
==== Front
Anal Chem
Anal Chem
ac
ancham
Analytical Chemistry
0003-2700
1520-6882
American Chemical Society

37495553
10.1021/acs.analchem.3c01504
Article
Proton-Coupled Photochromic Hemithioindigo: Toward Photoactivated Chemical Sensing and Imaging
Li Jing †#
Ma Xueqing †#
Wang Yifu †
Cheng Yu †
Qin Yuemin †
Zhai Jingying ‡
https://orcid.org/0000-0003-2629-8362
Xie Xiaojiang *†
† Department of Chemistry, Southern University of Science and Technology, Shenzhen 518055, China
‡ Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology, Shenzhen 518055, China
* Email: xiexj@sustech.edu.cn.
26 07 2023
08 08 2023
26 07 2024
95 31 1166411671
06 04 2023
08 07 2023
© 2023 The Authors. Published by American Chemical Society
2023
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

We report photoswitchable fluorescent hemithioindigos (HTIs) where the metastable E isomers were stabilized by the proton-bridged intramolecular hydrogen bond. Titration experiments and computational analysis indicated that the E isomers were much more basic than the Z isomers, which enabled photoactivated colorimetric and fluorescent pH response in solvents and polypropylene films. The HTIs exhibited reversibly switchable fluorescence with the Z isomers being the most fluorescent. Moreover, the HTIs were lysosomotropic and the kinetic fluorescence evolution during photoswitching was able to differentiate subcellular compartments with different pH. The combination of photoenhanced basicity, switchable fluorescence, and proton-coupled photochromism lay the groundwork for a broad range of chemical and biological applications.

National Natural Science Foundation of China 10.13039/501100001809 22274070 document-id-old-9ac3c01504
document-id-new-14ac3c01504
ccc-price
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pmcPhotoswitchable small molecules and fluorescent proteins are paving the way for cutting-edge technologies, including optogenetics,1,2 photopharmacology,3−6 optical information processing,7−9 energy conversion,10,11 super-resolution microscopy,12−15 and 3D printing.16 Among different photochromic and photoswitchable compounds, hemithioindigos (HTIs) are highly promising for chemical and biological applications due to their properties resulting from the geometric change.17 The photoswitching of HTIs can be mediated by a tunable spectrum of light in the visible and even near-infrared regions.18−20 In addition, HTIs exhibited other attractive properties such as ultrafast photoisomerization,21,22 excellent fatigue resistance,19 high light absorption,22 and photochromism.19

On the one hand, while excellent fundamental research on HTIs continues, the applications of this family of photoswitchable compounds just started to draw more attention recently. In previous works, Dube and coworkers pioneered the controlling of molecular motion,23−25 photoisomerization rate,22,26 and spectroscopic fine-tuning of HTIs.20,27 They also demonstrated HTI tweezers, which could relocate guest molecules under photoswitching.24 Also, the photoinduced geometry changes of HTIs were successfully relayed into the catalytic efficiency of hydrogen-bonding organocatalysts, achieving more than 10-fold modulation upon photoisomerization.28 Furthermore, Thorn-Seshold and coworkers designed photopharmaceutical HTIs to mimic colchicine structurally and successfully achieved the photo-controlled inhibition of the cytoskeletal protein tubulin with single-cell precision.29 In addition, HTI-containing peptides and nucleic acid binders were developed to regulate biological signaling,30,31 peptide folding,32,33 and nucleic acid recognition.34 Undoubtedly, more research is expected to fully realize the power of HTIs in different areas.

