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. 2023 Jul 10;1(4):395–402. doi: 10.1021/cbmi.3c00055

Benzothiazole-Based Fluorescent Probe as a Simple and Effective Platform for Functional Mitochondria Imaging

Kyeong-Im Hong †, Younghun Kim †, Jeong Heon Lee †, Kang Ho Chu †, Woo-Dong Jang †,*
PMCID: PMC11503673  PMID: 39473939

Abstract

graphic file with name im3c00055_0009.jpg

Mitochondrial imaging is crucial for studying disease pathogenesis and diagnosis. However, commercially available mitochondria-specific fluorescent probes are limited due to the difficulty in modifying cationic probes for functional bioimaging of mitochondria. To address this, we prepared a hydroxythiophene-conjugated benzothiazole (BzT-OH) as a platform for the design of functional fluorescent probes. Two probes, BzT-OAc and BzT-OAcryl, were synthesized by substituting the hydroxy group in BzT-OH with acetate and acrylate groups, respectively. BzT-OAc demonstrated blue fluorescence that shifted to green emission after enzymatic cleavage of the acetate group, allowing for monitoring of endogenous esterase activity. BzT-OAcryl showed high selectivity for cysteine at pH > 8.0, owing to its pH-responsive property, and could detect pH perturbations caused by mitochondrial dysfunction. Both probes exhibited high biocompatibility, quantum yield, and large Stokes shifts in mitochondria. BzT-OH can be easily conjugated with other functional groups and substrates of various enzymes for designing fluorescent probes for functional mitochondrial imaging. The photofunctional property of the probe can be changed due to the involvement of the hydroxyl group in the excited state intermolecular proton transfer.

Keywords: fluorescent probe, cysteine, esterase, mitochondria, ESIPT

Introduction

In eukaryotic cells, mitochondria are crucial organelles, which participate in several anabolic functions such as cell respiration, redox homeostasis, cell survival, and death.1,2 Mitochondrial dysfunction is closely related to a variety of diseases, including cancer, neurological disorders, and vascular diseases.3−8 Therefore, mitochondrial imaging is important to study disease pathogenesis and for diagnosing these diseases,9,10 and several mitochondria-specific fluorescent probes are currently commercially available. Most of these probes suffer from unavoidable potential-dependent changes in their properties, owing to the negative potential gradient in the mitochondrial membrane, which affects the ammonium or phosphonium moieties in the probes that are necessary for organelle staining.11,12 These cationic fluorescent probes have drawbacks such as poor biocompatibility—for example, the binding of these probes to mitochondrial DNA can accelerate cellular oncosis.13,14 Furthermore, structural modifications of these conventional probes for functional bioimaging are difficult, which is a critical disadvantage. In this context, a fluorescent probe having modifiable functional groups with a simple molecular structure would be useful for functional mitochondrial imaging.15−18 In this study, we designed a hydroxythiophene-conjugated benzothiazole derivative (BzT-OH; Scheme 1), which performed as an excellent mitochondria-specific fluorescent probe and showed successful biocompatibility, high quantum yield for fluorescence emission, and a large Stokes shift.19 The hydroxyl group in BzT-OH can be easily conjugated to other functional groups for functional mitochondrial imaging. Furthermore, because the hydroxyl group is essentially involved in the ESIPT phenomena, the photofunctional properties of probes can be greatly change. Therefore, we can easily obtain functional mitochondria imaging probes by introduction of substrates for enzymatic reaction or chemo-selective functional groups.

Scheme 1. Synthesis of BzT-OAc and BzT-OAcryl.

Scheme 1

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Reagents and conditions: (i) acetyl chloride, trimethylamine, tetrahydrofuran, 25 °C, (ii) acryloyl chloride, trimethylamine, CH2Cl2, 25 °C.

