A dual-lock fluorescent probe for detecting H2S and H2O2, its preparation method and application

Abstract

The application discloses a double-lock fluorescent probe for detecting H2S and H2O2, a preparation method and application thereof, and belongs to the field of fluorescent probes. The double-lock fluorescent probe for detecting H2S and H2O2 is P1, and the P1 takes 1,8-naphthalene dicarboxylic imide as a fluorescent parent body. In the application, phenyl borate and ferrocene containing a disulfide bond are bonded to the 1,8-naphthalene dicarboxylic imide fluorescent parent body to obtain the double-lock fluorescent probe P1. Under the double quenching action of the phenyl borate and the ferrocene, the fluorescence of the P1 is turned off. When only H2S exists, the disulfide bond is cleaved, and the ferrocene group falls off from the fluorescent parent body, but due to the presence of the phenyl borate, the fluorescence of the probe is still limited. Similarly, when only H2O2 exists, the phenyl borate is oxidatively degraded, but the fluorescence of the fluorescent parent body is still weak due to the PET effect of the ferrocene. Only when H2S and H2O2 exist simultaneously, the P1 can turn on green fluorescence emission. The probe P1 has short response time and excellent recognition effect on H2S and H2O2.

Description

Technical Field

[0001] This invention belongs to the field of organic chemical synthesis and fluorescence detection technology, specifically relating to a dual-lock fluorescent probe for detecting H2S and H2O2, its preparation method, and its application. Background Technology

[0002] Normal physiological activities of living organisms require a certain supply of oxygen. Hypoxia leads to cellular dysfunction, causing abnormal accumulation of reactive oxygen species (ROS) or imbalance in antioxidant metabolism, resulting in oxidative stress. Prolonged exposure to this state can cause cellular inactivation and tissue damage, leading to a range of diseases such as inflammation, neurological disorders, and cancer. Hydrogen sulfide (H2S) and hydrogen peroxide (H2O2), as important redox signaling molecules, participate in the complex redox regulation of various biological processes and disease-related oxidative stress. Studies have shown that intracellular H2S and H2O2 are closely interconnected to maintain cellular homeostasis, and their levels directly reflect the degree of oxidative stress and disease status. Therefore, accurate and sensitive monitoring of real-time H2S and H2O2 concentration fluctuations in cells to reflect their redox state is of great significance.

[0003] In recent years, fluorescent probes have attracted widespread research interest from scientists due to their superior sensitivity, selectivity, and biocompatibility compared to traditional detection methods. Currently, numerous fluorescent probes for detecting H2S or H2O2 have been developed. However, these probes are primarily designed based on a strategy of one sensor per analyte, thus failing to meet the requirement of simultaneous detection of hydrogen peroxide and hydrogen sulfide. Using two independent fluorescent probes in a single system to simultaneously detect these two analytes complicates instrument setup, leading to spectral overlap and crosstalk, uneven fluorescence intensity distribution, difficulties in achieving simultaneous sensing, and potentially reduced temporal resolution of fluorescence imaging. Therefore, there is an urgent need to design a fluorescent probe that can synchronously respond to H2S and H2O2 under the same testing environment to better elucidate the interaction between these two biological species in signal transduction and redox homeostasis regulation.

[0004] The "double-lock, double-key" probe is developed based on a synergistic two-factor stimulation design. In a "double-lock, double-key" fluorescent probe system, the presence of a single stimulus is insufficient to activate the probe's fluorescence; only when both stimuli (the two keys) are present simultaneously can the two "locks" be opened, ultimately resulting in the observed fluorescence signal. The multi-signal "double-lock, double-key" probe exhibits high accuracy and precision during identification, and avoids false positive / false negative results. These advantages perfectly compensate for the shortcomings of de-superimposed fluorescent probes and allow for real-time monitoring of hydrogen sulfide and hydrogen peroxide concentration fluctuations. Summary of the Invention

