A Near-Infrared Fluorescent Probe for Recognition of Hypochlorite Anions Based on Dicyanoisophorone Skeleton

A novel near-infrared (NIR) fluorescent probe (SWJT-9) was designed and synthesized for the detection of hypochlorite anion (ClO−) using a diaminomaleonitrile group as the recognition site. SWJT-9 had large Stokes shift (237 nm) and showed an excellent NIR fluorescence response to ClO− with the color change under the visible light. It showed a low detection limit (24.7 nM), high selectivity, and rapid detection (within 2 min) for ClO−. The new detection mechanism of SWJT-9 on ClO− was confirmed by 1H NMR, MS spectrum, and the density functional theory (DFT) calculations. In addition, the probe was successfully used to detect ClO− in HeLa cells.


Introduction
Neutrophils, known as polymorphonuclear cells, are the largest number of white blood cells in the body [1][2][3]. They are the main immune cells that protect the body from microbial infection and eliminate pathogens [4]. In neutrophils, hydrogen peroxide (H 2 O 2 ) reacts with chloride ions to generate hypochlorite anions [5][6][7] under the catalysis of myeloperoxidase. Hypochlorite anions plays a very important role in the human body [8,9]. However, excessive hypochlorite anion in the body will oxidize biological molecules, such as protein, cholesterol, DNA, and RNA in living cells, which will lead to cardiovascular disease, inflammatory disease, cancer, and so on [10][11][12][13][14][15]. Therefore, it is necessary to monitor hypochlorite anions in vitro and in vivo.
At present, many analytical methods have been applied to the detection of hypochlorite anions, such as colorimetry, luminescence, electrochemistry, and chromatography [16][17][18]. In addition, the fluorescence probing method was paid more attention as an excellent detection tool, which has realized the detection of many active species [19][20][21][22][23]. Fluorescence probes for the detection of hypochlorite anions have been reported continuously in recent years . Among them, some chemodosimeters using a diaminomaleonitrile group as the reaction site were used to detect hypochlorite anions, which had many advantages, such as specific recognition, high selectivity, and fast response. However, most chemodosimeters for the hypochlorite anion could hardly achieve near-infrared fluorescence emission and did not have a large Stokes shift (Table S1) [34][35][36][37][38][39][40][41][42][43][44][45][46]. It is well known that NIR fluorescent probes with a large Stokes shift have more advantages due to good applications in biological systems [47]. Therefore, it is necessary to design a near-infrared fluorescent probe with a large Stokes shift for the detection of hypochlorite anions with high selectivity and rapid response.
In connection with our previous work [26,48,49], herein, we reported a novel chemodosimeter (SWJT-9), which had a large Stokes shift and emitted near-infrared fluorescence due to using a dicyanoisophorone skeleton. It can specifically recognize a hypochlorite

Design SWJT-9
Through the Duff reaction, an aldehyde group was generated at the ortho position of the hydroxyl group in a dicyanoisophorone skeleton. The diaminomaleonitrile group was then used as the recognition group [36,51] to obtain the probe SWJT-9 (Scheme 1). The structure of SWJT-9 was confirmed by NMR and MS (Figures S1-S3, Supplementary Materials). The fluorescence of SWJT-9 would reduce by the rotational isomerization of the C=N double bond [52]. After the addition of hypochlorite anions, a reaction between SWJT-9 and ClO − would occur to obtain compound 3, which had no C=N bond; therefore, the fluorescence would be enhanced [53][54][55]. In connection with our previous work [26,48,49], herein, we reported a novel chemodosimeter (SWJT-9), which had a large Stokes shift and emitted near-infrared fluorescence due to using a dicyanoisophorone skeleton. It can specifically recognize a hypochlorite anion based on the inhibition of C=N rotational isomerization [50]. Moreover, SWJT-9 was successfully applied to HeLa cell imaging.

Design SWJT-9
Through the Duff reaction, an aldehyde group was generated at the ortho position of the hydroxyl group in a dicyanoisophorone skeleton. The diaminomaleonitrile group was then used as the recognition group [36,51] to obtain the probe SWJT-9 (Scheme 1). The structure of SWJT-9 was confirmed by NMR and MS (Figures S1-S3, Supplementary Materials). The fluorescence of SWJT-9 would reduce by the rotational isomerization of the C=N double bond [52]. After the addition of hypochlorite anions, a reaction between SWJT-9 and ClO − would occur to obtain compound 3, which had no C=N bond; therefore, the fluorescence would be enhanced [53][54][55].