For our proof-of-concept study, we prepared two fluorescent probes, acetylate- and acrylate-conjugated BzT-OH (BzT-OAc and BzT-OAcryl, respectively; Scheme 1), for functional mitochondrial imaging. Since acetylate is a common substrate for esterase, BzT-OAc can be used for monitoring esterase activity.20,21 Consequently, after treatment with porcine liver esterase (PLE), the blue emission of BzT-OAc shifted to green. On the other hand, BzT-OAcryl was used for biothiol detection because the acrylate group is an excellent Michael acceptor for the detection of nucleophilic substances. A fluorescence-based molecular screening test revealed that BzT-OAcryl is able to selectively detect cysteine (Cys) at high pH (>8.0). This pH-responsive property allows BzT-OAcryl to react with Cys to produce BzT-OH. Furthermore, cell experiments confirmed that both BzT-OAc and BzT-OAcryl could successfully stain the mitochondria, thus establishing that BzT-OAc and BzT-OAcryl can be used to monitor esterase activity and fluctuations in mitochondrial pH, respectively.

Results and Discussion

As shown in Scheme 1, BzT-OAc and BzT-OAcryl were synthesized by esterification of BzT-OH with acetyl chloride and acryloyl chloride, respectively. The obtained products were unambiguously characterized by 1H and 13C NMR, FT-IR, and MALDI-TOF-MS measurements (Figures S1–S5). Figure 1a shows the absorption and emission spectra of BzT-OH, BzT-OAc, and BzT-OAcryl in dimethyl sulfoxide (DMSO). The maximum absorption wavelengths (λabs) were 390, 381, and 381 nm for BzT-OH, BzT-OAc, and BzT-OAcryl, respectively (Table 1). Upon excitation at λabs (390 nm), BzT-OH showed a strong fluorescence emission with a maximum emission wavelength (λem) of 472 nm, where the fluorescence quantum yield (ΦF) was 50%. In contrast, λem values for BzT-OAc and BzT-OAcryl were 460 nm (ΦF = 52%) and 468 nm (ΦF = 16%), respectively. The absorption and emission of BzT-OH, BzT-OAc, and BzT-OAcryl in 10 mM phosphate buffered saline (PBS) containing 10% DMSO were again measured to investigate the photochemical properties in the aqueous phase (Figures 1b,c and S6). A pH-dependent change in the fluorescence emission intensity was observed for BzT-OH. When the pH was lower than 7.0, BzT-OH showed a very weak fluorescence emission (ΦF = 2.5% at pH 5.0). The emission intensity of BzT-OH was greatly enhanced with increasing pH (>8.0) (ΦF = 56% at pH 8.3) because the ESIPT process was activated at this condition. Such pH-responsive emission changes enable the use of BzT-OH as a fluorescent probe for mitochondrial imaging since the mitochondrial matrix has a basic microenvironment.22

Figure 1.

Figure 1

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(a) Absorption and emission spectra of BzT-OH, BzT-OAc, and BzT-OAcryl in DMSO. Emission spectra of (b) BzT-OH and (c) BzT-OAc and BzT-OAcryl in PBS buffer (10 mM) solution containing 10% DMSO. (d) pH-dependent changes in the emission intensity of BzT-OH, BzT-OAc, and BzT-OAcryl at 520 nm. The concentrations of BzT-OH, BzT-OAc, and BzT-OAcryl were adjusted to 10 μM in all solutions.

Table 1. Photophysical Properties of the Fluorescence Probes under Different Conditions.

  DMSO solution
 PBS buffer
  λabs (nm) λfl (nm) λfl (nm)
BzT-OH 390 472 (0.50)a 520 (0.56 in pH 8.0)
BzT-OAc 381 460 (0.52) 474 (0.21 in pH 7.4)
BzT-OAcryl 381 468 (0.16) 507 (0.03 in pH 7.4)
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a

Values in parentheses present the absolute quantum yields.

