The chloro-substituted cyanine (100.0 mg, 0.16 mmol) and NaH (60% in mineral oil, 8 mg, and 0.33 mmol) were placed in a flask containing DMF (2 ml), and the mixture was stirred at room temperature under nitrogen atmosphere for 10 min. 3,5-dihydroxybenzaldehyde (43.2 mg, 0.32 mmol) in DMF (1.0 ml) was introduced to the mixture via a syringe, and the reaction mixture was heated at 50°C for 2 h. The solution was then removed under reduced pressure. The crude product was purified by silica-gel flash chromatography using CH₂Cl₂/EtOH (20: 1) as the eluent to give compound Cy-Cys-OH, a blue-green solid (35 mg, yield 41.0%).¹H NMR (500 MHz, DMSO-d ₆) δ 10.22 (s, 1H), 8.56 (d, J = 15.1 Hz, 1H), 8.26 (s, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 7.3 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 6.61 (d, J = 15.1 Hz, 1H), 4.46 (q, J = 7.2 Hz, 2H), 2.73 (dt, J = 5.8 Hz, 4H), 1.87–1.81 (m, 2H), 1.76 (s, 6H), and 1.39 (t, J = 7.2 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d ₆) δ 193.21, 177.66, 174.83, 159.52, 154.86, 145.19, 142.76, 141.46, 132.26, 130.11, 127.74, 123.32, 119.12, 114.55, 113.66, 107.68, 105.35, 70.24, 51.02, 35.58, 31.74, 29.48, 27.67, 22.55, 20.40, 14.41, and 13.20. HRMS (ESI) calculated for C₂₈H₂₈NO₃ ⁺ ([M]⁺): 426.2064, found: 426.2051.

Compound Cy-Cys-OH (100 mg, 0.23 mmol) and Et₃N (15 μL, 0.1 mmol) was dissolved in CH₂Cl₂ (10 ml), and then acryloyl chloride (41.4 mg, 0.46 mmol) was added. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 3 h, and then the solution was extracted in a separatory funnel and evaporated under reduced pressure to give the crude product. The crude product was further purified by silica column chromatography with CH₂Cl₂/EtOH (120:1) to yield compound Cy-Cys-pH as a blue solid (65 mg, 70%). ¹H NMR (500 MHz, DMSO-d ₆) δ 8.58 (d, J = 15.3 Hz, 1H), 8.21 (s, 1H), 7.86–7.78 (m, 3H), 7.72 (d, J = 2.3 Hz, 1H), 7.62–7.58 (m, 1H), 7.54 (t, J = 7.5 Hz, 1H), 6.76 (d, J = 15.3 Hz, 1H), 6.67–6.63 (m, 1H), 6.50 (dd, J = 17.3, 10.4 Hz, 2H), 6.28 (dd, J = 10.4, 1.0 Hz, 1H), 5.33 (t, J = 4.7 Hz, 1H), 4.54 (q, J = 7.1 Hz, 2H), 2.82–2.76 (m, 2H), 2.74–2.70 (m, 2H), 1.89–1.84 (m, 2H), 1.77 (s, 6H), and 1.67 (dd, J = 13.0, 6.8 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d ₆) δ 192.43, 157.67, 151.76, 145.77, 143.34, 141.27, 135.24, 130.11, 127.60, 123.07, 115.25, 114.41, 107.82, 51.56, 35.59, 31.75, 29.55, 27.46, 25.58, 22.56, 14.41, and 13.48. HRMS (ESI) calculated for C₃₁H₃₀NO₄ ⁺ ([M]⁺): 480.2169, found: 480.2161.

Ultraviolet spectroscopy was carried using a Labtech UV Power PC spectrometer, and the fluorescence monitoring was done using a HITACHI F4600 fluorescence spectrophotometer. Cy-Cys-pH was dissolved in DMSO at a concentration of 1 mM as the stock solution and 30 µL of the resulting solution was transferred into a quartz fluorescence cell to obtain a final concentration of 10 µM. Then, 2 µL of a 50 mM solution of Cys was added to obtain a final Cys concentration of 0–120 μM, and the solution was mixed as the monitoring proceeded. pH titration of Cy-Cys-OH (10 µM) was performed in a DMSO/buffer solution (v/v, 3/7). The pH values were adjusted with a buffer pair. The mixture was equilibrated at room temperature for 2 min before measurement.

