A Zero Cross-Talk Ratiometric Two-Photon Probe for Imaging. of Acid ph in Living Cells/Tissues and Early Detection of Tumor.

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1 A Zero Cross-Talk Ratiometric Two-Photon Probe for Imaging of Acid ph in Living Cells/Tissues and Early Detection of Tumor in Mouse Model Di Xu, a, Yinhui Li, b,, * Chung-Yan Poon, a Hei-ga Chan, a Hung-Wing Li, a, * and Man Shing Wong a, * a Department of Chemistry, Hong Kong Baptist University, Hong Kong, , SAR China b Key Laboratory for Green Organic Synthesis and Application of Hunan Province, Key Laboratory of Environmentally Friendly Chemistry, Application of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, , China. mswong@hkbu.edu.hk; hwli@hkbu.edu.hk; yinhuili16@163.com. Contents: (1) Synthesis of PSIOH, SIOH and PSIBOH. S2 (2) Spectroscopic data and calculation results S4 (3) Experimental for imaging. S9 (4) Ratiometric data analysis and standard curve. S13 (5) References S13 S1

2 1. Synthesis of PSIOH,SIOH and PSIBOH. Compound 2 and 3 were prepared by the modified literature method. 1 Synthesis of PSIOH, SIOH and PSIBOH is shown below. a 74% 1 Cl OH b 85% 2 Cl OH Br CHO R c d 63% R 57% 3 4 R SIOH HO Cl e 78% R O PSIOH f g 4 63% 85% O R R = CH 3 OCH 2 CH 2 OCH 2 CH 2 PSIBOH Reagents and Conditions: a, ClCH 2 CH 2 OH, sealed tube; b, ClCH 2 CH 2 OH, sealed tube; c, (i) n-buli, THF, -78 C (ii) DMF; d, 1, EtOH, reflux; e, aoh, r.t.; f, 2, EtOH, reflux; g, aoh, r.t. Scheme S1. The synthetic route of SIOH, PSIOH and PSIBOH. Synthetic Procedures 1-(2-Hydroxyethyl)-2,3,3-trimethyl-3H-indol-1-ium chloride (1). A solution mixture of 2,3,3- trimethyl-3h-indole (0.30 g, 2.0 mmol) and 2-chloroethan-1-ol (0.16 g, 2.0 mmol) was heated to reflux in a sealed tube overnight. After cooling to room temperature, methanol was removed under vacuum. The resulting solid was washed with acetone and dried to afford 1 (0.42 g) in 87% yield. 1 H MR (400 S2

