Constructing Near-Infrared Dyes with D-A-D Type and large Stokes Shift Based on Asymmetric Electron Donating Properties and their Applications in biological systems

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Data may be preliminary. 3 June 2025 V1 Latest version Share on Constructing Near-Infrared Dyes with D-A-D Type and large Stokes Shift Based on Asymmetric Electron Donating Properties and their Applications in biological systems Authors : Mao-Hua Wang , Yun-Hao Yang , and Jian-Yong Wang 0000-0002-5254-1525 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174894144.45479136/v1 150 views 125 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract As one of the important atmospheric pollutants, sulfur dioxide and its derivatives are widely used in the production of additives and chemicals, as well as being one of the important factors affecting physiological activities. To achieve the goal of detection, we have developed a new near-infrared fluorescent probe Cou-Oxo , which can be used to ratiometric SO 2 derivatives in cells, zebrafish, and plant rhizomes. A novel near-infrared dye with a large π-system has been created by combining two small π-dyes. The dye Stokes shifts across was also expand by its structure (321 nm) largely. The NIR ratiometric fluorescence properties of the probe are achieved through the addition reaction of SO 2 derivatives (HSO 3 - ) to the C=C double bond on the Cou-Oxo probe. It showed a detection line of 0.942 μM, a short response time (within 30 s), good biocompatibility, excellent anti-interference, and pH stability. The probe was successfully used to detect changes in exogenous/endogenous SO 2 levels in cancer cells and living zebrafish. More interestingly, we successfully accomplished the visualization of SO 2 in normal and SO 2 -poisoned seedlings using tobacco seedlings as a model. not-yet-known not-yet-known not-yet-known unknown Cite this paper: Chin. J. Chem. 2024, 42, XXX—XXX. DOI: 10.1002/cjoc.202400XXX Constructing Near-Infrared Dyes with D-A-D Type and large Stokes Shift Based on Asymmetric Electron Donating Properties and their Applications in biological systems Mao-Hua Wang,a Yun-Hao Yang, a and Jian-Yong Wang*, a a a Faculty of Light Industry, State Key Laboratory of Green Papermaking and Resource Recycling, Key Laboratory of Pulp and Paper Science & Technology of Ministry of Education, Qi Lu University of Technology (Shandong Academy of Sciences), Jinan, 250353, P. R., China. No.3501, Daxue Road, Changqing District, Jinan, 250353, Shandong Province, PR China Keywords Sulfur dioxide | Near-infrared | Ratiometric | Fluorescent probe Comprehensive Summary As one of the important atmospheric pollutants, sulfur dioxide and its derivatives are widely used in the production of additives and chemicals, as well as being one of the important factors affecting physiological activities. To achieve the goal of detection, we have developed a new near-infrared fluorescent probe Cou-Oxo , which can be used to ratiometric SO 2 derivatives in cells, zebrafish, and plant rhizomes. A novel near-infrared dye with a large π-system has been created by combining two small π-dyes. The dye Stokes shifts across was also expand by its structure (321 nm) largely. The NIR ratiometric fluorescence properties of the probe are achieved through the addition reaction of SO 2 derivatives (HSO 3 - ) to the C=C double bond on the Cou-Oxo probe. It showed a detection line of 0.942 μM, a short response time (within 30 s), good biocompatibility, excellent anti-interference, and pH stability. The probe was successfully used to detect changes in exogenous/endogenous SO 2 levels in cancer cells and living zebrafish. More interestingly, we successfully accomplished the visualization of SO 2 in normal and SO 2 -poisoned seedlings using tobacco seedlings as a model. Background and Originality