Development of Sulfite‐Specific Fluorescent Probes Based on Isolongifolanone and Their Applications in Biological Imaging

ABSTRACT

Isolongifolanone, a natural product, offers several advantages over traditional small organic molecules, including excellent biocompatibility, high reactivity, low cytotoxicity, affordability, and environmental friendliness. While numerous compounds derived from isolongifolanone have been developed, most research has focused primarily on its antitumor properties, with limited exploration of its potential applications as a fluorescent probe. In this study, we designed and synthesized a series of novel natural small‐molecule fluorescent probes based on isolongifolanone, utilizing pyrazole compounds as the fluorophore, vanillin or p‐hydroxybenzaldehyde as the linker, and levulinate as the sulfite‐specific recognition group. The probes react with sulfite through a nucleophilic addition reaction, resulting in a strong fluorescence signal, demonstrating high sensitivity and good selectivity for sulfite detection. Quantitative detection of sulfite can thus be achieved by monitoring the fluorescence intensity. The chemical structure and reaction mechanism of the fluorescent probes were characterized using NMR, LC‐MS, and HRMS. Additionally, the isolongifolanone‐based fluorescent probes were successfully applied for the detection of sulfites in tumor cells, significantly broadening the application scope and value of isolongifolanone.

Isolongifolanone‐based natural small‐molecule fluorescent probes are designed and synthesized using pyrazole fluorophores, aldehyde linkers, and levulinate recognition units for selective sulfite detection. The probes exhibit strong fluorescence enhancement via a nucleophilic addition reaction, enabling sensitive quantitative detection and cellular imaging of sulfite in tumor cells, thereby expanding the application scope of isolongifolanone.

1. Introduction

Sulfite (SO₃ ²⁻), as a common chemical substance, exists widely in nature and industry, and its detection has significant biological implications in various fields. First, in the food industry, sulfites are commonly used as preservatives and bleaches, but excessive use will cause harm to human health, so its content monitoring is crucial to ensure food safety [1, 2]. Second, sulfite is an important indicator of environmental pollution; through the detection of sulfite content in water, atmosphere, and soil, it can effectively evaluate and control environmental pollution [3]. In addition, sulfites also play a key role in the metabolic process in organisms [4, 5, 6], and detection of their levels is helpful to study biological metabolic pathways and regulatory mechanisms. In the medical field, abnormal sulfite levels in the body are associated with a variety of diseases [7, 8, 9], so detecting sulfite content in the body can assist in disease diagnosis and treatment. In summary, sulfite detection is essential for ensuring food safety, environmental protection, and biomedical research. Therefore, it is of great significance to develop a rapid, sensitive and specific method for the determination of sulfite.

At present, traditional methods for detecting sulfite include colorimetry [10, 11], titration [12], chromatography [13, 14], spectrophotometry [15, 16], electrochemical analysis [17], and capillary electrophoresis [18, 19]. However, these methods usually have some problems such as complicated sample pretreatment, expensive equipment, cumbersome operation, long detection time, and low sensitivity, which makes it difficult to realize real‐time sample detection. In contrast, the fluorescence probe technique has the advantages of simple sample preparation, easy operation, fast detection speed, high sensitivity, and good selectivity [20, 21, 22, 23]. Therefore, it is of great significance to develop fluorescent probes with fast response speed, high sensitivity, and real‐time monitoring of sulfite.

Isolongifolanone, as a natural product derived from turpentine, has many advantages such as good biocompatibility, high reactivity, low price, and green environmental protection compared with traditional organic small molecules [24, 25]. Additionally, due to the carbonyl and active hydrogen in the structure of isongifolanone, a variety of new derivatives can be synthesized after chemical modification. As a result, isolongifolanone is widely used in the pharmaceutical and fragrance fields [26, 27, 28]. But so far, most of them are still used in the research of antitumor drugs; the development of organic small molecules with fluorescence properties has rarely been reported. Therefore, the synthesis of fluorescent probes with good biocompatibility based on isongifolanone has positive significance for the development and utilization of turpentine.