3 MHz, DMSO-d 6 ) δ (m, 1H), (m, 1H), (m, 2H), 4.61 (t, J = 4.0 Hz, 2H), 3.86 (t, J = 4.0 Hz, 2H), 2.85 (s, 3H), 1.55 (s, 6H). 13 C MR (100 MHz, DMSO-d 6 ) δ 197.7, 141.8, 141.2, 129.3, 128.8, 123.4, 115.7, 57.7, 54.3, 50.4, 22.1, (2-Hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indol-3-ium chloride (2). A solution mixture of 1,1,2-trimethyl-1H-benzo[e]indole (0.42 g, 2.0 mmol) and 2-chloroethan-1-ol (0.16 g, 2.0 mmol) was heated to reflux in a sealed tube overnight. After cooling to room temperature, methanol was removed under vacuum. The resulting solid was washed with acetone and dried to afford 2 (0.49 g) in 87% yield. 1 H MR (400 MHz, DMSO-d 6 ) δ 8.38 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 9.2 Hz, 1H), (m, 1H), (m, 1H), 4.27 (t, J = 5.2 Hz, 2H), 3.93 (t, J = 5.2 Hz, 2H), 2.94 (s, 3H), 1.78 (s, 6H). 13 C MR (100 MHz, DMSO-d 6 ) δ 197.7, 138.7, 136.8, 133.0, 130.5, 129.7, 128.3, 127.2, 127.2, 123.4, 113.6, 57.9, 55.6, 50.6, 21.6, (E)-1-(2-Hydroxyethyl)-2-(2-(9-(2-(2-methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-3,3-dimethyl- 3H-indol-1-ium (SIOH). A mixture of 3 (0.6 g, 2 mmol) and 1 (0.5 g, 2 mmol) was dissolved in absolute EtOH under nitrogen. The mixture was heated to reflux overnight. After cooling down to room temperature, the organic solvent was removed. The residue was purified by precipitation from methanol to afford SIOH (0.55 mg) in 57% yield. 1 H MR (400 MHz, DMSO-d 6 ) δ 9.13 (s, 1H), 8.65 (d, J = 16.0 Hz, 1H), (m, 1H), 8.28 (d, J = 8.0 Hz, 1H), (m, 2H), (m, 2H), 7.74 (d, J = 8.0 Hz, 1H), (m, 3H), (m, 1H), (m, 2H), (m, 2H), 3.94 (d, J = 4.0 Hz, 2H), 3.84 (t, J = 4.0 Hz, 2H), (m, 2H), (m, 2H), 3.08 (s, 3H), 1.86 (s, 6H). 13 C MR (100 MHz, DMSO-d 6 ) δ 182.5, 155.2, 143.9, 143.4, 141.2, 128.8, 128.7, 128.6, 126.9, 125.9, 124.9, 123.1, 122.9, 122.3, 120.7, 120.6, 115.0, 110.9, 110.8, 109.9, 71.3, 69.8, 68.8, 58.7, 58.1, 51.9, 48.9, 43.1, HRMS (MALDI-TOF) m/z Calcd for C 31 H 35 2 O Found [M + ]. 9a-(2-(9-(2-(2-Methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-9,9-dimethyl-2,3,9,9atetrahydrooxazolo[3,2-a]indole (PSIOH). A mixture of SIOH (1 g, 2 mmol) and aoh (0.2 g, 5 mmol) was dissolved in CH 2 Cl 2 and stirred at ambient temperature overnight. The resulting solution was washed with water and brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed. The crude product was purified by silica gel chromatography to afford PSIOH (0.8 g) in 78% yield. 1 H MR (400 MHz, CDCl 3 ) δ 8.17 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.62 (dd, J = 8.5 Hz, J = 1.5 Hz, 1H), (m, 3H), (m, 1H), (m, 1H), (m, 2H), (m, 1H), 6.84 (d, J = 7.8 Hz, 1H), 6.32 (d, J = 15.8 Hz, 1H), 4.52 (t, J = 6.2 Hz, 2H), 3.87 (t, J S3

4 = 6.2 Hz, 2H), (m, 2H), (m, 4H), (m, 2H), 3.33 (s, 3H), 1.51 (s, 3H), 1.24 (s, 3H). 13 C MR (100 MHz, CDCl 3 ) δ 151.0, 141.2, 140.7, 140.2, 133.2, 128.1, 127.8, 126.1, 124.8, 123.4, 123.1, 122.6, 121.8, 120.6, 119.5, 119.0, 112.3, 110.4, 109.3, 72.2, 71.1, 69.5, 63.8, 59.3, 50.4, 48.2, 43.5, 28.7, HRMS (MALDI-TOF) m/z Calcd for C 31 H 34 2 O Found [M + ]. 10a-(2-(9-(2-(2-Methoxyethoxy)ethyl)-9H-carbazol-3-yl)vinyl)-11,11-dimethyl-8,9,10a,11- tetrahydrobenzo[e]oxazolo[3,2-a]indole (PSIBOH). A mixture of 4 (0.6 g, 2 mmol) and 2 (0.6 g, 2 mmol) was dissolved in absolute EtOH under nitrogen. The mixture was heated to reflux overnight. After cooling down to room temperature, the organic solvent was removed. The residue and aoh (0.2 g, 5 mmol) were dissolved in CH 2 Cl 2 and stirred at ambient temperature overnight. The resulting solution was washed with water and brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed. The residue was purified by silica gel chromatography to afford PSIBOH (0.9 g) in 85% yield. 1 H MR (400 MHz, CDCl 3 ) δ 8.25 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), (m, 4H), 7.35 (d, J = 7.6 Hz, 1H), (m, 1H), (m, 2H), 6.46 (d, J = 15.6 Hz, 1H), 4.54 (t, J = 6.0 Hz, 2H), (m, 4H), (m, 2H), (m, 2H), (m, 2H), 3.36 (s, 3H), 1.91 (s, 3H), 1.50 (s, 3H). 13 C MR (100 MHz, CDCl 3 ) δ 147.8, 141.1, 140.6, 133.5, 130.9, 130.5, 130.2, 129.5, 129.2, 128.0, 126.4, 126.1, 124.8, 123.4, 123.1, 122.9, 122.3, 120.5, 119.4, 119.0, 114.2, 110.7, 109.2, 72.1, 71.0, 69.4, 63.5, 59.2, 50.3, 49.8, 43.4, 26.7, HRMS (MALDI-TOF) m/z Calcd for C 35 H 36 2 O Found [M + ]. 2. Spectroscopic data Table S1. Photophysical properties of PSIOH, SIOH and PSIBOH. cmpds Solvent λ abs /nm d λ em /nm e Φ f pk a g PSIOH Buffer a DMSO SIOH Buffer b DMSO PSIBOH Buffer a DMSO Buffer c a The deprotonated forms were tested in 0.2 M ah 2 PO 4 solution (ph = 9.2) containing 10% DMSO. b The protonated forms were tested in 0.1 M a 2 HPO 4 solution (ph = 4.5) containing 10% DMSO. c The protonated forms were tested in 0.2 M Citric Acid solution and 0.2 M a 2 HPO 4 solution (ph = 3.4) con- S4