Content As one of the major pollutants in the atmosphere, sulfur dioxide (SO 2 ) was emitted mainly from the mining of sulfur-containing minerals, chemical production activities and volcanic eruptions [1-3] . Sulfur dioxide (SO 2 ) was generally present in organisms in the form of its metabolic derivatives (sulfites and bisulfites) [4-6] . Studies had shown that sulfur dioxide derivatives could be present in the body through the respiratory tract or food [7-9] . Excessive inhalation of SO 2 might cause respiratory damage and even inflammation, neurological damage and cardiovascular disease [10-12] . In addition, sulfur dioxide derivatives could be endogenously produced through enzyme catalysis and intracellular oxidative stress reactions [13-16] . They were involved in regulating processes such as redox in organisms [9,17,18] . That is to say, SO 2 derivatives, an endogenous signaling molecule, played an important regulatory role in various physiological and pathological processes [19-21] . Therefore, the development of a rapid and accurate method to detect sulfur dioxide derivatives at the cellular level and in living organisms has become a challenge of practical importance. Various techniques have been reported for detecting sulfur dioxide (SO 2 ), including infrared spectroscopy [22] , electrochemical potential difference [23] , gas chromatography [24] , capillary chromatography [25] , etc. However, these methods exhibited certain limitations, such as time-consuming, complex operation, high instrument cost, and inability to detect in situ in real-time. The sensing technology was favored in biomedical, environmental and food testing fields because of easy operation, real-time in situ imaging and nondestructive detection [26-37] . Near-infrared fluorescent probes have been widely used in biomedical fields due to their long emission wavelength, high tissue penetration, and high signal background ratio, in comparison to conventional fluorescent dyes [38-43] . Therefore, it is necessary to develop a novel NIR fluorescent probe for the detection of SO 2 in organisms and real samples. The design and construction of conventional NIR molecular fluorophores can be broadly categorized into the following major types: Cyanine NIR dyes, Si-Rhodamines and donor-acceptor-donor (D-A-D) fluorophores [44,45] . Conventional Cyanine NIR dyes have been heavily used in the design of NIR molecules. However, they usually have a symmetric structure, which is not conducive to molecular orbital polarisation and are subject to significant solvent discolouration in polar solvents [46] . In general, these compounds suffer from low photostability, susceptibility to bleaching, small Stokes shifts, and severe self-absorption [47] . After structural optimization, some new asymmetric Cyanine analogues have been constructed. These compounds have been reported to have more favorable properties [48] . However, most of the products are difficult to prepare or separate from their analogues (Figure 1). Herein, we constructed a fluorescent molecule of D-A-D structure by molecular design strategy with coumarin and xanthene units as the building blocks, malononitrile as the electron-absorbing group, and dimethylamine on both sides as the electron-donating group. The probe possessed a large conjugated backbone and a favorable ICT process, allowing it to emit in the near-infrared band (around 800 nm). The detection of SO 2 was achieved through nucleophilic addition reactions to unsaturated double bonds exclusively. Meanwhile, the probe was successfully employed to monitor sulfur dioxide and its derivatives in HeLa cells and zebrafish due to its high precision (ratiometric response), large Stocke shifts (321 nm) and good selectivity. For the more, we explored inflammation models in tobacco seedling roots and used the Cou-Oxo probe to detect sulfur