On the other hand, reversibly switchable fluorophores have drawn significant attention in fluorescence microscopy due to their application in super-resolution imaging and their enormous potential in multiplexed and high-contrast imaging.12,35−37 Multiplexed fluorescence imaging of up to 20 fluorophores became possible in a single emission channel based on different switching kinetics of photoswitchable proteins.38 However, high-contrast imaging based on synthetic photoswitches was mainly reported on spiropyran, which requires ultraviolet (UV) light and suffers from poor photostability.39 In fact, spiropyran is one of the most extensively studied and applied photoswitchable compounds.40 The ring-opened merocyanine form is not only fluorescent but also much more basic than the ring-closed spiro-form, which has led to impressive applications including photoelectric conversion and chemical sensing.10,41,42 The variety of photoswitchable compounds with photoenhanced acidity or basicity is indeed very limited apart from compounds such as spiropyran, carbamates, and diarylethene.43−49

Furthermore, for biological applications, red-shifting the photoactivation wavelength from the UV region could reduce photodamage to biological samples, making HTIs highly promising. However, to become a photoswitchable fluorophore, the compound should reversibly respond (e.g., on and off) to different excitation or activation light. Since photon emission directly competes with isomerization in the excited state,36 it has been challenging to obtain photoswitchable compounds with high-fluorescence quantum yield during photoisomerization. Although nanoassemblies and molecular dyads are developed based on energy transfer,13,15,50−52 small-molecule photoswitches exhibiting both strong fluorescence and high photoswitching efficiency remain an appealing rarity.

Herein, we report on a variety of HTIs containing the basic pyridine or imidazole moieties that could form intramolecular hydrogen bonds upon Z/E isomerization and protonation. With systematic characterizations in various solvents, polypropylene films, and live HeLa cells, the HTIs were found to combine several intriguing properties together, including reversibly switchable fluorescence, significant photoenhanced basicity, photochromism at acidic conditions, and lysosomotropic subcellular accumulation for fluorescence imaging.

Experimental Section

Reagents and Materials

Cell Counting Kit-8 (CCK-8) was purchased from GlpBio. LysoTracker Deep Red was obtained from Thermo Fisher Scientific. Tetrahydrofuran (THF), toluene, dimethylsulfoxide (DMSO), piperidine, methanol, chloroform, ethanol (EtOH), chloroform-d (CDCl3), dichloromethane (DCM), isopropanol (i-PrOH), hexane, dichloroethane (DCE), sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaBARF), and dioctyl sebacate (DOS) were purchased from J&K Scientific Ltd. in China. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin solution (100×), and sterile phosphate-buffered saline (PBS) were purchased from Corning. Polypropylene films (Celgard 2400, 25 μm thickness, 41% porosity) was purchased from a local distributor of Celgard, USA.

Instrumentation and Measurements

Nuclear magnetic resonance spectra were recorded on Bruker AVANCE NEO 400 and Bruker AVANCE NEO 600. Electrospray ionization mass spectrometry (ESI-MS) data were acquired on a Thermo LTQ Orbitrap XL Instrument. HPLC spectra were determined through Shimadzu LC-20AD with an SPD-20AV detector. Confocal laser scanning fluorescence microscopy (CLSM) was performed on the LSM-900 microscope from Carl Zeiss. De-ionized water was purified by Millipore Integral 5. Ultraviolet–visible (UV–vis) absorption spectra were recorded on an absorption spectrometer (Evolution 220, Thermo Fisher Scientific), and temperature was controlled with water-cooled Peltier accessories. Steady-state fluorescence spectroscopy was performed on a modular fluorescence spectrometer (Fluorolog-3, Horiba Jobin Yvon). Fluorescence lifetime was acquired on the photoluminescence spectrometer FLS1000 with picosecond pulsed diode laser EPL-510 (Edinburgh Instruments, England). The light (365, 455, 470, 530, 565, 617, and 625 nm) for photoswitching was obtained from light-emitting diodes (LEDs) from Thorlabs, USA (LED4D, driven by the controller DC4104), through a liquid light guide (3 mm in diameter). The power of the light was measured with an analog handheld laser power meter console equipped with the standard photodiode sensors (PM100A, Thorlabs).

Preparation of Photochromic Polypropylene Films Based on HTI-Py

0.8 mg of HTI-Py, 3.9 mg of NaBARF, and 82 mg of the plasticizer DOS were dissolved in 100 μL of THF, and then the volatile solvent was evaporated with compressed air. Polypropylene films (14 mm x 14 mm) were immersed in the mixture to soak the components, and the excess amount of liquid was removed with a tissue paper.

