聚集诱导发射光笼分子的设计合成及原位光激活成像研究

doi:10.6023/A23100457

摘要/Abstract

通过将1,2,4,5-四氮嗪基团引入到聚集诱导发射(AIE)荧光团上, 制备了两个光激活的荧光小分子化合物TPA-Tz1和TPA-Tz2. 光激活后, TPA-Tz1和TPA-Tz2的荧光强度分别增强了167倍和100倍. 光激活的原理是光照导致四氮嗪基团转化为氰基, 从而恢复荧光发射. 通过密度泛函理论(DFT)计算, TPA-Tz1的猝灭机理为能量转移至暗态(ETDS). TPA-Tz1的激活产物TPA-CN1具有独特的AIE特点, 其聚集态分子间发生的电荷转移是导致聚集态荧光蓝移的主要原因. 通过溶剂挥发法获得的TPA-Tz1晶体结构揭示了其内部复杂的堆叠结构, 氢键和C—H…π作用代替π-π相互作用是同时维持晶格稳定和AIE特性的重要原因. TPA-Tz1具有良好的生物相容性, 其最高的细胞抑制率仅有25.3%. 因此, TPA-Tz1在细胞水平和多细胞生物秀丽隐杆线虫体内实现了原位的光激活成像.

关键词: 光笼, 聚集诱导发光, 密度泛函理论, 光激活荧光, 荧光成像

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

引用此文

赵玉强, 张霞, 杨芸如, 朱立平, 周莹. 聚集诱导发射光笼分子的设计合成及原位光激活成像研究[J]. 化学学报, 2024, 82(3): 265-273.

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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图/表 8

图1. (a) TPA-Tz1和TPA-Tz2的合成路线示意图; (b) TPA-CN1和TPA-CN2的合成路线示意图; (c) TPA-Tz1和TPA-Tz2的光反应示意图

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

图2. (a) 溶解在乙醇中的TPA-Tz1 (10 μmol/L)的荧光发射光谱; (b) TPA-Tz1在478 nm处荧光强度随时间的增长曲线; (c) 溶解在乙醇中的TPA-Tz2 (10 μmol/L)的荧光发射光谱; (d) TPA-Tz2在475 nm处荧光强度随时间的增长曲线激发波长: 365 nm; 插图为激活前后溶液的照片

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

图3. (a) 365 nm光源不同时间下TPA-Tz1 (10 μmol/L)的荧光发射光谱; (b) 365 nm光源下TPA-Tz1在478 nm处荧光强度随时间的增长曲线; (c) 395 nm光源不同光照时间下TPA-Tz1 (10 μmol/L)的荧光发射光谱; (d) 395 nm光源下TPA-Tz1在478 nm处荧光强度随时间的增长曲线

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

图4. (a) TPA-Tz1光激活前后的高分辨质谱; TPA-Tz1、光激活的TPA-Tz1和TPA-CN1的紫外吸收光谱(b)和荧光光谱(c) (溶剂: 无水乙醇, 浓度: 10 μmol/L); TPA-Tz1的S3 (d)和S11 (f)空穴-电荷等值面分布; TPA-CN1的S1 (e)和S8 (g)空穴-电荷等值面分布绿色等值面表示电荷, 蓝色等值面表示空穴

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

图5. (a) TPA-CN1和TPA-Tz1优化后的分子结构示意图; (b) TPA-Tz1的前线轨道电子云分布图和相应能量; (c) TPA-Tz1的激发态信息和其对应的前线轨道电子云分布图; (d) TPA-CN1激发态信息和其对应的前线轨道电子云分布图

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

图6. (a)~(c)不同含水量体系中TPA-CN1的荧光光谱; (d)不同含水量体系中TPA-CN1的荧光照片; (e)不同含水量体系中TPA-CN1的最大荧光强度; (f) TPA-CN1的S1空穴-电荷等值面分布绿色等值面表示电荷, 蓝色等值面表示空穴

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

图7. TPA-Tz1沿着x (a), y (b), z (c)不同方向的堆积模式; (d) TPA-Tz1头对头的堆积模式和面之间距离; (e) TPA-Tz1晶体中的C—H…π作用; (f) TPA-Tz1晶体中的氢键

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

图8. (a) TPA-Tz1在HepG2细胞上的光激活成像照片; (b) TPA-Tz1在秀丽隐杆线虫体内的光激活成像照片; (c) 40 μmol/L TPA-Tz1的细胞抑制率; (d) 细胞光激活成像照片的平均荧光强度, 比例尺: 10 μm; (e) 线虫光激活成像照片的平均荧光强度, 比例尺: 200 μm (**: P<0.01, ***: P<0.001)

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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