dioxide derivative flux in situ. not-yet-known not-yet-known not-yet-known unknown Figure 1 (a)Structure of the reported NIR cyanines. (b) Rational design of the novel asymmetric structures NIR fluorophores Cou-oxo. Results and Discussion Design strategy and proposed SO 2 response mechanism for the probe Cou-Oxo To achieve this goal, we suggest an alternative approach for producing new NIR dyes with larger π systems from smaller π dyes (Figure 1). This involves connecting two different dyes with a double bond, where the rotational substituent of the double bond significantly enhances the geometrical relaxation of the fluorophore in the excited state, resulting in a larger Stokes shift [49] . Coumarin was a traditional fluorescent compound that has attracted significant attention from researchers due to its excellent photostability, biocompatibility, and ease of modification. Xanthene, on the other hand, exhibited long emission wavelengths and high quantum yields. The combination of coumarin with the xanthene fluorescence platform offers a promising approach for developing fluorescent probes with extensive conjugated structures that are sensitive to sulfur dioxide. The NIR probe Cou-Oxo with a D-A-D structure was synthesized by coupling the compounds Cou-CN and 3 through Knoevenagel condensation. As shown in Figure 2, the acrylonitrile component of the probe molecule functions as a potent electron acceptor, while the diethylamine group on the fluorophore acts as a strong electron donor, resulting in a typical highly conjugated structure. The compound exhibits a greatly enhanced intramolecular charge transfer (ICT) process, resulting in relatively large Stokes shifts and near-infrared emission (781 nm). In the presence of sulphur dioxide derivatives, nucleophilic addition occurs at the double bond of Cou-Oxo , disrupting the conjugate structure of the entire compound and causing changes in the spectrum. Figure 2 Structural design and reaction mechanism of the fluorescent probe Cou-Oxo and its colorimetric effect. Optical responses of the probe Cou-Oxo to SO 2 derivatives The absorption properties of the Cou-Oxo probe were tested in pure solvent. The results showed a broad single absorption peak at approximately 600 nm, which was less affected by changes in the solvent (Figure S1). We then investigated the spectral properties of the Cou-Oxo probe in a solution of sulfur dioxide. Figure S2 demonstrates that Cou-Oxo maintains a single broad absorption peak at 600 nm, which is affected by its large conjugate structure. The absorption peak at 600 nm disappears upon the addition of the sulfur dioxide derivative. Instead, an obvious narrow absorption peak appears near 400 nm. The blue shift of the absorption peak indicates that the conjugated structure of Cou-Oxo was damaged. The responsiveness of the Cou-Oxo probe to SO 2 derivatives (HSO 3 - ) was tested. Upon addition of NaHSO 3 (the main form of SO 2 present in aqueous solution), the fluorescence of the Cou-Oxo probe at 781 nm decreased at an excitation wavelength of 460 nm. A new emission peak at 550 nm gradually appeared and increased（Figure 3a, b and S3）indicating a clear ratiometric characteristic. This suggested that the conjugated structure of the probe has been broken. Subsequently, before and after the addition of SO 2 derivatives (HSO 3 - ), the fluorescence colour changed from non-emitting to producing a bright yellow colour (under 365 nm UV light) (Figure 3a inset). When SO 2 derivatives (HSO 3 - ) were added in 10 equal amounts, the ratio of the emission intensity increased 100-fold from the original (Figure 3c). The above results indicated that SO 2 derivatives (HSO 3 - ) disrupted the conjugated structure of Cou-Oxo , producing a clear ratiometric fluorescence signal. The correlation between the fluorescence ratio and the concentration of sulphur