In this work, we designed and synthesized a series of novel natural organic small molecule fluorescence probes with specific response to sulfites, using isongifolanone as the starting skeleton and levulinate as the sulfite recognition group. When the sulfite ions were added to the probe, the nucleophilic reaction occurred between the sulfite ions and the probe molecules, and then the levulinate was shed from the probe, the hydroxyl group was exposed, and the intramolecular charge transfer (ICT) effect was formed, resulting in an enhanced fluorescence signal. Thus, the quantitative detection of sulfite can be realized by detecting the change in fluorescence intensity. The response mechanism of the probe to sulfite was verified by mass spectrometry and fluorescence spectrum, and the linear relationship and detection limit between the fluorescence intensity and the concentration of sulfite were established. Besides, these probes exhibit good cell permeability, enabling the detection and imaging of sulfites in living cells. This study not only expands the application field of isongifolanone, but also provides a new idea for the rapid detection of sulfites.

2. Results and Discussion

2.1. Synthesis and Characterization

Firstly, four sulfite‐specific fluorescent probes (IFA‐1, IFA‐2, IPM‐1, and IPM‐2) were synthesized from isolongifolanone through aldol condensation, cyclization, and addition, as shown in Scheme 1. In the synthetic design, isolongifolanone was employed as the core scaffold. The phenolic hydroxyl group of vanillin or p‐hydroxybenzaldehyde was first protected with a MOM group, followed by an aldol condensation reaction to construct the key intermediate. Subsequently, pyrazole derivatives bearing different substituents were introduced as fluorophoric units to further build the key fluorescent intermediates [29, 30]. After removal of the MOM protecting group, the corresponding fluorescent dyes were obtained. Finally, levulinic acid was used as the sulfite‐responsive moiety and was conjugated with the hydroxyl group of the fluorescent dyes via a condensation reaction, affording fluorescent probes with sulfite‐specific response characteristics. Each step of the synthesis was carefully monitored to ensure the formation of the desired products. After synthesis, all compounds, including intermediates and the final probes, were thoroughly characterized using various analytical techniques. We employed ¹H NMR and ¹³C NMR to confirm the structural integrity and purity of the synthesized compounds. Additionally, high‐resolution mass spectrometry (HRMS) was utilized to verify the molecular weights and structural confirmations of the probes. The results of these characterizations are presented in Figures S1–S15, providing a comprehensive overview of the synthesized compounds and their structural features. To further evaluate the stability of the developed probes for practical applications, the stability of probe IPM‐2 was systematically investigated. HPLC analysis showed that the chromatographic profile of IPM‐2 remained essentially unchanged after prolonged light irradiation and long‐term storage in PBS (pH 7.4), with no detectable degradation observed (Figure S16). These results indicate excellent photostability and storage stability, confirming its suitability for biological applications.

SCHEME 1.

Synthesis route of sulfite‐specific fluorescent probe: IFA‐1, IFA‐2, IPM‐1 and IPM‐2. (Reaction conditions: (I) tetrahydrofuran, sodium hydride, 0°C; (II) ethanol, sodium hydroxide, 88°C; (III) ethanol, potassium tert‐butanol, 88°C; (IV) methanol, hydrochloric acid, 70°C; (V) dichloromethane, EDCl, DMAP, r.t.).

