Xanthene-based fluorescent probe and application thereof in phosgene detection

Abstract

The invention provides a xanthene-based fluorescent probe and application thereof in phosgene detection, and belongs to the technical field of phosgene detection. The probe provided by the invention shows excellent sensitivity and selectivity for phosgene detection, has remarkable spectral change, rapid response and wide practicability, shows huge potential in monitoring phosgene and gaseous phosgene in a solution, and more importantly, can also effectively realize phosgene biological imaging in a living body, and has wide application prospects. Furthermore, real-time in-vivo monitoring is realized, and a powerful and effective new way is provided for phosgene detection.

Description

Technical Field

[0001] The present invention relates to the technical field of phosgene detection, and in particular to a xanthene-based fluorescent probe and a preparation method thereof and application thereof in phosgene detection. Background Art

[0002] Phosgene (COCl2) is a toxic gas with a distinctive odor that poses a major threat to human health due to its high reactivity. Inhalation of phosgene can cause respiratory and lung damage, ranging from mild physiological phenomena such as difficulty breathing, chest pain, and decreased blood pressure to severe cases leading to coma or even death. Historically, phosgene has been used as a chemical weapon, causing heavy casualties during World War I. Despite its lethality, phosgene is an important industrial raw material that is widely used in industries such as plastics, pharmaceuticals, dyes, and pesticides. Given the extreme toxicity and significant industrial uses of phosgene, rapid, sensitive, and selective detection methods are critical to reducing the risks of phosgene leaks or potential chemical threats.

[0003] Traditional phosgene detection methods, such as chromatography and spectroscopy, although highly selective and sensitive, usually require complex procedures, specialized equipment, and trained technicians, making real-time and on-site monitoring difficult to achieve. Organic molecules based on fluorescent probes are promising alternatives with the potential for simple, low-cost, and real-time in-situ analysis.

[0004] Although many fluorescent probes for phosgene detection have been reported, most of them operate within the visible spectrum. The scarcity of long-wavelength fluorescent probes poses a significant limitation that hinders their effective application in vivo. In addition, most existing probes are limited to imaging studies at the cellular level, and real-time in vivo detection, such as monitoring phosgene levels in living mice, remains a challenging goal. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a xanthene-based fluorescent probe, which can selectively respond to phosgene and emit fluorescence at a long wavelength of 625nm, and has the characteristics of fast response time and low detection limit, and can be effectively used for in vivo phosgene detection.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a xanthene-based fluorescent probe, referred to as RNP, whose structural formula is shown below: .

[0007] In a second aspect, the present invention provides a method for preparing a probe RNP, comprising the following steps: S1. Add compound 1 (3-amino-10-(diethylamino)-7-(2-(methoxycarbonyl)phenyl)-5,6-dihydrobenzo[c]chromene-12-ammonium), tert-butyl(2-bromophenyl)carbamate, BINAP, palladium(II) acetate and Cs2CO3 into toluene, and stir to react under nitrogen protection; S2, adding water to the mixture obtained in step S1, extracting with CH2Cl2, then adding trifluoroacetic acid (TFA) to the CH2Cl2 solution and stirring to react; S3. After removing the organic solvent, the residue is purified by silica gel column chromatography.

[0008] Preferably, in the above preparation method, the molar ratio of compound 1, tert-butyl (2-bromophenyl) carbamate, BINAP, palladium (II) acetate, and Cs2CO3 is (0.8~1.2): (1.2~1.7): (0.08~0.12): (0.08~0.12): (2.5~3.5).

[0009] Preferably, in the above preparation method, the reaction temperature of step S1 is 90-120°C.

[0010] Preferably, in the above preparation method, the purification in step S3 is eluted using CH2Cl2: MeOH = 100:1 to 20:1.

[0011] In a third aspect, the present invention provides an application of the probe RNP, specifically any of the following: 1) Detection of gaseous phosgene in the environment; 2) Qualitative and / or quantitative detection of phosgene in solution; 3) Phosgene imaging in vivo; 4) Prepare phosgene detection products.