dioxide derivatives (HSO 3 - ) was found to be reliable (Y = 0.12321 + 0.74827X，R 2 = 0.998). The lower limit of detection for Cou-Oxo was 0.942 μM (based on standard method of 3σ/k) (Figure 3d). These results indicate that Cou-Oxo is effective in detecting sulphur dioxide derivatives and has potential for quantitative detection. Figure 3 Fluorescence spectra of the probe Cou-Oxo (10 μM) at 550 nm (a) and 781 nm (b) after the addition of HSO 3 - (0 - 100 μM) in DMSO/PBS buffers (v/v = 1/1). Inset: Photographs of Cou-Oxo solution with (right) or without (left) the addition of 50 μM NaHSO 3 in natural light and under UV light. (c) Plot of the fluorescence intensity ratio (I 550 nm/I 781 nm) versus NaHSO 3 (0-100 µM). (d) Linearity of fluorescence intensity ratio (I 550 nm/I 781 nm) versus NaHSO 3 (0-40 µM). λ ex = 460 nm. not-yet-known not-yet-known not-yet-known unknown Study of the reaction time of Cou-Oxo pH is a crucial parameter for maintaining the physiological activity of cells and tissues in living systems. Accurately detecting the sample to be tested in complex biological samples is also important for evaluating a probe’s ability to resist interference. Therefore, we evaluated the probe Cou-Oxo for pH tolerance and immunity. As illustrated in Figure 4a, the probe was insensitive to HSO3- at pH < 6. This was due to the fact that the Michael addition reaction required the production of carbon negative ions, which was catalyzed by a base. The process was inhibited by the presence of a large amount of H+ ions in an acidic environment. The fluorescence emission of Cou-Oxo at 550 nm was observed when the pH was between 7 and 10, and the detection ability in alkaline environments remained unchanged. The experimental results indicated that Cou-Oxo might have potential value under physiological conditions (pH=7.4). The selectivity of Cou-Oxo at 550 nm was then tested. Figure 4b showed that Cou-Oxo exhibited significant fluorescence enhancement only in systems containing HSO3- and SO32- among the various analytes tested (1. Probe 2. K+ 3. CO32- 4. Na+ 5. SO42- 6. Mg2+ 7. Ca2+ 8. CI- 9. Al3+ 10. NO3- 11. Fe2+ 12. NO2-13. Fe3+ 14. PO43- 15. Arg 16. H2O2 17. L-Arg 18. TBHP 19. Val. 20. GSH 21. Gly 22. SO32- 23. HSO3-), while it remained inert under other guest analytes. This demonstrates that the probe has some anti-interference ability in complex environments. Figure 4 (a) Fluorescence intensity of Cou-Oxo (10 µM) and the same equivalent of HSO 3 - out at 550 nm under different pH conditions. (b) Fluorescence emission of the probe Cou-Oxo (10 µM) at 550 nm in the presence of different analytes in DMSO/PBS buffers (v/v = 1/1). λ ex = 460 nm. Study of the reaction time of Cou-Oxo The fluorescence kinetics of Cou-Oxo were tested. Figure S4 showed that Cou-Oxo remained stable for 90 minutes with minimal fluctuation before the addition of the respondent. However, in the presence of HSO 3 - , the fluorescence emission of Cou-Oxo decreased instantly within 30 s (at 781 nm), demonstrating the rapid response of the probe to HSO 3 - . Additionally, the fluorescence intensity at 550 nm continued to increase and levelled off. The probe Cou-Oxo demonstrated inherent stability and responsiveness to HSO 3 - , making it a suitable option for real-time tracking of respondents over extended periods. not-yet-known not-yet-known not-yet-known unknown The mechanism of Cou-Oxo recognition of SO2 To verify the proposed reaction mechanism (Figure 1, 2), we optimized the structures of Cou-Oxo and the adduct Cou-Oxo-HSO3- using the density functional theory (DFT) method. As shown in Figure 5a, the oxanthene ring and the acrylonitrile moiety of Cou-Oxo were connected by π bonds and were coplanar, with a dihedral angle of about 63 degrees with the coumarin moiety. However, upon addition of HSO3-, the coplanar structure was partially destroyed