Supporting Information

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1 Supporting Information Two-Photon Absorbing Dyes with Minimal Autofluorescence in Tissue Imaging: Application to in vivo Imaging of Amyloid- Plaques with a Negligible Background Signal Dokyoung Kim,, Hyunsoo Moon,, Sung Hoon Baik,, Subhankar Singha,, Yong Woong Jun, Taejun Wang, Ki Hean Kim,, * Byung Sun Park, Junyang Jung, Inhee Mook-Jung,, * and Kyo Han Ahn, * Department of Chemistry, Center for Electro-Photo Behaviors in Advanced Molecular Systems, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyungbuk , Republic of Korea Department of Biochemistry and Biomedical Sciences, College of Medicine, Seoul National University, 13 Daehak-Ro, Jongro- Gu, Seoul , Republic of Korea Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang, Gyungbuk , Republic of Korea Department of Anatomy and Neurobiology, School of Medicine, Biomedical Science Institute, Kyung Hee University, 26 Kyungheedae-Ro, Dongdaemun-Gu, Seoul 13-71, Republic of Korea Table of Contents 1. Scheme S2 2. Supporting Figures S2 1) Figure S1. Fluorescence emission spectra of acedan and 1 4 in. S2 2) Figure S2. Fluorescence emission spectra of 1 4 in various solvents. S3 3) Figure S3. Fluorescence emission spectra of acedan and 1 in various solvents. S3 4) Figure S4. Two-photon action spectra of 1, 5, acedan in various solvents. S3 5) Figure S5. UV/Vis absorption spectra of 1 4 in PBS buffer. S4 6) Figure S6. UV/Vis absorption spectra of 5 in PBS buffer. S4 7) Figure S7. UV/Vis absorption and emission spectra of 5 in various solvents. S5 8) Figure S8. Plot of fluorescence intensity of 5 in various phs. S5 9) Figure S9. In vitro assay of 5 with A plaques. S5 1) Figure S1. TPM images of tissues with acedan and 1 at a lower laser power. S6 11) Figure S11. TPM autofluorescence images of tissues depending on the emission windows and excitation wavelengths S7 12) Figure S12. TPM images of tissues with acedan and 1 at different emission windows and excitation wavelengths. S8 13) Figure S13. Imaging for site-specificity of 1 in living cells. S8 14) Figure S14. In vivo photo-bleaching test of 5. S9 15) Figure S15. Cell viability test of 1 and 5. S9 4. NMR spectra S1 5. HRMS spectra S15 S1

2 1. SCHEME Scheme S1. Synthesis of -extended acedan derivatives 1 5. Reagent and Conditions: 2-cyclohexen-1-one, 1,4-diazabicyclo [2.2.2]octane (DABCO), H 2 O/1,4-dioxane (1:2, v/v), sonication, 45 5 C, 48 h, 38%. 2-cyclopenten-1-one, 1,4- diazabicyclo[2.2.2]octane (DABCO), H 2 O/1,4-dioxane (1:1, v/v), sonication, 45 5 C, 48 h, 22%. (c) 3-buten-2-one, MgI 2, tetramethylethylenediamine (TMEDA), 4-dimethylaminopyridine (DMAP), MeOH, 25 C, 24 h, 28%. (d) Propargyl bromide, K 2 CO 3, DMF, 25 C, 12 h, quant. (e) CuI, CH 3 CN, 82 C, 71%. (f) malononitrile, CuI, Et 3 N, CH 3 CN, 75 C, 4 h, 8%. 2. SUPPORTING FIGURES Acedan Figure S1. Comparison of fluorescence emission spectra of 1, 2, 3, 4, and acedan in acetonitrile. All the measurements were carried out at 25 ºC at 1 M concentration by irradiating at the maximum absorption wavelength of each compound PhCH 3 Dioxane EtOAc CH 2 Cl 2 CH 3 CN DMF DMSO PBS 9% /PBS 8 6 PhCH 3 Dioxane EtOAc CH 2 Cl 2 CH 3 CN 4 2 DMF DMSO PBS 9% /PBS S2