[0012] In the above applications, the organism includes cells and living animals. For example, in some embodiments of the present invention, the cells are HeLa cells. For example, in other embodiments of the present invention, the living animal is a living mouse.

[0013] In a fourth aspect, the present invention provides a test strip having a probe RNP attached thereto, which can achieve specific detection of low-dose gaseous phosgene in the environment.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The novel fluorescent probe RNP provided by the present invention has excellent sensitivity and specificity for phosgene detection. After phosgene exposure, the probe exhibits rapid fluorescence onset within a short response time (6 seconds), and can be effectively used for the detection of phosgene in solution and gaseous phosgene, and can be used to monitor phosgene leakage in the environment. In addition, the present invention has also developed a portable detection paper strip embedded with RNP, which can quickly and easily visually detect phosgene vapor.

[0015] Given the longer emission wavelength of the probe RNP and its excellent performance in cell imaging, the probe also has the ability to image phosgene in living animals (such as living mice); experimental data show that living mice exposed to trace amounts (1 ppm) of phosgene can be effectively monitored. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a schematic diagram of the principle of the probe RNP of the present invention for selectively detecting phosgene; Figure 2 A roadmap for synthesizing probe RNP in an embodiment of the present invention; Figure 3 is the probe RNP in the embodiment of the present invention 1 HNMR spectrum; Figure 4 is the probe RNP in the embodiment of the present invention 13 CNMR spectrum; Figure 5 This is a high-resolution mass spectrum of the probe RNP in the embodiment of the present invention; Figure 6 : The UV-visible absorption spectrum and fluorescence emission spectrum of the probe RNP in the embodiment of the present invention, wherein a is the UV-visible absorption spectrum of the RNP in the presence of phosgene, b is the fluorescence emission spectrum of the RNP in the presence of phosgene, and c is the detection result diagram of the reaction time of RNP to different concentrations of phosgene; Figure 7 Graphs showing the selectivity and anti-interference test results of the probe RNP for phosgene in an embodiment of the present invention, wherein a and b are the selectivity test results of RNP for other potential interferents, and c is a graph showing the anti-interference test results of RNP and phosgene in the presence of other interferents; Figure 8Graph showing the fluorescence intensity changes of the probe RNP under different concentrations of phosgene in the embodiment of the present invention, wherein a is the fluorescence titration spectrum of the probe RNP in the range of 0-20 μM, and b is a linear relationship graph between the fluorescence intensity of RNP and 0-20 μM phosgene; Fig. 9 This is a diagram showing the results of the photostability test of the probe RNP in an embodiment of the present invention; Fig.10 The HPLC-MS analysis results of the probe RNP before and after the reaction with phosgene in the embodiment of the present invention; Fig.11 This is a result diagram of DFT calculation of the probe RNP response mechanism based on the B3LYP / 6-31G method in an embodiment of the present invention; Fig.12 This is a detection diagram of different concentrations of phosgene imaged by the probe RNP in HeLa cells in an embodiment of the present invention; Fig.13 This is a detection diagram of different concentrations of phosgene imaged by the probe RNP in a living mouse in an embodiment of the present invention; Fig.14 This is a comparison diagram of the test strips in the embodiment of the present invention exposed to 1-10 ppm phosgene vapor; Fig.15 This is a diagram showing the detection results of phosgene and other interfering substances by the test strip based on the probe RNP in an embodiment of the present invention. DETAILED DESCRIPTION

[0018] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs; the terms "including" and "having" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0020] In view of the problems that most of the probes for detecting phosgene in the prior art work in the visible spectrum, and that the biological detection of phosgene mainly focuses on the cell system, and there is little research on animal in vivo imaging, the present invention provides a xanthene-based fluorescent probe RNP with a longer emission wavelength, and its reaction mechanism with phosgene is as follows Figure 1 In the probe RNP, the o-phenylenediamine moiety is used as the recognition group, and the fluorescence emission is weak due to the PET effect. However, after exposure to phosgene, the probe exhibits significant fluorescence enhancement.