Photoswitching of HTI-Py in Live Cells

To record the photoswitching dynamics of HTI-Py- and HTI-EI-labeled cells, HeLa cells were first stained with 20 μM HTI-Py for 5 min. The cells were scratched with a cell scraper such that the density was very low. Then, after three times rinsing with PBS, the stained cells were imaged under LSM 900. Thirty frames were successively captured under continuous irradiation of the 488 nm laser within 27.1 s. The laser was then switched off, and the sample was left in the dark. Time-lapse images were captured at different time points with the laser switched on temporarily to observe the thermal relaxation of HTI-Py in the cells.

Full experimental details are provided in the Supporting Information.

Results and Discussion

Proton-Coupled Reversible Photochromism and Switchable Fluorescence

As shown in Figure 1a, early-reported HTIs bearing a benzene ring in the stilbene part typically exhibited meager fluorescence quantum yield (ϕF < 1%) and moderate photochromism (shift of maximum absorbance around 24 nm).20,22 Zweig and Newhouse recently showed that incorporating a pyrrole hydrogen-bond donor (Figure 1b) could enhance the photochromic effect with the maximum absorbance red-shifted by 67 nm.19 Inspired by previous works, we designed the HTI bearing a pyridine group (HTI-Py, Figure 1c) and hypothesized that its Z/E isomerization should depend on the acid–base equilibrium. Among the four species, the EH+ form is stabilized by an intramolecular hydrogen bond, which is absent in the ZH+ form, indicating that the E isomer would be more basic than the Z isomer.

Figure 1 (a) Previously reported parent benzene substituted HTI. (b) Previously reported pyrrole substituted HTI. (c) The proton-coupled photoswitching of HTI-Py in this work, where the metastable E isomer is stabilized by H+, forming an intramolecular proton bridge in EH+. The Z/E isomerization is accompanied by significant fluorescence and basicity changes as indicated.

As shown in Figure 2a, the neutral HTI-Py appeared yellow in the dark and exhibited an absorbance peak at 449 nm in toluene. After irradiation at 455 nm, the absorption peak wavelength at 449 nm remained the same whereas the molar extinction coefficient decreased from 10,400 to 5696 L·mol–1·cm–1. However, for the protonated forms (Figure 2b), irradiation at 455 nm caused the absorption peak to shift from 462 to 513 nm, turning the color of the solution from yellow to red.

Figure 2 (a) UV–vis absorption spectra and color of the neutral forms of HTI-Py (50 μM, toluene, 25 °C) before and after 455 nm irradiation (24.5 mW/cm2). (b) UV–vis absorption spectra and color of the protonated HTI-Py (50 μM, toluene, 25 °C) before and after 455 nm irradiation (24.5 mW/cm2). Dashed: 50 mM TFA. Solid: 1.5 M TFA. (c) 1H NMR spectra of HTI-Py (1 mM, d8-toluene, −30 °C) under the indicated conditions: with and without TFA, before and after 455 nm illumination (12 mW/cm2, 15 min). (d) Kinetic monitoring of the absorbance at 450 nm of HTI-Py (50 μM, toluene, 40 μM TFA, 25 °C) with 455 nm light irradiation sequentially switched on and off as indicated. (e) Fluorescence emission spectra of HTI-Py in different forms (50 μM, toluene, 25 °C). Ex: 449 nm. (f) Kinetic monitoring of the fluorescence at 509 nm of HTI-Py (50 μM, toluene, 40 μM TFA, 25 °C) with external 455 nm light (12 mW/cm2) switched on and off as indicated. The shutters and excitation light at 449 nm in the spectrometer were kept online all the time.