The in vitro cytotoxicity of Cy-Cys-pH was evaluated by using the standard methyl thiazolyl tetrazolium (MTT) assay in Hela and Panc-1 cells. Briefly, the cells were seeded in 96-well plates at 5,000 cells/well and cultured for 24 h. The medium was washed three times with PBS and incubated with different concentrations of Cy-Cys-pH (0, 2, 4, 6, 8, and 10 μM) for another 12 h. Then, the new medium containing 0.5 mg/ml MTT was added to each well of the 96-well assay plate and incubated for an additional 4 h. Finally, the medium washed thrice with PBS and replaced with 150 μL DMSO to dissolve the precipitates. Infinite M1000 UV-vis microplate reader (TECAN, Austria) was used to measure the absorbance at 570 nm and estimate the viability of cells.

Panc-1 cells (1 × 10⁶) were seeded in the 35-mm glass-bottom culture dishes (Φ35 mm) and incubated for 24 h. After cells adhered to the culture dishes, the cells were washed with PBS and then incubated in culture medium supplemented with 10% fetal bovine serum; then, they were subjected to fluorescence imaging using a two-photon confocal scanning laser microscope (TCS SP8 DIVE).

Cy-Cys-OH (Shen et al., 2021) and Cy-Cys-pH were synthesized according to the synthetic route shown in Supplementary Scheme S1. Their ¹H NMR and ¹³C NMR are exhibited in the Supplementary Figures S6–S9. First, Cy-Cys-pH was made to react with Cys to test how the absorbance spectra changed. The absorption spectra of the Cy-Cys-pH solution at various concentrations of Cys from 0 to 120 μM displayed that the absorption intensity at 592 nm steadily dropped while a new absorption peak emerged at 704 nm (Figure 1A). Meanwhile, the fluorescence intensity of Cy-Cys-pH at 725 nm gradually increased (Figure 1B), and it fit the linear relationship well between the fluorescence intensity and the Cys concentrations (Figure 1D). These findings demonstrated that at physiological Cys concentrations (30–200 µM) (Baker et al., 1990), Cy-Cys-pH was highly sensitive to Cys changes. In addition, the probe entirely interacted with Cys in 100 s and the fluorescence intensity of the mixture remained constant for at least 7 min (Figure 1E). The results proved that Cy-Cys-pH can detect Cys quickly and has good photostability. The photophysical properties of Cy-Cys-OH and Cy-Cys-pH were also examined in DMSO (Supplementary Table S1). Meanwhile, we validated the mechanism of action of Cy-Cys-pH with Cys, in which the nucleophilic addition reaction of Cys to the C=C bond of acrylate, while part of the aldehyde group, was not involved (Supplementary Figures S10, S11). Then, standard fluorescence pH titrations were performed in buffer solutions at Cy-Cys-OH concentration of 10 µM. The fluorescence emission at 682 nm decreased as the pH value increased, while it significantly increased at 725 nm, exhibiting a ratio shift (Figure 1C), and the fluorescence intensity was linear with a pH of 4.5–6.5, indicating the ability of pH-sensing activity in the tumor microenvironment pH range (Figure 1F). Furthermore, the capacity of Cy-Cys-OH to retain fluorescence intensity through the three cycles of pH 4.0 to 7.4 makes Cy-Cys-pH an excellent choice for monitoring the dynamic pH changes in the cellular system (Supplementary Table S3). When Cy-Cys-pH reacted with Cys (0–120 μM) in different buffer solutions (pH 5.0–7.0), the fluorescence intensity at 682 nm decreased and intensified at 725 nm (Figures 2A–E). This proved that the probe can respond to Cys and pH in sequence.

Before investigating the cellular experiments, we tested the sensing performance of the probe Cy-Cys-pH for Cys in the solution. The fluorescence response of the probe to various analytes, including 10 intracellular analytes (amino acids, metal ions, reactive oxygen species (ROS), and reactive nitrogen species (RNS)) is shown in Supplementary Table S4A,B. Neither fluorescence amplification nor clear interference was produced by these analytes when Cys was being detected, suggesting that Cy-Cys-pH is a reliable fluorescent probe and can work well in physiological environments.

The cytotoxicity of Cy-Cys-pH on Hela and Panc-1 cells was tested using the MTT assay prior to utilizing Cy-Cys-pH for imaging in cells. Even when the concentration of Cy-Cys-pH reached 10 μM, cell viability remained greater than 85%. (Supplementary Table S5). It can be inferred that Cy-Cys-pH has low cytotoxicity and is suitable for use in subsequent cell studies.