[0021] The probe RNP provided by the present invention shows excellent sensitivity and selectivity for phosgene detection. The probe has significant spectral changes, rapid response and wide practicality, making it very effective in monitoring phosgene in solution. In addition, the present invention has also developed a portable detection paper strip embedded with RNP, which can quickly and easily visually detect phosgene vapor. Most notably, the probe RNP can also be used for biological detection of phosgene, and is effective in both cell and mouse experiments, providing a theoretical basis for phosgene-related research in living systems.

[0022] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0023] The compound 1 used in the following examples was prepared with reference to the literature A new xanthene-based fluorescent probe with a red light emission for selectively detecting glutathione and imaging in living cells (Y. Wan, Y. Li, Z. Liao, Z. Tang, Y. Li, Y. Zhao, B. Xiong, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 223 (2019) 117265). If no specific techniques or conditions are specified in the following examples, the techniques or conditions described in the literature in the field or the product instructions are used; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0024] Example 1 like Figure 2 As shown, this example provides a method for preparing a probe RNP, comprising the following steps: First, compound 1 (3-amino-10-(diethylamino)-7-(2-(methoxycarbonyl)phenyl)-5,6-dihydrobenzo[c]chromen-12-ammonium) (1 mmol), tert-butyl(2-bromophenyl)carbamate (1.5 mmol), BINAP (0.1 mmol), palladium(II) acetate (0.1 mmol) and Cs2CO3 (3 mmol) were added to toluene (25 mL). The reaction mixture was stirred at 100°C overnight under nitrogen protection. After cooling, 30 mL of water was added to the above mixture, and then extracted three times with 20 mL of CH2Cl2. Next, 20 mL of TFA was added to the CH2Cl2 solution, and stirring was continued for 2 hours (room temperature). Finally, the organic solvent was removed, and the residue was purified by silica gel column chromatography (CH2Cl2 / MeOH = 100:1 to 20:1, volume ratio) to obtain the probe RNP (143 mg, yield 26.28%, light blue solid).

[0025] The prepared probe RNP was tested as follows: Figure 3 The probe RNP in CDCl3 1 HNMR spectrum. Specifically, 1 HNMR (400MHz, CDCl3) δ 10.20(s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.88-8.03 (m, 1H), 7.81-7.63 (m, 3H),7.51-7.54 (m, 1H), 7.20-7.24 (m, 3.74 (s, 3H), 3.53 (q, J = 7.1Hz, 4H), 2.79 (t, J = 7.5Hz, 2H), 2.46 (t,J = 7.4Hz, 2H), 2.04 (s, 1H), 1.71 (q, J = 6.9Hz, 1H), 1.49-1.32 (m, 4H), 1.27 (d, J = 13.2Hz, 25H), 0.96 (t, J = 7.4Hz, 2H), 0.88 (t, J = 6.5Hz, 2H).

[0026] Figure 4 The probe RNP in CDCl3 13CNMR spectrum. Specifically, 13 CNMR (101MHz, Chloroform-d) δ165.59, 152.75, 146.21, 143.16, 135.18, 133.29, 131.32, 129.91, 129.19,129.10, 128.86, 128.61, 127.38, 126.51, 124.61, 119.28, 117.72, 117.02,114.43, 113.74, 96.34, 65.60, 52.61, 45.40, 30.58, 29.72, 27.44, 24.16,22.72, 14.15, 13.76, 12.53.

[0027] Figure 5 This is the high-resolution mass spectrum of the probe RNP. Molecular weight: C 35 H 34 N3O3, 544.2595; Found value: 544.2591.

[0028] Example 2 This example demonstrates the feasibility of using probe RNP to detect phosgene in vitro through the following optical experiments. All optical experiments in this example were performed in dichloromethane (CH2Cl2) solution, with an excitation wavelength of 560nm and a slit width of 10nm.