According to 1H NMR spectroscopy (Figure 2c), the Z isomer of HTI-Py was the thermally stable and predominant form in the dark. After illuminating with 455 nm light at −30 °C, the chemical shift (δ) of the hydrogen labeled 1 to 5 changed significantly (Figures S1 and S2 for full spectra), indicating almost complete photoisomerization to the E form. Adding excess TFA to a solution of HTI-Py in d8-toluene in the dark resulted in ZH+ and changed the chemical shifts significantly. Similarly, illuminating the ZH+ form caused notable changes in the chemical shift of the hydrogens, corresponding to the generation of EH+. Calculated from the integrals of the hydrogens, the photoisomerization yield for Z to E and ZH+ to EH+ in d8-toluene was determined to be 96.6 and 95.5% mole percentage, respectively. Quantitative photoisomerization was also observed in different solvents and temperatures and is summarized in Table S1. In addition to NMR, the quantitative photoisomerization was also confirmed with high-performance liquid chromatography (Table S2).

By monitoring the absorbance at 450 nm, Figure 2d shows the reversible switching of HTI-Py in toluene where the E isomer could spontaneously relax back in the dark. However, different kinetics of the reversible switching was observed in different solvent environments (Figure S3). Figure S4 shows that increasing the temperature was able to accelerate the thermal E to Z isomerization. Figure S5 shows that the relaxation of EH+ could be strongly accelerated by illumination at 530 or 565 nm, which was not observed for the neutral E isomer.

Similar to other reported HTIs, the photoswitching of HTI-Py was very solvent dependent. Tables S3 to S6 summarize the photophysical properties in different solvents, including the maximum wavelength for fluorescence emission and light absorption, the isosbestic points, the half-life values corresponding to the thermal isomerization, and the influence of irradiation wavelength on the isomerization yields. During the Z–E isomerization, HTI-Py exhibited the solvatochromic effect with isosbestic points ranging from 467 to 497 nm. Also, the photochromism and the amount of TFA needed for protonation both were solvent dependent (Figures S6–S8). As shown in Figures S9–S12, photostationary states (PSS) were readily reached in different solvents upon increasing the light intensity at 455 nm and the absorbance of HTI-Py followed nicely the Lambert–Beer law in a wide range (20 to 500 μM), indicating the absence of molecular aggregation.

Unlike previously reported HTIs, the Z isomer of HTI-Py was quite fluorescent (ϕF = 20%) despite a high quantum yield of photoisomerization (ϕZE = 21.4%, Figure S13) in toluene. Figure 2e shows the fluorescence emission spectra of the different forms in toluene where the emission maximum of the Z isomer located around 508 nm. Upon protonation, emission intensity of ZH+ became much lower and was red-shifted to 553 nm (ϕF = 0.3%). The E isomers, protonated or not, were also much less fluorescent. Time-resolved fluorescence spectroscopy (Figure S14a) revealed a fluorescence lifetime of 6.1 ns for the Z isomer.

Figure 2f shows the reversible photoswitching of HTI-Py in the fluorescence mode by monitoring the emission intensity at 509 nm. The sample (45 μL) was entirely illuminated with an external liquid light waveguide on the top, where 455 nm light was switched on (20 s) and off (70 s). The results indicated that the switching of HTI-Py was reversible with negligible fatigue under these conditions.

Photoenhanced Basicity and pH Response

Computational analysis was performed to help evaluate the different basicity of the isomers. Figure 3a shows the Gibbs free energy change during the isomerization and the acid–base reactions with reference to the Z isomer. The structures and energy levels were calculated with density function theory (DFT) on the B3LYP-D3BJ/6-311++G(d,p)-PCM level.53 According to the computational results, the Gibbs free energy of the E isomer was higher than that of the Z isomer by 6.8 kcal/mol. The energy of EH+ was higher than that of ZH+ by 3.7 kcal/mol. These energy differences indicated a theoretical basicity increase of 7.7 orders of magnitude from Z to E (Supporting Information, page S-11), which could be more significant than previously reported spiropyrans (6.3 orders of magnitude).54 However, it should be emphasized that these results are calculated based on pure organic solvents with fixed dielectric constants.