3 (c) 8 6 PhCH 3 Dioxane EtOAc CH 2 Cl 2 CH 3 CN 4 2 DMF DMSO PBS 9% /PBS (d) PhCH 3 Dioxane EtOAc CH 2 Cl 2 CH 3 CN 4 2 DMF DMSO PBS 9% /PBS Figure S2. Fluorescence emission spectra of 1, 2, (c) 3, and (d) 4 in various solvents. All the measurements were carried out at 25 C in each of the solvents at 1 M concentration except PBS buffer (1 M). The fluorescence emission spectra were measured at an each absorption maximum CH 2 Cl 2 CH 3 CN CH 2 Cl 2 CH 3 CN Figure S3. Fluorescence emission spectra of acedan and 1, measured in different solvents (dichloromethane, acetonitrile, and ethanol) at 1 M concentration in each solvent and 25 ºC by irradiating at the maximum absorption wavelength in each solvent. F TP (GM) Toluene DMF F TP (GM) Ethanol (c) 12 F TP (GM) water Acetonitrile Figure S4. Two-photon action spectra of 1 (1 M) in toluene, DMF, and ethanol. Two-photon action spectra of 5 (1 M) in ethanol. (c) Two-photon action spectra of acedan (1 M) in water and acetonitrile. The two-photon action cross-section values were determined by following the literature (Lee, S. K.; Yang, W. J.; Choi, J. J.; Kim, C. H.; Jeon, S. J.; Cho, B. R. Org. Lett. 25, 7, 323. Kim, H. M.; Kim, B. R.; Hong, J. H.; Park, J. S.; Lee, K. J.; Cho, B. R. Angew. Chem., Int. Ed. 27, 46, 7445). Note: We were able to obtain two-photon action cross-section (TPACS) value only up to 94 nm, because of instrumental limitations. S3

4 Absorbance Absorbance Absorbance Absorbance Absorbance M 2 M 5 M 1 M M 2 M 5 M 1 M (c) M 2 M 5 M 1 M (d) M 2 M 5 M 1 M Figure S5. UV/Vis absorption spectra of 1, 2, (c) 3, and (d) 4 measured at different concentrations (1, 2, 5, and 1 M) in PBS buffer (1 mm, ph 7.4, containing <1% DMSO) at 25 ºC M 5 M 1 M 15 M 2 M Figure S6. UV/Vis absorption spectra of 5 measured at different concentrations (1, 5, 1, 15, and 2 M) in PBS buffer (1 mm, ph 7.4 containing <2% DMSO) at 25 ºC. S4

5 Absorbance PhCH 3 Dioxane EtOAc CH 2 Cl 2 DMF DMSO PBS 35 PhCH Dioxane EtOAc CH 2 Cl 2 DMF DMSO PBS Figure S7. UV/Vis absorption and fluorescence emission spectra of 5. All the measurements were carried out at 25 C and 1 M concentration by irradiating at the maximum absorption wavelength in each of the solvents ph Figure S8. Plot of fluorescence intensity change of 5 (1 M) at different phs, based on three different measurements. Excitation wavelength was 5 nm Concentration of A Concentration of compound 5 [ M] Figure S9. Plot of fluorescence intensity change of dye 5 (1 M) in the presence of different concentrations ( 5 M) of amyloid- peptide 42-mer (A 42). The intensity represents the peak height at the maximum emission wavelength, from three different measurements. Excitation wavelength was 5 nm. Saturation binding curves of A 42 aggregates (15 M) with different concentration of compound 5 (.1 2 M). S5