[0029] (1) UV-visible absorption spectrum.

[0030] Figure 6 a is the UV-visible absorption spectrum of 10μM RNP solution before and after the introduction of phosgene. As can be seen from the figure, the probe initially showed a maximum absorbance at 596nm, but after the introduction of 100μM phosgene, an obvious red shift occurred, the absorption peak moved to 583nm, and the test solution changed from red to light blue, indicating that there was an interaction between the probe and phosgene.

[0031] (2) Fluorescence emission spectrum.

[0032] Figure 6 b is the fluorescence emission spectrum of the 10 μM RNP solution before and after the introduction of phosgene. As can be seen from the figure, before the addition of phosgene, the RNP showed minimal fluorescence; however, the introduction of 100 μM phosgene resulted in a substantial increase in the fluorescence intensity at 625 nm, exceeding the original intensity by about 16 times. At the same time, the quantum yield increased from 2.1% to 28.6%. This longer wavelength enhanced fluorescence is beneficial for in vivo bioimaging because it allows greater tissue penetration and is less susceptible to interference from autofluorescence.

[0033] In addition, under the irradiation of a handheld 365 nm UV light source, the reaction solution exhibited strong red fluorescence, further confirming the ability of RNP to detect phosgene.

[0034] (3) Effect of phosgene concentration on fluorescence response.

[0035] The reactivity of the probe RNP to phosgene was observed by monitoring the change in fluorescence intensity over time after the introduction of 10, 20, and 30 μM phosgene into a 10 μM RNP solution. Figure 6 As shown in Figure c, after 10, 20, and 30 μM phosgene was introduced into the 10 μM RNP solution, the fluorescence intensity increased rapidly and reached saturation in a short time; specifically, the saturation time for 10 μM phosgene was 13 s and for 30 μM phosgene was 6 s. These results indicate that there is a direct correlation between the probe RNP and phosgene concentrations, and that the higher the concentration, the faster the response time.

[0036] (4) Selectivity for phosgene.

[0037] In this case, various potential interfering substances such as CH3COCl, ClCH2COCl, ClCOCOCl, SOCl2, (CH3CO)2O, POCl3, SO2Cl2, diethyl chlorophosphite (DCP) were used for selective experiments. Specifically, 100 μM of these interfering substances and phosgene were introduced into 10 μM RNP solution, and the fluorescence emission intensity was detected. The results are shown in Figure 2. Figure 7 a and 7b, where 1 to 9 are CH3COCl, ClCH2COCl, ClCOCOCl, SOCl2, (CH3CO)2O, POCl3, SO2Cl2, DCP and phosgene, respectively. The test results show that the introduction of 100 μM of these interferents into the 10 μM RNP solution did not cause a significant change in the probe fluorescence emission. Although DCP caused a slight increase in fluorescence at 633 nm, this effect was weak and different from the 625 nm peak observed for phosgene, indicating that the effect of DCP on the selectivity of RNP for phosgene is negligible.

[0038] Furthermore, 50 μM phosgene and another interfering substance (50 μM) were simultaneously introduced into the 10 μM RNP solution, and the changes in fluorescence emission intensity before and after the introduction were compared. Figure 7 c, where 1 to 9 are CH3COCl, ClCH2COCl, ClCOCOCl, SOCl2, (CH3CO)2O, POCl3, SO2Cl2, DCP and phosgene, respectively. The results show that the introduction of various interfering substances will not interfere with the fluorescence response of RNP to phosgene.

[0039] The above results indicate that the probe RNP exhibits excellent selectivity and anti-interference ability for phosgene, enabling it to specifically detect phosgene in complex environments.

[0040] (5) Quantitative detection of phosgene.