and the conformation was distorted. The electron clouds of Cou-Oxo were partially delocalized on the LUMO and HOMO, primarily concentrated in the oxygenated anthracene ring and the acrylonitrile plane. Due to the planar difference between the oxygenated anthracene and coumarin moieties, the compound exhibited only partial intramolecular charge transfer (ICT) effect, corresponding to the 600 nm wide absorption peak of Cou-Oxo . Conversely, upon introduction of HSO3-, the distribution of the frontier molecular orbital (FMO) on the HOMO was predominantly dispersed on the oxygenated anthracene ring, while the benzopyran and acrylonitrile portions contributed to the distribution of FMO on the LUMO. The charge transformation induced by the HOMO-LUMO transition of Cou-Oxo-HSO3- . Additionally, the π bond between the acrylonitrile and oxygenated anthracene molecules was disrupted, inhibiting the previous partial electron transfer. With the complete intramolecular charge transfer from the coumarin acrylonitrile portion to the oxygenated anthracene ring, this was attributed to the PET channel, thereby quenching the emission of Cou-Oxo in the near-infrared [18,41]. Furthermore, the energy gaps of Cou-Oxo and Cou-Oxo-HSO3- levels were calculated to be 2.35 eV and 2.19 eV, respectively. Cou-Oxo-HSO3- exhibited a new peak at m/z 677.2405/677.2422 as determined by high-resolution mass spectrometry. These results supported the aforementioned speculation being consistent with experimental outcomes. Figure 5 Optimized models of the Cou-Oxo and Cou-Oxo-HSO 3 - geometry (a) and of the frontier molecular orbitals on the basis of B3LYP/6-31G(d) (b). Imaging of endogenous/exogenous SO 2 detection in cells The results above demonstrated the excellent detection capability of Cou-Oxo , which prompted us to used it for cell imaging tests. Prior to this, we tested its cytotoxicity in HeLa cells using the MMT assay. Figure S5 shows that the cell survival rate remained consistently above 80% in the presence of different concentrations of the Cou-Oxo probe, indicating low cytotoxicity. To investigate the response of Cou-Oxo to exogenous SO 2 , Cou-Oxo was co-incubated with HeLa cells for 30 minutes. Subsequently, it was incubated with different concentrations of NaHSO 3 (0, 10, 20, 30 μM) for 30 minutes. Figure 6 shows that the yellow channel signal was weak when HSO 3 - was not added. However, the fluorescence intensity of the yellow channel gradually increased with the increase in HSO 3 - concentration. The experiment indicated that Cou-Oxo had the ability to detect alterations in external SO 2 levels at a cellular level. Figure 6 Imaging of exogenous SO 2 in HeLa cells with probe Cou-Oxo (10 µM). (a) HeLa cells treated with probe Cou-Oxo only for 30 min; (b, c) HeLa cells treated with probe Cou-Oxo for 30 min followed by addition of NaHSO 3 (0, 10, 20, 30 µM) and incubation for 30 min. (λ ex = 460 nm, λ em = 510 - 570 nm) Scale bar: 10 µm. Figure 7 Imaging of endogenous SO 2 in HeLa cells with the probe Cou-Oxo (10 µM). (a) HeLa cells treated with probe Cou-Oxo only for 30 min; (b, c) HeLa cells treated with probe Cou-Oxo for 30 min followed by incubation with 2,4 -dinitrophenylsulfonamide (0, 10, 20, 30 µM) for 30 min. (λ ex = 460 nm, λ em = 510 - 570 nm) Scale bar: 10 µm. Then, the response status of Cou-Oxo to endogenous SO 2 was assessed in HeLa cells. According to the literature, 2,4 -dinitrobenzenesulfonamide responds to intracellular sulfhydryl groups and releases endogenous SO 2 [18,50] . HeLa cells were incubated with Cou-Oxo for 30 min, then different concentrations of 2,4-dinitrobenzenesulfonamide (0, 10, 20, 30 µM) were added and incubation was continued for 30 min. As shown in Figure 7, the yellow channel signal was weak in the group without the addition of 2,4-dinitrobenzenesulfonamide. The fluorescence intensity of the