The overlapping of absorption and emission poses a persistent problem during microscopic imaging owing to reabsorption, crosstalk, and inner-filter effects.23 A common solution to reduce such inherent problems with general fluorescent probes is the excited-state intramolecular proton transfer (ESIPT).24 Since the nitrogen atom in benzothiazole forms intramolecular hydrogen bonds with the hydroxy group in the adjunct thiophene unit, BzT-OH shows a large Stokes shift through the ESIPT process. Above pH 8.0, the maximum wavelengths of BzT-OH in absorption and emission spectra were located at 420 and 520 nm, respectively. Large Stokes shift of BzT-OH at pH 8.0 was calculated at approximately 100 nm (Figure 1b). Considering the high quantum yield for fluorescence emission and large Stokes shift, BzT-OH is an excellent fluorescent probe for mitochondrial imaging. In contrast, BzT-OAc in 10 mM PBS containing 10% DMSO showed blue fluorescence emission (λem = 474 nm, ΦF = 21% at pH 7.4) over a wide pH range.25 The emission color of BzT-OAc in PBS solutions showed almost uniform behavior, since the hydroxy group of BzT-OH was protected by the acyl group. In contrast to BzT-OAc, BzT-OAcryl showed very weak fluorescence emission (λem = 507 nm, ΦF = 3.0%) owing to the intramolecular charge transfer (ICT) process. When the pH was increased to 12.0, both BzT-OAc and BzT-OAcryl showed a strong emission band at approximately 520 nm, owing to the generation of BzT-OH by ester group cleavage at high pH (Figure S6).

Because the cleavage of the acetylate group caused an emission change of BzT-OAc, the enzymatic cleavage of acetylate was examined by using PLE (Figure S7). When BzT-OAc was treated with PLE, the fluorescence intensity at 523 nm increased rapidly at pH 7.4 and 8.0. The time taken to reach the maximum fluorescence intensity at pH 7.4 was less than 1 min, following which the intensity decreased with time, which may be attributed to photobleaching. At pH 8.0, the reaction rate was slow, whereas the reaction hardly proceeded at pH 5.0 because of the stability of the protein. The spectral changes of BzT-OAc were monitored in the presence of other interferences such as metals, Reactive oxygen species (ROSs), amino acids, and other enzymes (Figure S8). In the presence of other interferences, the acetyl group of BzT-OAc was selectively cleaved by acety cleavable enzymes (PLE and BChE). Next, we performed in vitro imaging experiments using HeLa cells. Initially, the emission in the blue channel rapidly disappeared owing to acetyl group cleavage by the endogenous esterase. The time-course measurements suggest that the acetyl group cleavage in BzT-OAc was completed in approximately 10 min (Figure 2). Moreover, as the emission in the blue channel decreased, the emission in the green channel gradually increased and reached its maximum within approximately 10 min. However, it was difficult to observe a dramatic change in the green channel because BzT-OAc also showed a weak fluorescence emission in the green region. HeLa, B16, and A549 cells were treated with BzT-OAc and MTR to confirm intracellular localization. Consequently, the emission from BzT-OAc overlapped well with the emission from MTR for all cell lines (Pearson Correlation Coefficient, PCC = 0.80, 0.76, and 0.84 for HeLa, B16, and A549 cells, respectively), indicating selective staining of mitochondria using BzT-OAc (Figure 3).

Figure 2.

Figure 2

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Time-dependent imaging of HeLa cells by BzT-OAc staining. The cells were treated with 10 μM BzT-OAc and imaged in 2 min intervals using confocal microscopy (scale bar: 20 μm). Blue channel: λex = 405 nm, λem = 400–450 nm, and green channel: λex = 405 mm, λem = 570–617 nm.

Figure 3.

Figure 3

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Confocal microscope images of HeLa, B16, and A549 cells stained with BzT-OAc (10 μM) and MTR (100 nM). Scale bar: 20 μm.

