(19) C(14)-C(2)-H(69) C(19)-C(2)-H(69) C(19)-N(21)-O(22) 9.74 C(19)-N(21)-O(23) O(22)-N(21)-O(23) C(26)-P(24)-C(27) C(26)-P(24)-C(28) C(26)-P(24)-C(29) C(27)-P(24)-C(28) C(27)-P(24)-C(29) C(28)-P(24)-C(29) N(7)-C(25)-C(26) N(7)-C(25)-H(73) N(7)-C(25)-H(74) C(26)-C(25)-H(73) C(26)-C(25)-H(74) H(73)-C(25)-H(74) P(24)-C(26)-C(25) P(24)-C(26)-H(66) P(24)-C(26)-H(67) C(25)-C(26)-H(66) C(25)-C(26)-H(67) H(66)-C(26)-H(67) P(24)-C(27)-C(35) P(24)-C(27)-C(39) C(35)-C(27)-C(39) P(24)-C(28)-C(3) P(24)-C(28)-C(34) S-14

15 C(3)-C(28)-C(34) P(24)-C(29)-C(4) P(24)-C(29)-C(44) C(4)-C(29)-C(44) C(28)-C(3)-C(31) C(28)-C(3)-H(65) C(31)-C(3)-H(65) C(3)-C(31)-C(32) C(3)-C(31)-H(63) C(32)-C(31)-H(63) C(31)-C(32)-C(33) C(31)-C(32)-H(62) C(33)-C(32)-H(62) C(32)-C(33)-C(34) C(32)-C(33)-H(61) C(34)-C(33)-H(61) C(28)-C(34)-C(33) C(28)-C(34)-H(64) C(33)-C(34)-H(64) C(27)-C(35)-C(36) C(27)-C(35)-H(6) C(36)-C(35)-H(6) C(35)-C(36)-C(37) C(35)-C(36)-H(58) C(37)-C(36)-H(58) C(36)-C(37)-C(38) C(36)-C(37)-H(57) C(38)-C(37)-H(57) C(37)-C(38)-C(39) C(37)-C(38)-H(56) C(39)-C(38)-H(56) C(27)-C(39)-C(38) C(27)-C(39)-H(59) C(38)-C(39)-H(59) C(29)-C(4)-C(41) C(29)-C(4)-H(55) C(41)-C(4)-H(55) C(4)-C(41)-C(42) C(4)-C(41)-H(53) C(42)-C(41)-H(53) C(41)-C(42)-C(43) C(41)-C(42)-H(52) C(43)-C(42)-H(52) C(42)-C(43)-C(44) C(42)-C(43)-H(51) C(44)-C(43)-H(51) C(29)-C(44)-C(43) C(29)-C(44)-H(54) C(43)-C(44)-H(54) S-15