Figure 3 (a) Ground-state Gibbs free energy change and optimized structures of the different forms of HTI-Py from computational DFT calculation on the B3LYP-D3BJ/6-311++G (d, p)-PCM level of theory with DCE as the solvent. (b) UV–vis absorption spectra of HTI-Py (200 μM, DCE, 25 °C) with different TFA concentrations in the dark. (c) UV–vis absorption spectra of HTI-Py (200 μM, DCE, 25 °C) upon reaching the PSS under 455 nm light (7 mW/cm2).

To confirm if there is indeed photoinduced molecular basicity change, acid–base titrations were performed in DCE. Figure 3b,c show the comparison of the UV–vis absorption spectra during the titration of HTI-Py in DCE in the dark and under 455 nm light, respectively. When the titration was performed in the dark (Figure 3b), a new absorption peak eventually appeared at 472 nm. Under 455 nm illumination, the absorption peak at 449 nm gradually decreased with a new peak arising at 512 nm due to the formation of EH+ (Figure 3c). The presence of the isosbestic points indicated a clean binary transformation between the deprotonated and protonated forms. The pKa of the Z isomer in DCE was determined to be 1.2, indicating a rather low basicity. Under 455 nm light in this experiment, the apparent pKa was increased to 3.9 due to the more ready protonation of E to EH+. Therefore, the E isomer of HTI-Py was indeed more basic than the Z isomer.

However, the experimentally observed pKa increase was much less than that of the computational results. Further experiments were performed to seek an explanation. Although the thermal relaxation from E to Z in pure toluene and DCE was very slow, a small amount of TFA in the solution was found to accelerate the E to Z relaxation (Figures S15 and S16). This unusual effect was even observed during the 1H NMR titration at −30 °C. However, with more TFA added under 455 nm light irradiation, the overall equilibrium eventually became dominated by the protonation to form EH+. Thus, protons in the solvents are assumed to play two competing roles: (1) decreasing the stability of E by accelerating the E to Z isomerization and (2) protonating E to the more stable EH+ form. Experimental determination of the true basicity of the E isomer may require more sophisticated techniques such as transient optical spectroscopy since contribution of the E/Z isomerization is difficult to be separately determined.

Figure 4a shows the fluorescence change of HTI-Py upon 455 nm irradiation in aqueous solutions at different pH. The more acidic the solution, the more the fluorescence intensity decay and the higher the decay lifetime τ (Figure 4b). The results indicated faster reverse E/Z isomerization at neutral pH, which was also observed in UV–vis absorption spectroscopy (Figure S17). Therefore, the fluorescence decay was attributed to the formation of EH+. Figure 4c shows the color of the HTI-Py solutions at different pH. Before illumination, the color at different pH values was not much different. However, after 455 nm light illumination, the color of the more acid solutions changed gradually from yellow to magenta, which also confirmed the presence of EH+.

Figure 4 (a) The evolution of fluorescence at 579 nm of HTI-Py (50 μM, DMSO-H2O (1:4, v/v), 25 °C) at the indicated pH with activation light at 455 nm (17 mW/cm2) switched on from the fourth second. (b) The fluorescence decay lifetime (τ) of HTI-Py (50 μM, DMSO-H2O (1:4, v/v), 25 °C) in (a) as a function of pH. (c) Pictures of the HTI-Py (50 μM, DMSO-H2O (1:4, v/v), 25 °C) at different pH before and after 455 nm light illumination. (d) Pictures of the photochromic polypropylene films containing HTI-Py and cation exchange NaBARF (1:1.4 molar ratio). 1: as prepared. 2: after 455 nm light illumination. 3: after contacting an aqueous solution at pH 4.6 and illuminating by 455 nm light. All aqueous buffers were prepared from 10 mM of sodium phosphate, boric acid, and acetic acid and adjusted to desired pH values.