Further, the emission change of BzT-OAc was monitored again in the stressed cells. When HeLa cells were treated with H2O2, a well-known acidifying agent of intracellular pH, blue emission disappeared, but the generation of green emission was not observed (Figure S9).26 These results indicate that the acetate groups in BzT-OAc can be rapidly cleaved by the endogenous esterase; however, generated compound BzT-OH does not exhibit fluorescence emission owing to acidic pH conditions. In contrast, when HeLa cells were pretreated with cisplatin, blue emission was sustained for 40 min in the mitochondrial compartment, indicating that cisplatin inhibits the endogenous esterase (Figure 4).

Figure 4.

Figure 4

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Confocal microscope images of HeLa cells pretreated with cisplatin and stained with BzT-OAc (10 μM) and Mitrotracker Deep Red (MTR) (100 nM). Scale bar: 20 μm.

Because the acrylate group is an excellent Michael acceptor, the acrylate group cleavage in BzT-OAcryl was examined using 10 equiv of various amino acids (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val), homocysteine (Hcy), and glutathione (GSH). Various amino acids and biothiols were added to the solution of BzT-OAcryl in 10 mM PBS containing 10% DMSO under various pH conditions, and the solution was incubated for 10 min at 37 °C. As shown in Figure 5, the emission intensity of BzT-OAcryl increased more than 10 times with the addition of Cys at pH 8.0, whereas none of the other amino acids or GSH affected its fluorescence emission intensity. In the case of Hcy, a slight fluorescence enhancement of BzT-OAcryl was observed. In the case of Cys, at pH 8.0, owing to the Michael reaction between Cys and acrylate, BzT-OAcryl was converted to BzT-OH, thus transforming its emission spectrum to that of BzT-OH. At pH 5.0, BzT-OAcryl did not react with any biothiols, because biothiol nucleophilicity was insufficient under these conditions to cause the Michael reaction (Figure S10a). In contrast, at pH 7.4, the acrylate group in BzT-OAcryl was cleaved by Cys to generate BzT-OH; however, the emission intensity of BzT-OH at pH 7.4 was very low (Figure S10b). Because the fluorescence turn-on response of BzT-OAcryl requires both basic conditions and the presence of Cys, BzT-OAcryl can successfully detect Cys in a basic environment such as the mitochondria but not in acidic or neutral environments.

Figure 5.

Figure 5

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Emission intensity (at 526 nm) of BzT-OAcryl containing various amino acids (10 equiv) including cysteine (10 equiv) in various pH conditions at 360 nm illumination. These are recorded in a 10% DMSO-containing buffer solution (citrate buffer for pH 4.0 and 5.0 and PBS buffer for pH 7.4 and pH 8.0); A: l-alanine, D: l-aspartic acid, E: l-Glutamic acid, F: l-phenylalanine, G: l-glycine, H: l-histidine, I: l-isoleucine, K: l-lysine, L: l-leucine, M: l-methionine, N: l-asparagine, P: l-proline, Q: l-glutamine, R: l-arginine, S: l-serine, T: l-threonine, V: l-valine, W: L-tryptophan, Y: l-tyrosine, GSH: l-glutathion, Hcy: l-homocysteine. Cys: l-cysteine.

Competitive experiments on acrylate cleavage by Cys were performed in the presence of various amino acids (10 equiv) and biothiols (10 equiv). The results showed that the other amino acids, GSH, and Hcy did not interfere with the Cys-sensitive fluorescence response of BzT-OAcryl (Figure S11). To determine the detection limit, fluorescence titration of BzT-OAcryl against Cys (up to 10 equiv) in 10 mM PBS containing 10% DMSO at pH 8.0 was performed (Figure S12). After 10 min of incubation at 37 °C, the fluorescence intensity of BzT-OAcryl showed good linear increments up to 40 μM and was saturated at 100 μM Cys, and the correlation coefficient of the linear regression was greater than 0.987. The detection limit (3s/k) for Cys was estimated to be 2.2 μM from the standard deviation of blank measurements (s) and the slope of the corresponding calibration line (k).27 Time-course measurements of the emission at 526 nm were performed to observe the reaction time between BzT-OAcryl and biothiols (10 equiv) in 10 mM PBS containing 10% DMSO at pH 8.0 (Figure S13). The emission intensity of BzT-OAcryl remained unchanged in the absence of biothiols or GSH. In the presence of Hcy, the emission intensity of BzT-OAcryl increased gradually, indicating a slow reaction between BzT-OAcryl and Hcy. In the presence of Cys, BzT-OAcryl exhibited remarkably rapid changes in the emission intensity, and the reaction was complete within 5 min.