[0041] The quantitative ability of the probe RNP to phosgene was demonstrated by fluorescence titration experiments, specifically: the fluorescence emission intensity at 625 nm after the reaction of 10 μM RNP with different concentrations of phosgene was detected under excitation at 560 nm. Figure 8 As shown, the fluorescence emission of 10 μM RNP at 625 nm gradually increased as the phosgene concentration increased from 1 to 20 μM ( Figure 8 a), when the phosgene concentration exceeds 16 μM, it reaches a saturation platform, indicating that there is a robust linear correlation between fluorescence intensity and phosgene concentration (R²=0.99858) ( Figure 8 b). This result indicates that the probe RNP can be used for quantitative detection of phosgene.

[0042] Further using the 3σ / k method, the detection limit (LOD) of the probe RNP was calculated to be 0.85nM, where k represents the slope of the calibration curve and σ represents the standard deviation of 10 blank measurements. This extremely low detection limit proves that the probe RNP has high sensitivity and is very suitable for detecting trace amounts of phosgene.

[0043] (6) Stability testing.

[0044] After introducing 50 μM phosgene into the 10 μM probe RNP solution, photostability was tested under 605 nm excitation from a 150 W xenon lamp. Fig. 9 ) showed that the probe RNP had good photostability.

[0045] Example 3 This case experimentally confirmed the detection mechanism of probe RNP for phosgene, including the following two aspects: (1) HPLC-MS analysis.

[0046] The reaction product of the probe RNP and phosgene was determined using HPLC-MS. Specifically, 500 μM of the probe RNP was mixed with 1000 μM of phosgene and incubated for 5 minutes before detection.

[0047] The results are as follows Fig.10 As shown in Figure 1, after exposure to phosgene, the probe RNP underwent significant changes; specifically, the peak corresponding to the RNP at 4.49 minutes disappeared, while a new peak with a molecular weight of 571.09 appeared at 5.08 minutes. Figure 1 shown.

[0048] (2) DFT calculation.

[0049] The DFT calculations were performed using the B3LYP / 6-31G method. The results are as follows: Fig.11 As shown, it is shown that the LUMO of the probe RNP is mainly located on the oxygen heterocyclic ring with an energy level of -2.05eV, and the HOMO is mainly distributed in the o-phenylenediamine part, indicating that it has a strong oxidation potential, which is consistent with its electron-donating properties. This unique electron distribution promotes the PET effect, resulting in fluorescence quenching. After interacting with phosgene, both the LUMO and HOMO energy levels are reduced, and their spatial distribution becomes more delocalized in the entire molecular structure, which is conducive to the effective energy transfer of photoexcited excitons, and realizes the ICT effect and fluorescence emission recovery. Moreover, the energy gap between the LUMO and HOMO of the recognition product increases from 4.35eV to 4.62eV, which is consistent with the blue shift of the absorption spectrum observed in the experiment of Example 2.

[0050] Example 4 Taking HeLa cells as an example, this example first tested the cytotoxicity of the probe RNP and further demonstrated its ability to be used for imaging in cells.

[0051] Cytotoxicity was evaluated in the HeLa cell line using the MTT method. Specifically, the cell survival rate was calculated after HeLa cells were treated with different concentrations of probe RNP for 24 hours. The results showed that cells treated with 10 μM RNP for 24 hours showed a cell survival rate of more than 80%, indicating that the probe RNP has low cytotoxicity to living cells.

[0052] The fluorescence imaging of exogenous phosgene in living cells was performed using a confocal microscope. Specifically, the cells were pre-incubated with 5 μM probe RNP for 1 hour, and then different concentrations of phosgene (0-5 μM) were introduced for reaction and detection. Fig.12 (blue channel: λex=405nm, λem=430-500nm; red channel: λex=580nm, λem=620-660nm) As shown, incubating cells with 5μM RNPs will produce minimal fluorescence, indicating that the background signal in living cells is low, and the fluorescence intensity increases proportionally with the phosgene concentration, that is, it has a clear dose dependence. These results demonstrate that the probe RNP can safely and effectively image exogenous phosgene in living cells.

[0053] Example 5 Using BALB / c mice as an example, this example demonstrates the ability of the probe RNP to image phosphodiester in living organisms.