2.2. Optical Properties

In this section, the sensing property of the above four probes (IFA‐1, IFA‐2, IPM‐1, and IPM‐2) specifically for sulfite detection was investigated. The fluorescence spectra of each probe reacted with different concentrations of sulfite were investigated in phosphate‐buffered saline (PBS) solution. As illustrated in Figure 1 and Table 1, fluorescence spectroscopy revealed that the four probes exhibit similar excitation and emission characteristics. This finding suggests that they share certain structural similarities, which may contribute to a consistent fluorescence response mechanism. Additionally, the fluorescence intensity of the probes exhibited a gradual increase with the rising concentrations of sulfite (Figure 1A–H). This trend indicates that the probes effectively respond to sulfite, allowing for real‐time monitoring of its concentration. Moreover, there is a strong linear relationship between the fluorescence intensity and the concentration of sulfite within the range of 0–10 µM, as illustrated in Figure 1I–L: IFA‐1 (y = 4.2658x + 129.03, R ² = 0.9775), IFA‐2 (y = 7.5999x + 246.19, R ² = 0.9982), IPM‐1 (y = 8.2943x + 412.20, R ² = 0.9917) and IPM‐2 (y = 10.6541x + 264.22, R ² = 0.9952). This linearity underscores the probes' ability to quantitatively detect sulfite levels accurately. In the meantime, the detection limits of IFA‐1, IFA‐2, IPM‐1, and IPM‐2 for sulfite were calculated by the formula LOD = 3σ/k to be 2.135, 1.180, 0.886, and 0.729 µM (Table 1), respectively, all demonstrating the high sensitivity of all four probes in detecting sulfite at low concentrations. To further elucidate their photophysical properties, UV–vis absorption spectra of all probes were additionally recorded (Figure S17A). All probes exhibited strong absorption bands in the range of 270–330 nm, with molar extinction coefficients (ε) of (1.84–3.06) × 10⁴ M⁻¹ cm⁻¹ at 307.0 nm (Table 1). In addition, fluorescence quantum yields (Φ _f) were determined using the standard relative method with quinine sulfate as the reference standard under identical experimental conditions, and the corresponding values are summarized in Table 1. Furthermore, the UV–vis spectral changes of probe IPM‐2 before and after reaction with sulfite were investigated. As shown in Figure S17B, an increase in absorbance accompanied by a slight redshift was observed upon addition of SO₃ ²⁻. Notably, the resulting absorption profile closely resembles that of the parent compound IPM‐OH, suggesting the formation of the corresponding product upon reaction. Consequently, the findings suggest that these four probes are highly suitable candidates for rapid quantitative analysis of sulfite, offering reliable performance for applications requiring swift detection and measurement.

FIGURE 1.

The fluorescence emission spectra of the probes: (A) IFA‐1, (B) IFA‐2, (C) IPM‐1, and (D) IPM‐2, in response to increasing concentrations of sulfite in PBS buffer. The correlation curve between the fluorescence intensity of (E) IFA‐1, (F) IFA‐2, (G) IPM‐1, and (H) IPM‐2 at 480 nm and the sulfite concentration. The linear relationship between the fluorescence intensity of (I) IFA‐1, (J) IFA‐2, (K) IPM‐1, and (L) IPM‐2 at 480 nm and the sulfite concentration. The measurements were performed in PBS buffer (10 mM, pH 7.4) containing a probe concentration of 10 µM, and the samples were incubated with sulfite for 60 min prior to fluorescence measurements at 37°C. Data are presented as mean ± S.D. (n = 3).

TABLE 1.

Comparison of the sulfite‐specific fluorescent probes IFA‐2, IFA‐1, IPM‐1 and IPM‐2.

Probe	λ ex/λ em (nm)	Detection limit (µM)	Response time (min)	Stokes shift (nm)	ε (M−1 cm−1)	Φ f (reacting with SO3 2−)
IFA‐2	307/482	1.180	50	175	2.44 × 104	0.165
IFA‐1	304/485	2.135	30	181	2.65 × 104	0.056
IPM‐1	310/482	0.886	15	172	1.84 × 104	0.090
IPM‐2	310/488	0.729	50	178	3.06 × 104	0.049

2.3. Response Mechanism

Based on the observations from the fluorescence spectra, we proposed a potential reaction mechanism for the four isolongifolanone‐based fluorescent probes in response to sulfite, as illustrated in Scheme 2. Initially, the electron‐withdrawing nature of the levulinate moiety suppresses the ICT process, resulting in a weak fluorescence signal from the probes [31, 32]. However, upon the addition of sulfite, the nucleophilic nature of the sulfite leads it to actively attack the carbonyl carbon at the 4‐position of the levulinic acid, resulting in ester bond cleavage and changes in the electronic state of the molecule, which produce a substantial fluorescence enhancement. To further substantiate our proposed mechanism, we conducted detailed mass spectrometry analyses, the results of which are presented in Figures S18–S20. These analyses provided valuable insights into the reaction products formed after the probes interacted with sulfite. Notably, the products were consistent with the expected data for the fluorescent dye we designed, thereby confirming the validity of the mechanism outlined in Scheme 2. In summary, our proposed mechanism clarifies the interactions between the fluorescent probes and sulfite, not only reinforcing the chemical basis for the probes’ sensitivity but also opening avenues for further exploration in the development of efficient fluorescent sensors.