6 1 Control Acedan Control Brain Liver Kidney Spleen Lung Brain Liver Kidney Spleen Lung Figure S1. TPM images of tissues of different mouse organs, acquired with acedan and 1, respectively, at a middle depth (~2 μm) of the sectioned tisses (thickness ~5 μm) at a lower laser power of ~6 mw at the focal point. Images of tissues of brain, liver, kidney, spleen, and lung from the left, stained with acedan (1 M) and at 37 C for 1 min, observed under excitation at 78 nm. The tissue images obtained under otherwise the same conditions except by staining with 1 and irradiating at 9 nm. Dyeuntreated tissues were used as controls. Scale bar is 5 m. S6

7 Emission Channel 2 (> 56 nm) Emission Channel 1 (< 56 nm) TP λ ex Brain Liver Kidney Spleen Lung 78 nm 9 nm 78 nm 9 nm Channel 1 (TP ex = 78 nm) Channel 1 (TP ex = 9 nm) Channel 2 (TP ex = 78 nm) Channel 2 (TP ex = 9 nm) Brain Liver Kidney Spleen Lung (c) 1 9 TP ex = 78 nm TP ex = 9 nm Brain Liver Kidney Spleen Lung Figure S11. TPM autofluorescence images of tissues (dye-untreated) of different mouse organs depending on the emission S7

8 1 + MitoTracker 1 + LysoTracker 1 Acedan windows (channel 1, < 56 nm region; channel 2, > 56 nm region) and excitation wavelengths (78 nm and 9 nm). Laser power was ~6 mw at the focal point. Scale bar is 5 µm. The relative autofluorescence intensities of the respective TPM images shown in, depending on the channel and excitaiton wavelength. (c) The relative autofluorescence intensities of the merged TPM images collected from both the channels depending on the excitation wavelength, 78 nm and 9 nm, respectively. Brain Liver Kidney Spleen Lung TP λ ex = 78 nm; Emission Channel 1 (< 56 nm) 5 μm TP λ ex = 9 nm; Emission Channel 2 (> 56 nm) Acedan Brain Liver Kidney Spleen Lung Figure S12. TPM images of tissues of different mouse organs, acquired with acedan and 1 (1 μm each), respectively, at different emission windows and excitation wavelengths. Laser power ~6 mw at the focal point. The emission intensities of acedan and dye 1 treated tissues were collected at channel 1 (< 56 nm region) with excitation at 78 nm and channel 2 (> 56 nm region) with excitation at 9 nm, respectively. The relative fluorescence intensity data for of the TPM images shown in. Bright field Green Channel Red Channel Merged Images Intensity plots 3 25 Lysotracker ROI ( M) ROI ( M) Mitotracker 1 Figure S13. Bright field and confocal images of HeLa cells co-labeled with dye 1 (1 mm) and LysoTracker Green (1 nm) (upper row) or MitoTracker Green (2 nm) (lower row) for 3 min at 37 C. The fluorescence emission were collected in the green channel (48 5 nm) and red channel (62 7 nm), respectively. Excitation wavelengths were 45 nm for dye 1 and 488 nm for LysoTraker green and MitoTracker Green. The intensity plots represent the fluorescence intensities along with the solid lines (white S8

9 Cell Viability (%) Cell Viability (%) colored) in the corresponding images. These data show that dye 1 is not specifically localized in lysosome, mitochondria, and nucleus, but mainly localized in cytosol. Figure S14. In vivo photobleaching data: TPM images of 5 A plaque complex before and after irradiation at the depth of 5 m. The images were observed under excitation at 1, nm for 35 min and with approximately 5 mw laser power at the focal point. Scale bar is 5 m. Relative fluorescence intensity plot of before and after irradiation for 35 min, respectively Control Control M 2 M 5 M 1 M 2 M 5 M Figure S15. Cell viability of compound 1 and compound 5, measured with SHSY5Y cell lines. Cell cultures with DMSO solution were used as controls. S9

10 4. NMR SPECTRA Compound 1 S1

11 Compound 2 S11

12 Compound 3 S12

13 Compound 4 S13

14 Compound 5 S14

15 5. HRMS SPECTRA Compound 1 Compound 2 S15

16 Compound 3 Compound 4 S16

17 Compound 5 S17