Photoproperties of Probe
In order to study the effect of organic solvents on the probe, methanol, ethanol, acetonitrile, DMSO, and DMF were used ( Figure S4, Supplementary Materials). The effect of the buffer solution and pH on the probe were also studied ( Figure S5, Supplementary Materials). According to these results, and considering the solubility of the probe, the solution of ethanol and PBS (9/1, v/v) at pH 7.4 was selected as the test condition.
As shown in Figure 1a, the UV-Vis absorption spectrum of SWJT-9 showed an obvious absorption band centered at 430 nm. After the addition of ClO − to the solution, the absorbance red-shifted to about 550 nm. The color of the solution changed from yellow to pink (Figure 1a, inset). These results suggested that a reaction might occur between SWJT-9 and ClO − . In the fluorescence spectrum, SWJT-9 showed weak emission at about 667 nm (Φ = 0.018) under excitation at 550 nm ( Figure 1b). After the addition of ClO − , the fluorescence increased (Φ = 0.113) [56], and the fluorescence color of the solution was observed from red to deep red (Figure 1b, inset). These results indicated that SWJT-9 could detect hypochlorite anions by colorimetric and fluorescence turn-on responses. The fluorescence titration experiments were then conducted. As shown in Figure 1c, the fluorescence intensity enhanced with the increase of the ClO − concentration, indicating that SWJT-9 was a turn-on fluorescence probe. The detection limit was calculated as 24.7 nM Scheme 1. Synthesis of SWJT-9.

Photoproperties of Probe
In order to study the effect of organic solvents on the probe, methanol, ethanol, acetonitrile, DMSO, and DMF were used ( Figure S4, Supplementary Materials). The effect of the buffer solution and pH on the probe were also studied ( Figure S5, Supplementary Materials). According to these results, and considering the solubility of the probe, the solution of ethanol and PBS (9/1, v/v) at pH 7.4 was selected as the test condition.
As shown in Figure 1a, the UV-Vis absorption spectrum of SWJT-9 showed an obvious absorption band centered at 430 nm. After the addition of ClO − to the solution, the absorbance red-shifted to about 550 nm. The color of the solution changed from yellow to pink (Figure 1a, inset). These results suggested that a reaction might occur between SWJT-9 and ClO − . In the fluorescence spectrum, SWJT-9 showed weak emission at about 667 nm (Φ = 0.018) under excitation at 550 nm ( Figure 1b). After the addition of ClO − , the fluorescence increased (Φ = 0.113) [56], and the fluorescence color of the solution was observed from red to deep red (Figure 1b, inset). These results indicated that SWJT-9 could detect hypochlorite anions by colorimetric and fluorescence turn-on responses. The fluorescence titration experiments were then conducted. As shown in Figure 1c, the fluorescence intensity enhanced with the increase of the ClO − concentration, indicating that SWJT-9 was a turn-on fluorescence probe. The detection limit was calculated as 24.7 nM ( Figure S6, Supplementary Materials), which was far lower than the concentration of hypochlorite anions produced by cells [57].
Molecules 2023, 28, x FOR PEER REVIEW 3 ( Figure S6, Supplementary Materials), which was far lower than the concentration o pochlorite anions produced by cells [57]. In addition, it is well known that the reaction time is an important paramete examining intracellular hypochlorite anions [28]. The shorter the time, the bette recognition. As shown in Figure 1d, the fluorescence intensity tends to equilibrate w two minutes. These results showed that this probe has high sensitivity to hypochl anions. Moreover, the constant (kobs) of the pseudo-first-order reaction was calculat be 0.03041 s -1 , and the t1/2 was 23 s ( Figure S7, Supplementary Materials).

Competition Experiments
As show in Figure 2, when active oxygen species or other anions were added t solution of SWJT-9, the fluorescence intensity was still weak. The fluorescence inte at 667 nm significantly enhanced when ClO − was added to the above solution. Thes sults indicated that the presence of other reactive oxygen species or anions would interfere with the recognition of ClO − by SWJT-9. The probe had a good anti-interfer property and potential application in biological environments. In addition, it is well known that the reaction time is an important parameter for examining intracellular hypochlorite anions [28]. The shorter the time, the better the recognition. As shown in Figure 1d, the fluorescence intensity tends to equilibrate within two minutes. These results showed that this probe has high sensitivity to hypochlorite anions. Moreover, the constant (k obs ) of the pseudo-first-order reaction was calculated to be 0.03041 s −1 , and the t 1/2 was 23 s ( Figure S7, Supplementary Materials).

Competition Experiments
As show in Figure 2, when active oxygen species or other anions were added to the solution of SWJT-9, the fluorescence intensity was still weak. The fluorescence intensity at 667 nm significantly enhanced when ClO − was added to the above solution. These results indicated that the presence of other reactive oxygen species or anions would not interfere with the recognition of ClO − by SWJT-9. The probe had a good anti-interference property and potential application in biological environments.

Response Mechanism
In order to verify the possible reaction mechanism of SWJT-9 and ClO -, 1 H NMR titration ( Figure 3) and MS spectrum ( Figure S8, Supplementary Materials) were used. As shown in Figure 3, the proton signal of the C=N bond (Ha) appeared at 8.5 ppm. When ClOwas added, the peak (Ha) disappeared gradually while a new proton (Hb) appeared. Compared with two spectrum (SWJT-9 + ClO − and compound 3), they were basically the same, which indicated 3 was the product from a reaction of SWJT-9 and ClO − . In addition, as shown in Figure S8