Next, we investigated whether the probe might be used to monitor Cys in living cells. Panc-1 cells were incubated with Cy-Cys-pH at pH 7.4 in a neutral environment, resulting in considerable fluorescence, showing that Cy-Cys-pH can interact with endogenous Cys to produce fluorescence (Figure 3A). Meanwhile, there was a noticeable fluorescence quenching when the cells were treated with N-ethylmaleimide (NEM) (Figure 3B). When 100 μM Cys was added to the NEM-pretreated cells, strong fluorescence was observed once more (Figure 3C), and very little fluorescence could be induced by the Hcy and GSH treatments (Figures 3D,E). These demonstrated that Cy-Cys-pH is able to detect exogenous and endogenous Cys and is unaffected by the abundant Hcy and GSH in cancer cells.

Since Cy-Cys-pH first responds to Cys and then quantifies pH by the proton exchange of Cy-Cys-OH, we demonstrated the feasibility of Cy-Cys-pH in quantifying intracellular pH in living cells. Intracellular calibration experiments were conducted with Panc-1 cells in high K⁺ buffers of different pH using Cy-Cys-OH. As shown in Figure 4A, the fluorescence intensity of the red channel increased steadily, while the green channel diminished with pH from 5.0 to 8.0 following the successful establishment of a calibration curve, which demonstrated good linearity in the pH range of 5.0–8.0 (Figure 4C), indicating that the Cy-Cys-pH can effectively track intracellular pH.

Within the cells, imported cystine via the cystine/glutamate anti-porter system (system X_c ⁻) is reduced to Cys in a NADPH-dependent manner to replenish intracellular Cys, and intracellular Cys decreases with reducing cystine concentrations (Mak et al., 2015). Thus, we investigated whether Cy-Cys-pH can track the dynamic pH change in cells caused by Cys depletion. As a result, we first regulated Cys by directly controlling the extracellular cystine concentration. Panc-1 cells were pre-incubated with different concentrations of cystine for 12 h and then cultured with Cy-Cys-pH for 30 min. As depicted in CLSM images (Figure 4B), when the extracellular cystine contents were reduced from 100 mM to 10 μM, the fluorescence of the green channel was relatively enhanced, while the red channel weakened, exhibiting a change in ratio. Importantly, the ratio of fluorescence intensity (F_green/F_red) was displayed from 0.38 to 0.73 (Figure 4D), and the intracellular pH showed a decline from 7.30 to 6.78 compared to the previously calibrated graph which clearly demonstrated that the probe was successful in tracking the dynamic pH decline along with the concentration-reduced Cys conditions.

In fact, system X_c ⁻ is overexpressed in different tumor types, and the pancreatic cell line Panc-1 has high resistance to gemcitabine because it has a higher systemic X_c ⁻ expression than MIAPaCa-2 and BxPC-3, which makes it easier for cancer cells to obtain Cys through extracellular cysteine (Gout et al., 2008). In contrast, suppressing system X_c ⁻ activity is successful in preventing cystine uptake, which will result in a shortage of Cys and cause cell ferroptosis (Olive et al., 2020). Crucially, it has been found that restricting cystine uptake by inhibiting system X_c ⁻ increases the sensitivity of cancer cells to chemotherapy (Maher et al., 2013). As we all know, various anticancer drugs target cells through diverse signaling pathways, changing the pH in each condition. Simultaneously, encouraged by the aforementioned experimental findings, we explored how the intracellular pH of cells changes while being treated with various anticancer drugs. We employed three different anticancer drugs, all of which have been proven to inhibit system X_c ⁻ (erastin (Stockwell et al., 2012), sulfasalazine (Bruchovsky et al., 2001), and sorafenib (Wrosch et al., 2017)). As shown in Figures 5A,D, when Panc-1 cells were treated with erastin (0–400 μM) for 12 h and then with Cy-Cys-pH (5 μM) for 30 min, a rise in the fluorescence ratio values (F_green/F_red) was found and the intracellular pH decreased from 7.58 to 6.97. Similarly, the intracellular pH of Panc-1 cells was reduced from 7.54 to 6.89 after being exposed to sulfasalazine (0–400 μM) (Figures 5B,E). By comparison, cells were incubated with sorafenib at concentrations of 0–40 μM and Panc-1 cells had a decrease in the intracellular pH from 7.54 to 6.65 (Figures 5C,F), indicating that even though they are both system X_c ⁻ inhibitors, erastine and sulfasalazine are less capable of inhibiting system X_c ⁻ than sorafenib, as well as killing Panc-1 cells by depleting intracellular Cys. These results also demonstrated that Cy-Cys-pH can monitor intracellular pH during the therapeutic process with anticancer drugs. Meanwhile, the anticancer strategy by associating Cys and pH is, in part, an effective strategy as they target two features of the tumor microenvironment—oxidative stress and alkaline intracellular pH, both affecting cancer-cell progression.