Subsequently, HeLa, B16, and A549 cells were treated with MTR (100 nM) and BzT-OAcryl (10 μM) and incubated for 50 min. Figure 6 shows that the green emission of BzT-OH (generated by the reaction between BzT-OAcryl and Cys) overlaps well with the red emission from MTR, indicating that BzT-OAcryl could simultaneously image endogenous Cys and the mitochondria (PCC = 0.88, 0.81, and 0.82 for HeLa, B16, and A549 cells, respectively). To identify whether green emission in mitochondria was induced by Cys, HeLa cells were pretreated with N-ethylmaleimide (NEM), an endogenous biothiol removal agent.28 Green emission was scarcely visible, because BzT-OAcryl did not react with Cys (Figure S14). Additionally, biothiols (100 μM) such as Cys, Hcy, and GSH were incubated with HeLa cells pretreated with NEM. As shown in Figure S13, green emission was only visible from Cys treatment due to the reaction of Cys and BzT-OAcryl; treatment with GSH and Hcy did not show green emissions. A colocalization experiment with MTR showed that green emission induced by exogenous Cys was also colocalized with MTR (Figure 7a). As a result, the probe selectively detected intracellular and extracellular Cys over GSH and Hcy in living cells.

Figure 6.

Figure 6

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Confocal microscope images of HeLa, B16, and A549 cells stained with BzT-OAcryl (10 μM) and MTR (100 nM). Scale bar: 20 μm.

Figure 7.

Figure 7

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Efficient staining of Cys in mitochondria by BzT-OAcryl. (a) HeLa cells were treated with 100 μM of Cys after pretreatment with N-ethylmaleimide (NEM) (100 μM) for 30 min. Then BzT-OAcryl (10 μM) and MitoTracker Deep Red (100 nM) were incubated with HeLa cells for 50 min. (b) Acidification of intracellular pH; HeLa cells were exposed to pH 5.0 HEPES buffer containing 10 μM of nigericin. (c) HeLa cells were treated with MitoTracker and BzT-OAcryl, followed by treatment with H2O2. Cell images were obtained using confocal laser fluorescence microscopy (scale bar: 20 μm), BzT-OAcryl (green, λex = 405 nm, λem = 520–617 nm), and MitoTracker Deep Red (red, λex = 641 nm, λem = 650–700 nm).

Next, HeLa cells were incubated in a HEPES buffer (pH 5.0) containing 10 μM nigericin to acidify the mitochondrial matrix. As shown in Figure 7b, the green emission in mitochondria hardly overlapped with the red emission from MTR (P = 0.28) because BzT-OH cannot emit green fluorescence under acidic conditions. Furthermore, when HeLa cells were treated with H2O2, no green emission was observed (Figure 7c). Therefore, unlike MTR, BzT-OAcryl can detect pH perturbations caused by mitochondrial dysfunction.