yellow channel gradually increased with the increase of 2,4 -dinitrophenylsulfonamide concentration. The above results indicated that Cou-Oxo can detect endo/exogenous SO 2 at the cellular level and monitor changes in intracellular SO 2 concentration. not-yet-known not-yet-known not-yet-known unknown Imaging of SO2 detection in tobacco seedlings Studies have shown that sulfite and sulfite ions are formed in excess of SO2, which cause damage to plant tissues, leading to leaf loss and yellowing and, in severe cases, cell death [51]. Inspired by the aforementioned Cou-Oxo cell imaging, we tried to used Cou-Oxo to detect HSO3- in plants. We selected tobacco seedlings as the best model for HSO3- detection in plants due to their short seedling return cycle and high survival rate during cultivation. Firstly, Tobacco seedlings were cultured in soil contaminated with varying concentrations of HSO3- (0, 5, 10, 15, 20 µM) and then incubated with Cou-Oxo (10 μM) for 10 h. The roots of tobacco seedlings were sectioned and imaged. Figure 8 shows that sections of tobacco seedling rhizomes cultured in uncontaminated soil exhibit only faint fluorescence in the yellow channel. As the concentration of HSO3- increased, the fluorescence intensity in the yellow channel gradually increased. The results suggest that the Cou-Oxo probe is a useful tool for detecting HSO3- in plant tissues, indicating promise for detecting HSO3- content in tobacco. Figure 8 (a) Fluorescence imaging of the root diameter of tobacco seedlings with different concentrations of HSO 3 - by the probe Cou-Oxo ; (b) Fluorescence intensity plot in the yellow channel (A). (λ ex = 460 nm, λ em = 510 - 570 nm) Scale bar: 10 µm. Imaging for detection of endogenous/exogenous SO2 in zebrafish Building on the cellular and tobacco seedling rhizome experiments described above, we examined the ability of the probe to image exogenous/endogenous SO 2 in live zebrafish. The results, as depicted in Figure S6 and Figure 9, indicate that the probe Cou-Oxo (10 µM) exhibited feeble fluorescence in the yellow channel after incubation with zebrafish. However, the fluorescence signal in the yellow channel significantly increased with the addition of exogenous SO 2 (0, 10, 25, 50 μM) (Figure 9 b). The fluorescence signal was similarly released in zebrafish when endogenous SO 2 (0, 5, 10, 25 µM) was used for stimulation alone (Figure S6b). These imaging results confirm the ability of the Cou-Oxo probe to detect HSO 3 - in living organisms. Figure 9 Confocal imaging of Cou-Oxo with, SO 2 in zebrafish. (A) Probe Cou-Oxo (10 μM) control. (B) Zebrafish were treated with probe Cou-Oxo for 30 min followed by the addition of NaHSO 3 (0, 10, 25, 50 μM). (C) Merged channels for channels a and b. Conclusions In summary, we have developed a new near-infrared probe Cou-Oxo , through a molecular design concept. The probe’s Stokes shift was increased by constructing a D-A-D type asymmetric structure molecule through linking two common dyes, resulting in near-infrared emission at 781 nm. The probe’s unsaturated bonds react with SO 2 derivatives (HSO 3 - ) to disrupt the molecule’s π-conjugated structure, resulting in a ratiometric sensing property. The spectroscopic test revealed that the probe had a short response time ( within 30s), good biocompatibility, and a low detection limit of 0.942 μM. Additionally, the imaging results of HeLa cells and zebrafish demonstrated that Cou-Oxo was effective in detecting HSO 3 - in both cells and organisms. To validate the ability of Cou-Oxo to detect HSO 3 - in plant systems, tobacco seedlings were constructed as HSO 3 - plant models. The above results indicate the probe was expected to be a useful tool for future analysis and detection of SO 2 derivatives. Experimental Materials and Instruments The experiments used materials and reagents procured from commercial