Tuning the π-bridge of quadrupolar triarylborane chromophores for one- and two-photon excited fluorescence imaging of lysosomes in live cells

Abstract

A series of tetracationic quadrupolar chromophores containing three-coordinate boron π-acceptors linked by different π-bridges, namely 4,4'-biphenyl, 2,7-pyrene, 2,7-fluorene, 3,6-carbazole and 5,5'-di(thien-2-yl)-3,6-diketopyrrolopyrrole, were synthesized. While their neutral precursors 1-5 displayed highly solvatochromic fluorescence, the water-soluble tetracationic target molecules 1M-5M, did not, but their emission colour could be tuned from blue to pink by changing the π-bridge. Compound 5M, containing the diketopyrrolopyrrole bridge, exhibits the most red-shifted absorption and emission maxima and the largest two-photon absorption cross-section (4560 GM at 740 nm in MeCN). Confocal laser scanning fluorescence microscopy studies in live cells confirm localization of the dye at the lysosome. Moreover, the low cytotoxicity, and high photostability of 5M combined with two-photon excited fluorescence imaging studies demonstrate its excellent potential for lysosomal imaging in live cells.

Scheme 1. Previously reported quadrupolar chromophores for two-photon excited fluorescence (TPEF) imaging of lysosomes.–

Scheme 2. Previously reported quadrupolar chromophore for TPEF imaging 2TM and quadrupolar target molecules 1M–5M for cell imaging.

Scheme 3. Synthesis of compounds 1M–5M. (a) [Pd₂(dba)₃] (dba = trans,trans-dibenzylideneacetone), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (SPhos), KOH, toluene, H₂O, 85 °C; (b) MeOTf, CH₂Cl₂; (c) TfOH, MeOH.

Fig. 1. Absorption and emission spectra (top) and pictures of the solutions under UV irradiation (bottom) of 1 (left) and 5 (right) in various solvents (hexane: black, toluene: blue, diethylether: red, dichloromethane: green, MeCN: orange).

Fig. 2. DFT (CAM-B3LYP/6-31G(d))-calculated relevant orbitals for 1 and 5. Hydrogen atoms are omitted for clarity. Surface isovalue: ± 0.03 [ea₀^–3]^1/2. Orbital energy differences are not direct approximations of excitation energies. They are provided here for comparison between the compounds.

Fig. 3. Absorption (top, left) and emission spectra (top, right) of 1M–5M in water. Compound 2M was dissolved in 10% MeCN in water. Absorption and emission spectra of 1M in various solvents (EtOH: black, MeCN: blue, H₂O: red) (bottom, left) and picture of the solutions of 1M–5M in MeCN under UV irradiation.

Fig. 4. DFT (CAM-B3LYP/6-31G(d))-calculated relevant orbitals for 1M–4M. Hydrogen atoms are omitted for clarity. Surface isovalue: ± 0.03 [ea₀^–3]^1/2.

Fig. 5. DFT (CAM-B3LYP/6-31G(d))-calculated frontier orbitals for 5M. Hydrogen atoms are omitted for clarity. Surface isovalue: ± 0.03 [ea₀^–3]^1/2.

Scheme 4. Non-boron containing analogues; 1A was measured in acetone,2A in toluene,3A in ethyl acetate,4A in MeCN and 5A in DMSO.

Fig. 6. One-photon absorption (blue) and two-photon absorption spectra (red) of 1M (first row, left), 2M (first row, right), 3M (second row, left), 4M (second row, right) and 5M (third row, left) in MeCN.

Fig. 7. Two-photon absorption spectra (left) and two-photon brightness (σ₂Φ_f) (right) of 1M–5M in MeCN.

Fig. 8. Side view of the geometries of the DFT-optimized S₀ states of 1M–5M at the B3LYP/6-31G(d) level of theory. Atom colour code: carbon (grey), boron (pink), sulphur (yellow), nitrogen (blue), oxygen (red). Hydrogen atoms are omitted for clarity.

Fig. 9. Co-staining experiment of HeLa cells with 5M and LysoTracker™ Green. The cells were loaded with 5M (500 nM, 2 h) and LysoTracker™ Green (100 nM, 20 min) under 5% CO₂ at 37 °C. Fluorescence images of 5M (top, left, λ_ex = 559 nm; λ_em = 570–670 nm) and LysoTracker™ Green (top, right, λ_ex = 473 nm; λ_em = 490–540 nm). The merged image of the bright field image and both fluorescence images (bottom, left), and the correlation plot of the intensities (bottom, right, R_r = 0.81), show co-localization of the dye 5M in the lysosomes. Scale bar: 20 μM.

Fig. 10. Confocal microscope images of HeLa cells at 37 °C with 5M (500 nM). Merged bright field image with fluorescence image (λ_ex = 559 nm; λ_em = 570–670 nm) between 10 min and 120 min after staining. Scale bar: 20 μm.

Fig. 11. Comparison of the repeated fluorescence images of HeLa cells stained with (a) 5M and (b) LysoTracker™ Red under irradiation with a confocal laser at 561 nm (WLL, output power 70%, AOTF 2%). Each number indicates the number of recorded confocal images. The rectangle (bottom, left in each picture) is the cutout of the image at n = 1. Scale bar: 2 μm. (c) Plots of integrated fluorescence intensities (I) relative to the initial value (I₀) as a function of the number of recorded images.

Fig. 12. (a) Two-photon excited fluorescence and (b) bright field images of HeLa cells stained with 5M (500 nM). The TPEF image was recorded under excitation at 720 nm (AOTF 38%) using an HyD detector with a bandpass filter 650/50 and an HC Fluotar L 25×0.95 W VISIR objective. Scale bar: 20 μm.