Conclusion

We designed hydroxythiophene-conjugated benzothiazole derivative BzT-OH as a mitochondria-specific fluorescent probe, which has excellent biocompatibility, high quantum yield for fluorescence emission, and a large Stokes shift. To obtain a functional fluorescent probe for mitochondrial imaging, the hydroxy group of BzT-OH was substituted with acetate or acrylate groups. The acetate-bearing molecular probe BzT-OAc showed a blue fluorescence emission, which shifted to green in response to endogenous esterase activity. Acrylate-bearing BzT-OAcryl successfully stained mitochondria, with excellent selectivity for Cys at pH > 8.0, thus exhibiting pH-responsive property via its reaction with Cys. Treatment of HeLa cells with nigericin or H2O2 revealed that BzT-OAcryl could detect pH perturbations caused by mitochondrial dysfunction. Following the two examples illustrated in this proof-of-concept study in various cells, several novel fluorescent probes may be designed for functional mitochondrial imaging, as BzT-OH can accommodate diverse functionalization.

Methods

Materials and Measurements

All commercially available reagents were reagent grade and used without further purification. PLE was purchased from Sigma-Aldrich. UV/vis absorption spectra were recorded by using a JASCO V-660 spectrometer. Fluorescence emission spectra were recorded using a JASCO FP-6300 spectrophotometer equipped with a thermostatic cell holder (ETC-273T, JASCO) coupled to a controller (ETC-272T, JASCO). Fluorescence emission quantum yields were measured by using a spectrofluorometer (FluoroMax Plus, Horiba). All of the spectra were measured using a quartz cuvette with a path length of 1 cm. 1H and 13C NMR spectra were recorded on Bruker Advance DPX 400 spectrometers at 25 °C in CDCl3. MALDI-TOF-MS was performed on a Bruker Daltonics LRF20 instrument with dithranol (1,8,9-trihydroxyanthracene) as the matrix.

Synthesis

BzT-OAc

To a solution of BzT-OH (45 mg, 0.14 mmol) in tetrahydrofuran (3.0 mL) was added trimethylamine (0.3 mL) at 25 °C. Acetyl chloride (0.05 mL, 0.71 mmol) was then added to the solution and stirred at 25 °C. The reaction was monitored using silica thin-layer chromatography. After the completion of the reaction, the solution was extracted with H2O/CH2Cl2. The combined organic layer was concentrated and purified by silica column chromatography with 33% dichloromethane/hexane as the eluent to afford BzT-OAc as a yellow powder (36 mg, 70%). 1H NMR (400 MHz, CDCl3, 25 °C) δ = 8.05–8.03 (d, J = 8 Hz, 1 H), 7.89–7.87 (d, J = 8 Hz, 1 H), 7.51–7.47 (t, J = 8 Hz, 1 H), 7.40–7.36 (t, J = 8 Hz, 1 H), 7.33–7.31 (m, 2 H), 7.24 (s, 1 H), 7.08–7.06 (m, 1 H), 2.52 (s, 3 H) ppm; 13C NMR (100 MHz, CDCl3, 25 °C) δ = 167.66, 157.28, 152.46, 146.46, 138.68, 136.43, 134.87, 128.33, 126.67, 126.39, 125.23, 125.13, 122.95, 121.58, 121.40, 118.83, 21.51 ppm; MALDI-TOF-MS: [C17H11NO2S3]+ calcd. m/z 357.46, observed 357.6.

BzT-OAcryl

To a solution of BzT-OH (27 mg, 0.085 mmol) in CH2Cl2 (4.0 mL) were added three drops of trimethylamine at 0 °C. Subsequently, acryloyl chloride (0.013 mL, 0.171 mmol) was added, and the mixture was stirred at 25 °C. The reaction was monitored using silica thin-layer chromatography. After the reaction was completed, the solution was concentrated and purified by silica column chromatography with 25% dichloromethane/hexane as the eluent to afford BzT-OAcryl as a yellow powder (26 mg, 82%). 1H NMR (400 MHz, CDCl3, 25 °C) δ = 8.04–8.03 (d, J = 4 Hz, 1 H), 7.87–7.85 (d, J = 8 Hz, 1 H, 7.48–7.31 (m, 5 H), 7.07 (s, 1 H), 6.84–6.79 (d, J = 20 Hz, 1 H), 6.54–6.47 (m, 1 H), 6.22–6.19 (d, J = 12 Hz, 1 H) ppm; 13C NMR (100 MHz, CDCl3, 25 °C) δ = 162.71, 157.35, 152.54, 146.30, 138.76, 136.46, 135.02, 134.67, 128.44, 127.28, 126.57, 126.39, 125.16, 123.10, 121.75, 121.45, 118.81, 118.75 ppm; MALDI-TOF-MS: C18H11NO2S3 [M + H]+m/z calcd. 369.00, observed 369.93.