To further explore the pH response, HTI-Py was used to prepare photochromic polypropylene films (25 μm thick, Figure 4d). The polypropylene film 1 contained NaBARF as cation exchanger and a plasticizer DOS as solvent. Directly illuminating this film with 455 nm light caused no obvious color change (film 2), because there were no protons in the film to generate EH+. However, hydrogen ions could enter the films when the film contacted an acid aqueous solution (pH 4.6) according to the ion-exchange principle. Thus, after exposure to the sample solution, the color of the film turned from yellow to magenta (film 3) after 455 nm light illumination. The results indicate that HTI-Py is potentially promising for photoactivated chemical sensing.

Photoisomerization of HTI-EI

A similar photoswitch HTI-EI bearing an ethylimidazole moiety was also synthesized (Figure 5a), and it was expected to behave similarly to HTI-Py. Figure 5b shows the UV–vis absorption spectra of the different forms of HTI-EI with similar changes compared to those of HTI-Py. Additional photophysical properties and spectroscopic characterizations are presented and discussed in Figures S18–S24. Figure S18 shows the emission spectra of the different forms of HTI-EI. The Z isomer of HTI-EI was the most fluorescent species with ϕF of 17.6%. The absorption and emission peak wavelengths for the different species are listed in Table S3. Similar to HTI-Py, Figure 5c shows a much slower thermal relaxation from the protonated E form (EH+ to Z) compared with that of the deprotonated E form (E to Z), which verifies the stabilizing effect of the intramolecular hydrogen bond.

Figure 5 (a) The proton-coupled isomerization reaction of HTI-EI. (b) UV–vis absorption spectra of different forms of HTI-EI (50 μM, DCE, 25 °C). Z (black solid), E (black dashed), ZH+ (red solid, 130 mM TFA), and EH+ (red dashed, 3 mM TFA). (c) Kinetic monitoring of the absorbance at 459 and 454 nm of HTI-EI (50 μM, DCE, with or without 3 mM of TFA, 25 °C) to different light stimulation. The blue, gray, and green backgrounds represent 455 nm light irradiation (24.5 mW/cm2), darkness, and 530 nm light irradiation (19.0 mW/cm2), respectively.

Lysosome Accumulation and Photoswitching in HeLa Cells

Due to the basic pyridine moiety, HTI-Py could act as a lysosomotropic agent for live-cell imaging. Figure 6a shows the confocal laser scanning microscopic (CLSM) images of HeLa cells stained with 10 μM of HTI-Py in the culture medium. Colocalization with the commercial LysoTracker Deep Red revealed an excellent Pearson coefficient of 0.9, indicating that HTI-Py accumulated in the lysosomes.

Figure 6 (a) CLSM images of live HeLa cells stained by 10 μM HTI-Py and LysoTracker Deep Red. Green channel from HTI-Py (em: 500 to 630 nm, ex: 488 nm). Red channel from LysoTracker Deep Red (em: 650–700 nm, ex: 640 nm). (b) Subcellular distribution of 50 μM HTI-Py in HeLa cells pseudo-colored according to the fluorescence intensity. (c) Time-lapse CLSM images of a single HTI-Py-stained HeLa cell upon switching the 488 nm laser on and off. (d) Quantitative analysis and fitting of the fluorescence intensity changes of the images in (c) (upper row, laser on). (e) Quantitative analysis and fitting of the fluorescence intensity changes in (c) (lower row, laser off). (f) Repeated photoswitching of HTI-Py in a single HeLa cell with the 488 nm laser repeatedly switched on (13.1 s) and off (420 s). All scale bars: 20 μm.

Similar to other cell staining dyes, the intracellular distribution of HTIs could be very sensitive to the dye concentration in the culture medium. Figure 6b shows the fluorescence intensity distribution in cells cultured with 50 μM of HTI-Py. While the lysosomes were very bright (region A), staining of other subcellular structures (region B) was also observed. However, the two regions exhibited different kinetic fluorescence changes upon blue light irradiation. As shown in Figure S25, the fluorescence in the lysosomal region A changed much more significantly than that of region B, which could be explained by their different subcellular pH.