5 taining 10% DMSO. d λ abs of the one-photon absorption spectra in nm. e λ em of the one-photon absorption spectra in nm. f Fluorescence quantum yield. g pk a values measured by one photon mode. Figure S1. Fluorescence intensity of PSIOH (10 µm) in (a) MeC and (b) DMSO-buffer (1:9, v/v) at ph 7.0 excited at 320 nm over a period of 60 min. Figure S2. Absorption spectra of PSIOH (10 µm) at different ph values in DMSO-buffer solution (1:9, v/v). S5

6 Figure S3. (a) Absorption spectra of PSIBOH (10 µm) at different ph values in DMSO-buffer solution (1:9, v/v). (b) Emission spectra of PSIBOH (10 µm) at different ph values in DMSO-buffer solution (1:9, v/v) (λ ex = 380 nm). (c) Emission intensity ratio (F 619 /F 430 ) changes over the ph range of The red line represents the nonlinear fitting of the experimental data by Origin software. S6

7 Figure S4. DFT optimized molecular structures of PSIOH and SIOH as well as PSIBOH and its corresponding acidic form and their respective HOMO and LUMO plots in water. Figure S5. (a) The time courses of fluorescence intensity of PSIOH (10 µm) at different ph values (4.49, 6.64, and 8.04, respectively). (b) Changes in fluorescence intensity of PSIOH between ph 4.49 (λ ex = 602 nm) and 9.18 (λ ex = 435 nm). λ ex = 320 nm. (c) Fluorescence intensity changes of 10 µm PSIOH in DMSO/buffer (2/1 v/v) at ph 4.49, 6.64 and 8.04 in the presence of diverse metal ions and bioactive small molecules, respectively. 1, blank; 2, Mg 2+ (50 µm); 3, Pb 2+ (50 µm); 4, Cd 2+ (50 µm); 5, Ca 2+ (50 µm); 6, Hg 2+ (50 µm); 7, Co 2+ (50 µm); 8, K + (50 µm); 9, Cu 2+ (50 µm); 10, Ba 2+ (50 µm); 11, Ag + (50 µm); 12, Mn 2+ (50 µm); 13, Zn 2+ (50 µm); 14, Fe 3+ (50 µm); 15, H + 4 (50 µm); 16, H 2 O 2 ; 17, Aspartic acid (50 µm); 18, Cysteine (50 µm); 19, Phenylalanine (50 µm); 20, Arginine (50 µm); 21, S7

8 Lysine (50 µm); 22, Tryptophan (50 µm); 23, Glutamic acid (50 µm); 24, Methionine (50 µm); 25, Leucine (50 µm); 26, Glutamine (50 µm); 27, Valine (50 µm); λ ex = 320 nm (c) The time courses of fluorescence intensity of PSIOH (10 µm) at different ph values (4.49, 6.64, and 8.04, respectively) PSIOH Cytotoxicity log concentration (M) Figure S6. Cell viability values (%) estimated by MTT proliferation. HeLa cells were incubated with different concentrations of PSIOH at 37 C for 24 h. 140 TP Fluorescence Intensity / au ph 4.49 ph Wavelength / nm Figure S7. The changes of the TP fluorescence spectra of PSIOH (350 µm) at different ph values in DMSO-buffer (1:9, v/v) (λ ex = 750 nm). The TP fluorescence ph titrations of PSIOH excited at 750 nm were also carried out and the results are shown in Figure S7. The spectral emission wavelengths and behaviour upon titration are relatively comparable to those obtained by one-photon excitation shown in Figure 2a. Because of the poor detector sensitivity below 400 nm of the femtosecond laser system, it could not afford highly sensitive and accu- S8