SCHEME 2.

Reaction mechanism for probes to SO₃ ²⁻.

2.4. Response Time

Since the response time is a critical factor in evaluating the detection performance of a probe [33, 34], we systematically measured the time‐dependent fluorescence intensity of the probes in the presence of sulfite. As shown in Figure 2, the fluorescence intensity of the probes at 480 nm gradually increased over time following the addition of sulfite. Among the four probes tested, IPM‐1 exhibited the fastest response, reaching nearly saturation within just 15 min. This rapid increase in fluorescence intensity continued to stabilize within 20 min, showcasing its efficiency in detecting sulfite concentrations quickly. In comparison, the response times for the other three probes IFA‐1, IFA‐2, and IPM‐2 were 30, 50, and 50 min, respectively (Table 1). To quantitatively compare the reaction kinetics, the fluorescence evolution profiles were further analyzed using a pseudo‐first‐order kinetic model. The corresponding pseudo‐first‐order rate constants (k _obs) for IFA‐1, IFA‐2, IPM‐1, and IPM‐2 were determined to be 0.0934, 0.0571, 0.2159, and 0.0584 min⁻¹, respectively. These results are consistent with the observed response times, with IPM‐1 exhibiting the largest k _obs value, indicating the fastest reaction with sulfite among the four probes. Therefore, the four probes we designed exhibit moderate response times, with probe IPM‐1 emerging as an excellent candidate for the rapid quantitative detection of sulfite, offering both efficiency and reliability in practical applications.

FIGURE 2.

Fluorescence spectra and dynamic curves of the fluorescent probes (A) IFA‐1, (B) IFA‐2, (C) IPM‐1, and (D) IPM‐2 after reacting with SO₃ ²⁻ for different durations. The measurements were performed in PBS buffer (10 mM, pH 7.4) containing a probe concentration of 10 µM. Data are presented as mean ± S.D. (n = 3).

2.5. Selectivity and Specificity of the Probes

In order to further evaluate the selectivity and specificity of our synthesized probes (IFA‐1, IFA‐2, IPM‐1, and IPM‐2) toward SO₃ ²⁻, the fluorescence intensity of the probe in the presence of various potential interferents commonly found in biological and environmental samples was investigated in PBS buffer. As shown in Figures 3 and S21, only SO₃ ²⁻ induced a significant enhancement of the fluorescence signal, and the addition of the other interferents containing Br⁻, Cl⁻, Cys, F⁻, Gla, GSH, H₂O₂, NH₄ ⁺, NO²⁻, HS⁻, and SO₄ ²⁻ induced no significant change in the fluorescence intensity. This result indicates that these probes possess satisfactory specificity to SO₃ ²⁻. On the other hand, in a coexistence system including both the interferents and sulfite, the response of the probes toward sulfite was not significantly disturbed, suggesting that these probes are capable of specific sensing of SO₃ ²⁻ in most biological environments. In summary, our research findings confirm that these probes not only exhibit high selectivity for sulfite but also effectively resist interference from other common analytes. This characteristic significantly enhances their potential for reliable quantitative analysis in practical applications, making them valuable tools for monitoring sulfite levels in various environments.

FIGURE 3.

The fluorescence intensity of probe (A) IFA‐1, (B) IFA‐2, (C) IPM‐1, and (D) IPM‐2 in the presence of SO₃ ²⁻ and other relevant interferents. Black column: probe + interferent, red column: probe + interferent + SO₃ ²⁻. All fluorescence measurements were performed using a probe concentration of 10 µM in PBS buffer (10 mM, pH 7.4), and the concentrations of the interferents were as follows: Cl⁻, F⁻, Br⁻, NH₄ ⁺, NO₂ ⁻, and SO₄ ²⁻ at 3 mM; Cys and GSH at 2 mM; H₂O₂ at 200 µM. Data are presented as mean ± S.D. (n = 3).