Cell Cultures and Imaging

HeLa (human cervical cancer cells, ATCC CCL-2), B16 (human skin melanoma cells), and A549 (lung carcinoma epithelial cells) cells were cultured in Minimum Essential Medium (MEM; Invitrogen), Dulbecco’s modified Eagle’s Medium (DMEM; Invitrogen), and Ham’s F-12K (Kaighn’s) Medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO2, respectively. For the imaging, cells were treated with 100 nM MitoTracker Deep Red FM (MTR, Invitrogen) and 10 μM BzT-OH, BzT-OAc, or BzT-OAcryl. Cell images were obtained using confocal fluorescence microscopy (LSM 800, Zeiss, Germany) and analyzed using ZEN 2.3 software (Carl Zeiss). A light of wavelength 405 nm was used for the excitation of BzT-OH, BzT-OAc, and BzT-OAcryl, and 640 nm for the excitation of MTR. Fluorescence emission was collected through three channels: blue, green, and red. The emission from MTR was collected through a red channel set at 650–700 nm, those from BzT-OH and BzT-OAcryl through a green channel set at 520–617 nm, and those from BzT-OAc through blue and green channels at 400–469 and 517–620 nm, respectively.

To induce oxidative stress, HeLa cells were treated with H2O2 (16 mM). After 1 h of incubation at 37 °C, cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene). The medium was then replaced with MEM containing 10 μM BzT-OAc and 100 nM MTR, and cell images were recorded by using confocal fluorescence microscopy.

To inhibit endogenous esterase, the cells were pretreated with 1 μM cisplatin for 1 h at 37 °C. Then, cells were washed twice with DPBS and were further incubated with MTR (100 nM) for 1 h at 37 °C. The medium was then replaced with MEM containing 10 μM BzT-OAc, and cell images were recorded using confocal fluorescence microscopy.

To remove endogenous biothiols, HeLa cells were pretreated with 1 mM N-ethylmaleimide (NEM) for 0.5 h. Then, the cells were treated with BzT-OAcryl (10 μM) and MTR (100 nM) for 50 min, and cell images were recorded by using confocal fluorescence microscopy. Additionally, 100 μM of biothiols such as Cys, Hcy, and GSH were incubated for 0.5 h with NEM pretreated HeLa cells. The cells were treated with BzT-OAcryl for 50 min, and confocal images were recorded again.

For acidification of the intracellular pH, HeLa cells were treated with 10 μM nigericin as follows: cells were first treated with 10 μM BzT-OAcryl and 100 nM MTR for 50 min. Then, the medium was replaced with HEPES buffer (pH 5.0) containing 10 μM nigericin and incubated for 0.5 h at 37 °C. The cells were washed twice with DPBS, and cell images were recorded using confocal fluorescence microscopy.

Acknowledgments

This work was supported by the National Research Foundation (NRF) grants (2020R1A2C3004520 and 2022R1A4A1020543) funded by Ministry of Science, ICT & Future Planning (MSIP), Korea.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.3c00055.

  • Spectral data and additional experimental data (PDF)

Author Contributions

K.-I. Hong: conceptualization, investigation, writing - original draft; Y. Kim: investigation; J. H. Lee: investigation; K. H. Chu: investigation; W.-D. Jang: supervision, writing - review and editing.

The authors declare no competing financial interest.

Supplementary Material

im3c00055_si_001.pdf (1.1MB, pdf)

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