suppliers, which were used directly without further processing, unless otherwise stated. All reagents used were analytically pure, and ultrapure water was used for experimental tests. Information on the instrumentation involved in the study can be found in the supporting materials Systhesis of probe Cou-Oxo Synthesis of compound 1. Ethyl 3-oxobutanoate (1.30 g, 10.0 mmol), 4-(diethylamino)-2-hydroxybenzaldehyde (1.93 g, 10.0 mmol), and piperidine (0.26 g, 3.0 mmol) were dissolved in anhydrous ethanol (15.0 mL) and heated at reflux for 6 hours under a nitrogen blanket. After the reaction, the remaining solvent was removed by evaporation under reduced pressure. The reaction system was then treated with a mixed solution of hydrochloric acid and glacial acetic acid (v/v = 1/1, 30.0 mL) at 120℃. The reaction was continued for another 7 h. After the reaction, the resulting mixture was poured into deionized water (40.0 mL). The pH was then neutralized using a 40% solution of sodium hydroxide. The resulting yellow-brown precipitate was filtered and washed multiple times with water and ethanol to obtain the desired product. Synthesis of compound Cou-CN. Compound 1 (518.62 mg, 2.0 mmol) was reacted with malononitrile (198.19 mg, 3.0 mmol), piperidine (34.06. mg, 0.4 mmol), and glacial acetic acid (24.02 mg, 0.4 mmol) in anhydrous ethanol (10.0 mL) under nitrogen protection. The mixture was refluxed for 6 h. After the reaction, the solvent was removed by evaporation under reduced pressure. The residue was redissolved in dichloromethane and purified on a silica gel column using PE/EtOAc (v/v = 20/1) as eluent. Compound Cou-CN (399.56 mg, 1.3 mmol) was obtained. Yield: 65%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.89 (s, 1H), 7.37 (d, J = 9.0 Hz, 1H), 6.65 (dd, J = 9.0, 2.1 Hz, 1H), 6.49 (d, J = 1.8 Hz, 1H), 3.47 (q, J = 7.1 Hz, 4H), 2.66 (s, 3H), 1.25 (t, J = 7.1 Hz, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 173.18, 158.61, 157.92, 153.06, 145.05, 131.12, 114.82, 112.97, 110.22, 107.50, 97.11, 84.67, 45.41, 23.01, 12.54. not-yet-known not-yet-known not-yet-known unknown Synthesis of compound 2. A mixture of N, N-dimethylformamide (DMF) (306.0 mmol, 23.5 mL) and trichloromethane (100.0 mL) was added to an eggplant flask. Phosphorus tribromide (255.0 mmol, 24.2 mL) was added in small amounts several times under nitrogen at 0℃. After 1.5 h of reaction, cyclohexanone (102.0 mmol, 10.5 mL) was added and allowed to react for 12 h at room temperature, resulting in a clear orange solution. Sodium bicarbonate was added to the solution to neutralize the pH to 7. The extraction process was carried out using a mixture of dichloromethane and water. The water content in the solution was eliminated by adding anhydrous sodium sulfate. The resulting product was then purified through column chromatography, using petroleum ether as the eluent. The final product obtained was a clear, pale-yellow liquid. 1H NMR (400 MHz, Chloroform-d ) δ 10.01 (s, 1H), 2.73 (dt, J = 8.5, 3.1 Hz, 2H), 2.27 (td, J = 6.1, 2.9 Hz, 2H), 1.82–1.62 (m, 4H). Synthesis of compound 3. To the eggplant flask containing DMF (25.0 ml) as a solvent, add 4-(diethylamino)-2-hydroxybenzaldehyde (4.1 mmol, 792.3 mg) and cesium carbonate (8.2 mmol, 1.5630 g). Stir the mixture thoroughly to dissolve the solid, and then slowly add compound 2 (12.4 mmol, 2.3442 g). After 48 hours of reaction at room temperature, the solution was extracted with dichloromethane and water. The extracted solution was treated with sodium bicarbonate and purified by column chromatography using hexane and ethyl acetate (20:1-10:1, v/v) as the eluent. The final product was obtained as a yellow solid. 