2.6. Fluorescence Imaging in Living Cells

Motivated by the SO₃ ²⁻‐activated fluorescence enhancement of probes IFA‐1, IFA‐2, IPM‐1 and IPM‐2 in aqueous solution, we next investigated their capability for imaging sulfite levels in living cells. Prior to cellular imaging experiments, the cytotoxicity of the four probes and the fluorescent product IPM‐OH, formed from the reaction of IPM‐2 with sulfite, was evaluated through the widely used MTT (3‐(4,5‐ dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. The results demonstrated that all probes exhibited negligible cytotoxicity toward 4T1 cells at concentrations ranging from 0 to 40 µM (Figure S22). Notably, after 24 h of incubation, cell viability remained consistently above 80%, indicating the excellent biocompatibility of the probes. Subsequently, the exogenous sulfite in 4T1 cells was imaged through the fluorescence microscope. As shown in Figures 4 and S23–S25, the cells incubated exclusively with the probes at 37°C for 60 min displayed a rather weak fluorescence signal. Subsequently, the cells were pretreated with different concentrations of sulfite (0, 20, 40, and 80 µM) for 30 min before incubating them with each probe for another 60 min. As anticipated, a remarkable fluorescence signal was observed in the cells following sulfite treatment, indicating that the presence of sulfite in the cell effectively activated the probes. Notably, the intensity of the fluorescence gradually increased with higher concentrations of sulfite. Meanwhile, we evaluated the imaging capability of IPM‐2 for endogenous sulfite. To the best of our knowledge, endogenous sulfite in living cells can be generated upon stimulation with norepinephrine bitartrate [35, 36]. Compared with the control group, the cells pretreated with norepinephrine bitartrate for 30 min exhibited a distinct blue fluorescence signal (Figure S26). These findings indicate that the probes we designed and synthesized can effectively penetrate living cells and provide real‐time fluorescence imaging of both exogenous and endogenous sulfite levels in cells, highlighting their significant potential in biological research and medical diagnostics.

FIGURE 4.

(A) Fluorescent imaging of 4T1 cells pretreated with different concentrations of sulfite (0, 20, 40, and 80 µM) for 30 min and then further incubated with IPM‐2 for 60 min. (B) Quantification of fluorescence intensity of 4T1 cells in (A). Data are presented as mean ± S.D. (n = 3). The statistical significance level is ***p < 0.001.

3. Conclusions

In summary, we have developed a series of intelligent sulfite‐responsive fluorescent probes based on the framework of the natural product isolongifolanone. In this study, we conducted a comprehensive characterization of the chemical structures of these fluorescent probes and systematically investigated their optical properties, demonstrating high sensitivity and rapid response to sulfite. Compared with previously reported sulfite probes (Table S1), the probes developed in this study exhibit a notably larger Stokes shift, which helps to reduce spectral overlap, minimize background interference, and improve the reliability of fluorescence detection. Additionally, these probes exhibited excellent stability and selectivity for sulfite in the presence of various interferents. Notably, they were successfully employed to monitor exogenous sulfite levels in living cells. This research not only expands the novel application domains of isolongifolanone but also provides valuable insights for related fields of study. Future work will focus on further optimizing these probes to enhance sensitivity and specificity, as well as exploring their applicability in in vivo models.

4. Experimental Section

4.1. Materials and Instruments

Unless otherwise specified, chemical reagents were obtained from commercial vendors and used without further purification. Reactions were monitored using thin‐layer chromatography (TLC). High‐resolution mass spectrometry (HRMS) was conducted with an LC‐MS2010A instrument. Fluorescence spectra were recorded using a Hitachi F‐4500 fluorescence spectrophotometer with excitation and emission slit widths of 5 and 5 nm, respectively. Fluorescence images of the cells were captured with a Leica fluorescence microscope.

4.2. Optical Studies of Probes

Add 10 µL of the fluorescent probe stock solution and 90 µL of DMSO to each of the thirteen 10 mL centrifuge tubes. Then, add varying volumes of sodium sulfite stock solution and PBS buffer to prepare 13 test solutions, each containing different concentrations of sodium sulfite. Subsequently, measure the fluorescence emission spectra of each test solution and record the changes in fluorescence intensity.