[0054] BALB / c mice were given 100 μL of 50 μM RNP solution by tail vein injection, and then the mice were exposed to phosgene concentration of 1 or 2 ppm in a closed chamber for 10 minutes. Fluorescence imaging was performed immediately after the exposure, and the results are shown in Figure 2. Fig.13 Further results showed that the fluorescence signal was preferentially localized in mouse lung tissue, and there was a direct correlation between phosgene concentration and lung fluorescence intensity.

[0055] Example 6 Based on the highly specific fluorescence recognition between the probe RNP and phosgene, in this example, a test strip was prepared using the probe RNP in order to achieve rapid visual detection of gaseous phosgene in the environment.

[0056] The test strips are prepared by dipping the cut filter paper strips into a dichloromethane solution containing 10 μM RNP, soaking them fully and then taking them out to dry, thus obtaining a light blue test strip ( Fig.14 ).

[0057] After exposure of the test strips to 1 to 10 ppm of phosgene vapor, the strips exhibited a color change from light blue to red ( Fig.14 ), indicating a significant enhancement in fluorescence emission. Notably, even at extremely low concentrations (1 ppm), the test strips exhibited a clear color change and fluorescence enhancement, highlighting the excellent sensitivity of this detection method.

[0058] After exposing the test strip to other interfering substances (5ppm), the color is developed with a 365nm UV lamp, and the result is as follows Fig.15 As shown in the figure, from left to right are CH3COCl, phosgene, ClCH2COCl, ClCOCOCl, SOCl2, (CH3CO)2O, POCl3,SO2Cl2, DCP. From the test results, it can be seen that the colorimetric and fluorescence responses of the test strip to other interfering substances are negligible, and only phosgene causes significant colorimetric and fluorescence reactions.

[0059] The above results show that the test strip prepared in this example has excellent sensitivity and can meet the strict requirements of phosgene detection in environmental monitoring.

[0060] In summary, the novel fluorescent probe RNP provided by the present invention exhibits excellent sensitivity and selectivity for phosgene detection. The probe has significant spectral changes, rapid responsiveness and wide practicability, making it very effective in monitoring phosgene in solution. Moreover, the test strip based on the probe RNP also shows great potential in detecting phosgene gas. Most notably, the probe of the present invention can also realize in vivo phosgene bioimaging, providing an effective new tool and method for the study of the interaction between phosgene and organisms.

[0061] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A xanthene-based fluorescent probe, characterized in that: The structural formula is as follows: 。 2. Use of the xanthene-based fluorescent probe as claimed in claim 1 in phosgene detection.

3. Use of the xanthene-based fluorescent probe as claimed in claim 1 in in vivo phosgene imaging.

4. The use according to claim 3, characterized in that: The organisms include cells and animals.

5. The use according to claim 4, characterized in that: The animal is a mouse.

6. Use of the xanthene-based fluorescent probe as claimed in claim 1 in the preparation of a product for detecting phosgene.

7. A test strip, characterized in that: The test strip is attached with the xanthene-based fluorescent probe according to claim 1.

8. Use of the test strip as claimed in claim 7 in gaseous phosgene detection.

9. A method for preparing the xanthene-based fluorescent probe as claimed in claim 1, characterized in that: The following steps are involved: S1. Add 3-amino-10-(diethylamino)-7-(2-(methoxycarbonyl)phenyl)-5,6-dihydrobenzo[c]chromene-12-ammonium, tert-butyl(2-bromophenyl)carbamate, BINAP, palladium(II) acetate and Cs2CO3 into toluene, and stir to react under nitrogen protection; S2, adding water to the mixture obtained in step S1, extracting with CH2Cl2, then adding trifluoroacetic acid to the CH2Cl2 solution and stirring to react; S3. After removing the organic solvent, the residue is purified by silica gel column chromatography.

10. The method according to claim 9, characterized in that The reaction temperature in step S1 is 90-120°C.

Images