HTI-EI exhibited a similar lysosomotropic property (Figure S26). The HTIs showed low cellular toxicity according to the commercial CCK-8 assay (Figure S27). The cells exhibited above 80% of viability up to 100 μM of dye loading, which indicated excellent biocompatibility.

The successful photoisomerization in cells is critical for the applications of HTIs in areas like photopharmacology. Since HTI-Py is both fluorescent and photoswitchable, observing the fluorescence change during the photoswitching of HTI-Py in cells became possible. Since 488 nm laser was equipped on most CLSM, the fluorescence and photochromism of the HTIs under 488 nm irradiation was confirmed (Figure S28). Figure 6c shows the time-lapse CLSM images of the HTI-Py stained HeLa cells and the fluorescence intensity changes over time. The cells were continuously illuminated with the 488 nm laser for 27.1 s, and images were acquired every 0.93 s. Then, the laser was switched off and the cells were kept in the dark. Images were taken after 9, 36, 117, 203, 302, and 413 s, respectively, with minimum exposure in fast scan mode of the confocal microscope (LSM 900, Zeiss). To differentiate the photoswitching from the widely recognized fluorescence recovery after photobleaching (FRAP), cells were seeded far away from each other and rinsed thoroughly to remove excessive dyes before imaging. Figure 6d shows the quantitative analysis of the fluorescence decay, which was fit bi-exponentially to account both photoswitching and photobleaching. The decay time constant of 8.7 s for the photoisomerization indicated an acidic pH environment according to Figure 4b and agrees well with the average lysosomal pH of ca. 5.1 according to the literature.55 Monoexponential fluorescence recovery was observed in Figure 6e with a lifetime of 104.5 s, which is close to the lifetime in the kinetic study of HTI-Py in aqueous solutions (Figure S17). Figure 6f shows the multiple photoswitching cycles by turning the excitation laser on and off periodically. Overall, these results indicated that HTIs with proton-mediated photoswitching are very promising for cell imaging applications. Photoswitchable probes, compared with nonphotoswitchable fluorescent probes, could potentially be applied to high-contrast imaging and photo-actuated manipulation of the cellular environment.

Conclusions

In summary, photoswitchable HTIs were presented where the metastable E isomers could be arrested by protons through intramolecular hydrogen bonding. The Z isomers were highly fluorescent and yet switchable upon illumination around 455 nm. The fluorescence of the E isomers and the protonated forms (ZH+ and EH+) were very low compared with the neutral Z isomers. The photoisomerization of the HTIs was accompanied by photoenhanced basicity, making them promising for photoactivated pH sensing and potentially the chemical sensing of other species such as inorganic ions. The E isomer of HTI-Py was theoretically 7.7 orders more basic than the Z isomer, making it the first reported visible light-induced photobase with higher photoenhanced basicity than that of spiropyrans. Last but not least, the HTIs were lysosomotropic and remained photoswitchable in live cells. The temporal fluorescence evolution upon visible-light stimulation allowed the differentiation of subcellular regions with different pH. The unique combination of photochromism, reversibly switchable fluorescence, and basicity changes makes the HTIs promising for various chemical and biological applications.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c01504.Reagents and materials; synthesis of HTI-Py and HTI-EI; instrumentation and spectroscopic measurements; preparation of photochromic polypropylene films based on HTI-Py; cytotoxicity evaluation; colocalization with LysoTracker Deep Red; photoswitching of HTI-Py in live cells; determination of the fluorescence quantum yield; determination of the photoisomerization yield, quantum yield, and pKa difference; supplementary tables, figures and discussion; computational details (PDF)

Supplementary Material

ac3c01504_si_001.pdf

Author Contributions

# J.L. and X.M. contributed equally.

The authors declare no competing financial interest.

Acknowledgments

The authors thank the National Natural Science Foundation of China (22274070), the Shenzhen Science and Technology Program (202110293000007), and the National Key Research and Development Program of China (2021YFA0715900) for financial support.
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