9 rate measurements of the emission intensity of PSIOH below 400 nm (UV region) where the signal fluctuated. However, PSIOH works well with the two-photon confocal microscope (Olympus FV MPE) for two photon imaging studies, all the images were obtained from blue channel at nm and red channels at nm nm with λ ex = 750 nm. Active Cross-Section (GM) SPIOH Wavelength/nm Figure S8. Two-photon action cross-section spectrum of PSIOH measured in PBS (20 mm, ph 7.0) containing 20% DMSO. 3. Experimental for imaging. Two-Photon Properties and Fluorescence Microscopy Imaging in Cells. Fluorescence images of HeLa cells were obtained using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope. The PSIOH-loaded cells were incubated at 37 C for 15 min in high K + ion buffer (120 mm KCl, 30 mm acl, 1 mm CaCl 2, 0.5 mm MgSO 4, 1 mm ah 2 PO 4, 5 mm glucose, 20 mm HEPES) with various ph values in the presence of 10 µm nigericin, in which the use of nigericin was a standard protocol for homogenizing the ph of cells and culture medium to perform the intracellular ph calibration experiment. To explore the effect of oxidative stress on ph fluctuations, HeLa cells 1) were treated with redox active substance (H 2 O 2 /0.1 mm, glucose/10 mm, and H 4 Cl/1.0 mm), 2) kept under 20%, 10%, 5% and 0% oxygen content for 3 h, and 3) irradiated by UV light for 10 min and 30 min at 37 C, respectively. Then, two-photon confocal fluorescence imaging of HeLa cells was performed S9

10 under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope, with a mode-locked titanium-sapphire laser source (120 fs, pulse width, 80 MHz repetition rate) set at wavelength of 750 nm. Figure S9. Ratiometric TP excited images of PSIOH in HeLa cells at various ph: blue channel, emission at nm (TPM; λ ex = 750 nm); red channel, emission at nm (TPM; λ ex = 750 nm).the fourth column is the pseudo-coloured ratiometric images constructed from blue and red channels. S10

11 Figure S10. TP microscopic images of PSIOH loaded HeLa cells for 0.5 h at 37 C and then irradiated by UV light for 10 min and 30 min. The images were obtained from blue channel at nm and red channels at nm with λ ex = 750 nm. The fourth column is the pseudo-coloured ratiometric images constructed from blue and red channels. Figure S11. The 3D reconstitution of confocal XYZ scanning micrographs from 32 confocal Z-scan UCL imaging sections at different penetration depths both in normal and cancer liver tissues. S11

12 (A) (B) 0 min 15 min 30 min 45 min Mean Intensity min 15 min 30 min 45 min 0.85 (C) Wavelength / nm Ratio min Figure S12. (A) TP fluorescence images of PSIOH in HeLa cells monitoring over time. The HeLa cells were incubated with PSIOH for 30 min at ph 7.4, then imaged using TP fluorescence microscopy at different time points. Images were obtained from blue channel at nm and red channel at nm with λ ex = 750 nm, respectively. (B) The corresponding two-photon excited fluorescence spectra of PSIOH in HeLa cells over times with λ ex = 750 nm in DMSO-buffer. (C) The time course of fluorescence profile of PSIOH in HeLa cells was represented by a plot of I red /I blue ratio as a function of time. 4. Ratiometric data analysis and standard curve All the images were analyzed by an image processing software, Image J. 2 The fluorescence signals of the cells were obtained by measuring the net fluorescence intensity at the same position of the cell images obtained by both red and blue channels (emission at nm and nm, respectively). et Intensity = [(1 1 square pixel of individual position of cell) (1 1 square pixel of background area of the cell image)]. The ratio was obtained by dividing the average of the net intensity of cell images at red channel by that at blue channel. A ph standard curve was established by correlating the I red /I blue (average net intensity obtained at red channel /average net intensity obtained at blue channel) against dif- S12

13 ferent ph. The ph of cells at different spots of the tissue slice was determined by fitting I red /I blue in the standard curve Equation y = a + b Adj. R-Squ Value Standard Er B Intercept B Slope Ratio Figure S13. Fluorescence profile of PSIOH inside the HeLa cells in buffer at ph represented by a plot of I red /I blue as a function of cultural media ph. Blue channel, emission at nm red channel, emission at nm; TPM; λ ex = 750 nm. ph 5. References: 1. Yang,W. G.; Wong, Y.; g, O. T. W.; Bai, L. P.; Kwong, D. W. J.; Ke, Y.; Jiang, Z. H.; Li, H. W.; Yung, K. K. L.; Wong, M. S. Angew. Chem. Int. Ed. 2012, 51, Kardash, E.; Bandemer, J.; Raz, E. at. Protocols. 2011, 6, S13