1 H NMR (400 MHz, Chloroform- d ) δ 10.05 (s, 1H), 9.40 (s, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.67 (s, 1H), 3.07 (q, J = 6.9 Hz, 4H), 2.61 – 2.53 (m, 2H), 2.42 (td, J = 6.0, 2.2 Hz, 2H), 1.94 – 1.81 (m, 2H), 1.01 (t, J = 7.1 Hz, 6H). Synthesis of probe Cou-Oxo. Compound Cou-CN (68.98 mg, 0.224 mmol) and compound 3 (63.6 mg, 0.224 mmol) were dissolved in toluene (15 mL) and refluxed under N 2 atmosphere for 5 h. The reaction was catalyzed by the addition of piperidine (0.17 mg, 0.045 mmol). At the end of the reaction, the crude product obtained was purified on a silica gel column eluting with PE/EtOAc (v/v =15/1), yielding Cou-Oxo (16 mg, 0.028 mmol) 12.47%. 1 NMR (400 MHz, Chloroform- d ) δ 9.18 (s, 1H), 7.65(s, 1H), 7.37 – 7.30 (m, 2H), 7.01 (d, J = 8.3 Hz, 1H), 6.87 (d, J = 14.8 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.65 (dd, J = 8.9, 2.4 Hz, 1H), 6.59(s, 1H), 6.53 (d, J = 2.3 Hz, 1H), 3.48 (qd, J = 7.3, 3.3 Hz. 4H), 2.92 (qd, J = 6.8, 2.6 Hz, 4H), 2.58 (dt, J = 20.1, 5.8 Hz, 4H), 2.49 – 2.27 (m, 2H), 1.27 (d, J = 7.1 Hz, 6H), 0.91 (t, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, Chloroform- d ) δ 193.30, 164.45, 158.85, 157.62, 152.35, 150.49, 146.01, 139.73, 135.67, 130.30, 128.27, 126.48, 126.02, 120.72, 120.32, 116.68, 116.60, 115.08, 114.52, 113.23, 112.86, 109.85, 107.82, 97.21, 77.23, 46.00, 45.24, 31.28, 29.82, 25.71, 12.48, 12.15. HRMS (ESI) Found: 573.2870 [M+H] + ; Molecular formula C 36 H 37 N 4 O 3 requires [M+H] + 573.2861. Scheme 1 Synthetic route of probe Cou-Oxo Supporting Information The supporting information for this article is available on the WWW under https://doi.org/10.1002/cjoc.202400xxx. Acknowledgement This work was financially supported by Natural Science Foundation of China (21801145), Natural Science Foundation of Shandong Province (ZR2017BB012). References 51. Chen, W.; Fang, Q.; Yang, D.; Zhang, H.; Song, X.; Foley, J. Selective, Highly Sensitive Fluorescent Probe for the Detection of Sulfur Dioxide Derivatives in Aqueous and Biological Environments. Anal. Chem. 2014 , 87 , 609-616. Ba-Shammakh, M. S. A Multiperiod Mathematical Model for Integrating Planning and SO 2 Mitigation in the Power Generation Sector. Energy & Fuels 2011 , 25 , 1504-1509. van Thriel, C.; Schäper, M.; Kleinbeck, S.; Kiesswetter, E.; Blaszkewicz, M.; Golka, K.; Nies, E.; Raulf-Heimsoth, M.; Brüning, T. Sensory and pulmonary effects of acute exposure to sulfur dioxide (SO 2 ). Toxicol. Lett. 2010 , 196 , 42-50. 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Chem. 2011 , 55 , 553-557. Olszyk, D. M.; Tingey, D. T. Phytotoxicity of air pollutants: evidence for the photodetoxification of SO 2 but not O 3 . Plant Physiol., 1984 , 74 , 999-1005. Manuscript received: XXXX, 2024 Manuscript revised: XXXX, 2024 Manuscript accepted: XXXX, 2024 Version of record online: XXXX, 2024 The Authors Left to Right: Authors Names You will be invited to submit the most recent photos of all the authors upon acceptance of the manuscript Supplementary Material File (image13.emf) Download 173.75 KB Information & Authors Information Version history V1 Version 1 03 June 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords fluorescent probe near-infrared ratiometric sulfur dioxide Authors Affiliations Mao-Hua Wang Qilu University of Technology View all articles by this author Yun-Hao Yang Qilu University of Technology View all articles by this author Jian-Yong Wang 0000-0002-5254-1525 [email protected] Qilu University of Technology View all articles by this author Metrics & Citations Metrics Article Usage 150 views 125 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Mao-Hua Wang, Yun-Hao Yang, Jian-Yong Wang. Constructing Near-Infrared Dyes with D-A-D Type and large Stokes Shift Based on Asymmetric Electron Donating Properties and their Applications in biological systems. Authorea . 03 June 2025. DOI: https://doi.org/10.22541/au.174894144.45479136/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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