Response Mechanism
In order to verify the possible reaction mechanism of SWJT-9 and ClO -, 1 H NMR titration ( Figure 3) and MS spectrum ( Figure S8, Supplementary Materials) were used. As shown in Figure 3, the proton signal of the C=N bond (H a ) appeared at 8.5 ppm. When ClOwas added, the peak (H a ) disappeared gradually while a new proton (H b ) appeared. Compared with two spectrum (SWJT-9 + ClO − and compound 3), they were basically the same, which indicated 3 was the product from a reaction of SWJT-9 and ClO − . In addition, as shown in Figure S8

DFT Calculations
In order to investigate the relationship between the probe and the spectral changes, density functional theory (DFT, B3LYP/6-311G, Gaussian 09) calculations were performed [58]. As shown in Figure 4, the HOMO electron density of SWJT-9 was distributed on the dicyanoisophorone skeleton and diaminomaleonitrile group. However, for the LUMO level, the electron density was only located on the dicyanoisophorone skeleton, which meant that C=N isomerization occurred to lead to the weak fluorescence of SWJT-9 [59].

DFT Calculations
In order to investigate the relationship between the probe and the spectral changes, density functional theory (DFT, B3LYP/6-311G, Gaussian 09) calculations were performed [58]. As shown in Figure 4, the HOMO electron density of SWJT-9 was distributed on the dicyanoisophorone skeleton and diaminomaleonitrile group. However, for the LUMO level, the electron density was only located on the dicyanoisophorone skeleton, which meant that C=N isomerization occurred to lead to the weak fluorescence of SWJT-9 [59]. At the HOMO and LUMO levels, the electrons of compound 3 were all mainly distributed in the whole dicyanoisophorone group, indicating that compound 3 had a strong fluorescence emission. These results suggested that SWJT-9 could be considered as a turn-on probe for ClO − .

Cytotoxicity of SWJT-9 and Its Imaging in HeLa Cells
In order to further prove the excellent performance of the near-in of SWJT-9, HeLa cells were used ( Figure 5) to study bioimaging. Fi incubated with SWJT-9 (10.0 μM) for 30 minutes. As shown in Figure of SWJT-9 was very weak in the red channel. In the other group, SW were incubated at 37 °C for 30 minutes and then incubated with minutes. The fluorescence in the red channel improved obviously. Th that SWJT-9 has good cell membrane transparency and can detect Cl addition, the CCK test results showed that SWJT-9 did not produce city in HeLa cells ( Figure 6).

Cytotoxicity of SWJT-9 and Its Imaging in HeLa Cells
In order to further prove the excellent performance of the near-infrared fluorescence of SWJT-9, HeLa cells were used ( Figure 5) to study bioimaging. First, HeLa cells were incubated with SWJT-9 (10.0 µM) for 30 min. As shown in Figure 5b, the fluorescence of SWJT-9 was very weak in the red channel. In the other group, SWJT-9 and HeLa cells were incubated at 37 • C for 30 min and then incubated with ClOfor another 20 min. The fluorescence in the red channel improved obviously. These results indicated that SWJT-9 has good cell membrane transparency and can detect ClO − in HeLa cells. In addition, the CCK test results showed that SWJT-9 did not produce significant cytotoxicity in HeLa cells ( Figure 6). incubated with SWJT-9 (10.0 μM) for 30 minutes. As shown in Figure 5b, the fluoresc of SWJT-9 was very weak in the red channel. In the other group, SWJT-9 and HeLa were incubated at 37 °C for 30 minutes and then incubated with ClOfor anothe minutes. The fluorescence in the red channel improved obviously. These results indic that SWJT-9 has good cell membrane transparency and can detect ClO − in HeLa cel addition, the CCK test results showed that SWJT-9 did not produce significant cyto city in HeLa cells ( Figure 6).

Materials and Reagents
The materials, reagents, and detection methods are described in the Supplementary Materials.

Synthesis of Probe SWJT-9
Compound 1 and compound 2 were prepared according to the previously reported literature [60].
Compound 3 (90.10 mg, 0.26 mmol) and diaminomaleonitrile (32.01 mg, 0.30 mmol) were added to ethanol (10 mL). Then, the mixture was heated at 80 °C for two hours and then filtered with suction. After washing with ethanol, SWJT-9 can be obtained as a solid. Yield: 45.0 %. 1

Materials and Reagents
The materials, reagents, and detection methods are described in the Supplementary Materials.

Synthesis of Probe SWJT-9
Compound 1 and compound 2 were prepared according to the previously reported literature [60].
Compound 3 (90.10 mg, 0.26 mmol) and diaminomaleonitrile (32.01 mg, 0.30 mmol) were added to ethanol (10 mL). Then, the mixture was heated at 80 • C for two hours and then filtered with suction. After washing with ethanol, SWJT-9 can be obtained as a solid. Yield: 45.0 %. 1

Conclusions
In conclusion, a near-infrared fluorescent probe SWJT-9 for ClO − detection was developed. It exhibited a large Stokes shift (237 nm), high selectivity, good sensitivity, and fast response. An obvious color change was observed by the naked eye under visible light or ultraviolet light. SWJT-9 was a new near-infrared fluorescent probe using diaminomaleonitrile as the recognition group, and it can be successfully used in cells, indicating its potential use in biological analyses. This work could provide some inspiration for future research of near-infrared fluorescence probes.