Design and Synthesis of Aggregation-Induced Emission Photocage Molecules for In Situ Photoactivation Imaging Studies

doi:10.6023/A23100457

Abstract

Two photoactivated fluorescent small molecule compounds, TPA-Tz1 and TPA-Tz2, were synthesized by incorporating a 1,2,4,5-tetrazine group into an aggregation-induced emission (AIE) fluorogen. Upon continuous UV light exposure, the fluorescence intensity of TPA-Tz1 and TPA-Tz2 increased by 167-fold and 100-fold, respectively. The photoactivation mechanism of the TPA-Tz1 solution was confirmed using high-resolution mass spectrometry, both before and after illumination, as part of standard verification procedures. The photoactivation mechanism involves the conversion of the tetrazine group to the cyanide group upon light exposure, leading to the restoration of fluorescence emission. Density functional theory (DFT) calculations revealed that the quenching mechanism of TPA-Tz1 involves energy transfer to dark states (ETDS). The fluorescence assessment of photoactivated TPA-CN1 products in solutions with varying water contents revealed distinct AIE characteristics. Specifically, a decline in fluorescence was evident at water contents below 50%, attributable to the twisted intramolecular charge transfer (TICT) effect. Conversely, as water content increased from 60% to 95%, a conspicuous blue shift and enhanced fluorescence were observed. Analysis of the excited states of its dimer, employing time-dependent density functional theory (TDDFT) hole charge analysis, underscored that charge transfer within the aggregated state predominantly accounted for the observed blue shift. The crystal structure of TPA-Tz1, obtained using a solvent evaporation method, unveiled its intricate internal stacked structure. The replacement of π-π interactions by hydrogen bonds and C—H…π interactions played a crucial role in maintaining both lattice stability and AIE. Moreover, cytotoxicity assessments conducted across diverse cell lines demonstrated the biocompatibility of TPA-Tz1, revealing a maximum cell inhibition rate of only 25.3%. Ultimately, photoactivated fluorescence imaging experiments were conducted on cells and Caenorhabditis elegans, utilizing a laser confocal imager for cells and a fluorescence microscope for the organism. The findings illustrated the capability of TPA-Tz1 to facilitate in situ photoactivation imaging at both the cellular and in vivo levels in multicellular organisms.

Key words: photocage, aggregation-induced emission, density functional theory, photoactivatable fluorescence, fluorescence imaging

Cite this article

Yu-Qiang Zhao, Xia Zhang, Yunru Yang, Liping Zhu, Ying Zhou. Design and Synthesis of Aggregation-Induced Emission Photocage Molecules for In Situ Photoactivation Imaging Studies[J]. Acta Chimica Sinica, 2024, 82(3): 265-273.

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Fig. & Tab. 8

Figure 1. (a) Schematic representation of the synthesis route of TPA-Tz1 and TPA-Tz2; (b) schematic representation of the synthesis route of TPA-CN1 and TPA-CN2; (c) schematic representation of the photochemical reaction of TPA-Tz1 and TPA-Tz2

Figure 2. (a) Fluorescence emission spectra of TPA-Tz1 (10 μmol/L) dissolved in ethanol; (b) time-dependent growth curve of fluorescence intensity at 478 nm for TPA-Tz1; (c) fluorescence emission spectra of TPA-Tz2 (10 μmol/L) dissolved in ethanol; (d) time-dependent growth curve of fluorescence intensity at 475 nm for TPA-Tz2 Excitation wavelength: 365 nm; inset shows photos of the solution before and after activation

Figure 3. (a) Fluorescence emission spectra of TPA-Tz1 (10 μmol/L) at different time points under a 365 nm light source; (b) time-dependent growth curve of fluorescence intensity at 478 nm for TPA-Tz1 under a 365 nm light source; (c) fluorescence emission spectra of TPA-Tz1 (10 μmol/L) at different time points under a 395 nm light source; (d) time-dependent growth curve of fluorescence intensity at 478 nm for TPA-Tz1 under a 395 nm light source

Figure 4. (a) High-resolution mass spectra of TPA-Tz1 before and after photoactivation; UV-absorption (b) and fluorescence spectra (c) of TPA-Tz1, photoactivated TPA-Tz1, and TPA-CN1 (solvent: anhydrous ethanol, concentration: 10 μmol/L); hole-charge isosurface distribution of TPA-Tz1's S3 (d) and S11 (f); hole-charge isosurface distribution of TPA-CN1's S1 (e) and S8 (g) Green isosurfaces represent charge, while blue isosurfaces represent holes

Figure 5. (a) Schematic representation of the optimized molecular structures of TPA-CN1 and TPA-Tz1; (b) electron cloud distribution and corresponding energy of the frontier orbitals of TPA-Tz1; (c) excited state information of TPA-Tz1 and the corresponding electron cloud distribution of its frontier orbitals; (d) excited state information of TPA-CN1 and the corresponding electron cloud distribution of its frontier orbitals

Figure 6. (a)~(c) Fluorescence spectra of TPA-CN1 in different water content systems; (d) fluorescence photos of TPA-CN1 in different water content systems; (e) maximum fluorescence intensity of TPA-CN1 in different water content systems; (f) isosurface distribution of hole-charge for TPA-CN1's S1 state Green isosurfaces represent charge, while blue isosurfaces represent holes

Figure 7. (a), (b) and (c) Stacking modes of TPA-Tz1 in different directions along the x, y and z axes; (d) head-to-head stacking mode of TPA-Tz1 and interplanar distance; (e) C—H…π interactions in the TPA-Tz1 crystal; (f) hydrogen bonds in the TPA-Tz1 crystal

Figure 8. (a) Photoactivation imaging of TPA-Tz1 on HepG2 cells; (b) photoactivation imaging of TPA-Tz1 inside C. elegans; (c) cell inhibition rate at 40 μmol/L TPA-Tz1; (d) average fluorescence intensity of cell photoactivation imaging, scale bar: 10 μm; (e) average fluorescence intensity of nematode photoactivation imaging, scale bar: 200 μm (**: P<0.01, ***: P<0.001)

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