4.3. Response Time

Add 10 µL of the fluorescent probe stock solution and 90 µL of DMSO to a centrifuge tube, followed by 600 µL of sodium sulfite stock solution and 300 µL of PBS solution. This mixture yields a test solution with a final concentration of 10 µM for the fluorescent probe and 600 µM for sodium sulfite. Measure the fluorescence emission spectra at various time points (0, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50, and 60 min) to observe the reaction between the fluorescent probe and sulfite, and record the changes in fluorescence intensity over time. Ensure that three parallel samples are set up for each test condition to guarantee accuracy.

4.4. Selectivity of Probe for Sulfite

Prepare 10 10 mL centrifuge tubes and add 10 µL of the fluorescent probe stock solution along with 90 µL of DMSO to each tube. Then, add varying interferents to each tube to prepare test solutions containing 10 µM of the fluorescent probe along with different concentrations of interferents. The concentrations of the interferents are as follows: Cl⁻, F⁻, Br⁻, NH₄ ⁺, NO₂ ⁻, and SO₄ ²⁻ at 3 mM; Cys and GSH at 2 mM; H₂O₂ at 200 µM; and SO₃ ²⁻ at 600 µM. Measure the fluorescence emission spectra of each solution and record the fluorescence intensity ratio at 485 nm. For accuracy, prepare three parallel samples for each condition.

4.5. Specificity of Probe for Sulfite

Prepare 10 10 mL centrifuge tubes, each containing a test solution with the fluorescent probe (IFA‐1, IFA‐2, IPM‐1, and IPM‐2), sodium sulfite, and various interferents at different concentrations. The concentration of fluorescent probe was set to 10 µM, while sodium sulfite was added at 800 µM. The concentrations for each interferent are as follows: Cl⁻, F⁻, Br⁻, NH₄ ⁺, NO₂ ⁻, and SO₄ ²⁻ at 3 mM; Cys and GSH at 2 mM; H₂O₂ at 200 µM. Record the fluorescence emission spectra for each solution using a fluorescence spectrophotometer, noting the fluorescence intensity ratio at the maximum emission wavelength. To ensure reliable results, prepare three parallel samples for each condition.

4.6. Cell Culture and Imaging

The mouse breast cancer 4T1 cell line (fourth mammary tumor cell line 1) was purchased from Wuhan Servicebio Technology Co., Ltd. (catalogue number: CVCL_0125). 4T1 cells were cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified incubator. The atmosphere was maintained at 5% CO₂ and 95% air. When the cells reached approximately 80%–90% confluency, they were washed with PBS (pH 7.4) and treated with 0.25% trypsin‐0.9 mM EDTA solution to detach the cells. After detachment, the cells were subcultured, and the growth medium was replaced every two days to maintain optimal cell growth conditions.

To perform the cell imaging assay, 4T1 cells were seeded in a 12‐well plate and incubated in a cell culture incubator. After 24 h, the culture medium was removed, and fresh medium containing different concentrations of sodium sulfite (0, 20, 40, and 80 µM) was added to the cells and incubated for 30 min. Subsequently, the cells were incubated with 20 µM of the fluorescent probes (IFA‐1, IFA‐2, IPM‐1, and IPM‐2) for an additional 1 h. Then the medium was removed, and the cells were washed three times with PBS buffer. Finally, the cells were imaged by a fluorescence microscope.

4.7. Statistical Analysis

All quantitative data are presented as mean ± standard deviation (SD), with a minimum sample size of n ≥ 3 for each experiment. Statistical significance was evaluated using two‐tailed Student's t‐test. Statistical significance was indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001. All statistical analyses were performed using GraphPad Prism and Origin.

Author Contributions

Qi Zheng: investigation, performed the experiments, formal analysis. Anna Wang: writing – original draft. Zhongwen Sun: nvestigation, methodology. Yushun Yang: investigation, methodology. Juan Sun: conceived the study, supervision, investigation, funding acquisition, writing – review and editing. Yang Zhou: conceived the study, supervision, investigation, funding acquisition, writing – review and editing. Bo Chen: conceived the study, supervision, investigation, funding acquisition, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv71565‐sup‐0001‐SuppMat.docx

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.