[0005] This invention uses 1,8-naphthalenedimide as the fluorescent parent compound, selects phenylboronic acid ester as the H2O2 reaction site and fluorescence activity inhibitor on one side of the parent compound, and introduces a disulfide bond at the amine site as the H2S action site, and connects ferrocene to the other side of the disulfide bond as the fluorescence quenching group, thus obtaining a dual-locked fluorescent probe that can simultaneously detect H2S and H2O2. Therefore, this invention provides a dual-locked fluorescent probe for detecting H2S and H2O2, its preparation method and application.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides a dual-locked fluorescent probe for detecting H2S and H2O2. The dual-locked fluorescent probe for detecting H2S and H2O2 is P1, wherein P1 uses 1,8-naphthalenediamide as the fluorescent parent compound, and the chemical structural formula of P1 is as follows:

[0008]

[0009] Preferably, a disulfide bond is selected as the H2S response site on one side of the fluorescent matrix, and ferrocene connected to the disulfide bond is selected as the fluorescence quenching group. On the other side of the fluorescent matrix, a phenylboronic acid ester is selected as the H2O2 reaction site and the fluorescence activity inhibiting group.

[0010] In another aspect, this invention provides a method for preparing the aforementioned dual-lock fluorescent probe for detecting H2S and H2O2. The method involves amidation of 4-borate pinacol ester-1,8-naphthalenedicarboxylic anhydride with t-Boc-cystamine to obtain a first intermediate NA-SS-Boc, followed by deBoc reaction with trifluoroacetic acid to obtain a second intermediate NA-SS-NH2. Finally, NA-SS-NH2 and ferrocene carboxylic acid are amidated to obtain the target probe P1. The structures of NA-SS-Boc and NA-SS-NH2 are as follows:

[0011]

[0012] Preferably, it includes the following steps:

[0013] S1: Synthesis of the first intermediate NA-SS-Boc: The compound 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride and t-Boc-cystamine were dissolved in the first solvent, and the temperature was gradually increased to reflux under an inert atmosphere. After the reaction was completed, the mixture was cooled, washed with water, extracted and dried by rotary evaporation, and separated by column chromatography to obtain the light white solid NA-SS-Boc.

[0014] S2: Synthesis of the second intermediate NA-SS-NH2: Dissolve NA-SS-Boc from step S1 in the second solvent, add trifluoroacetic acid, and react at room temperature under an inert atmosphere. After the reaction is complete, wash with water, extract, evaporate to dryness, and then dry directly in an oven to obtain white solid NA-SS-NH2.

[0015] S3: Synthesis of dual-lock fluorescent probe P1: NA-SS-NH2 and ferrocene carboxylic acid from step S2 were dissolved in the second solvent. Dicyclohexylcarbodiimide was added as a dehydrating agent, and 4-dimethylaminopyridine was added as a catalyst. After the reaction was carried out completely at room temperature under an inert atmosphere, the mixture was washed with dilute hydrochloric acid and separated. After neutralization with alkali, the mixture was evaporated to dryness and separated by column chromatography to obtain a light brown solid P1.

[0016] The overall synthesis route is as follows:

[0017]

[0018] Preferably, the first solvent is acetonitrile, and the second solvent is dichloromethane.

[0019] Preferably, in step S1, the molar ratio of the raw material compound 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride to t-Boc-cystamine is 1:1-1.5; in step S2, the molar ratio of NA-SS-Boc to trifluoroacetic acid is 1:1-3; and in step S3, the molar ratio of NA-SS-NH2 to ferrocene carboxylic acid is 1:1-2. The equivalents of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to NA-SS-NH2 are 1-1.8 and 0.5-1, respectively. The inert atmosphere in steps S1, S2, and S3 is a nitrogen atmosphere.

[0020] Preferably, the post-treatment method in step S1 is water washing, extraction and separation, rotary drying, column chromatography and drying; the post-treatment method in step S2 is water washing, extraction multiple times and direct rotary drying, and drying in an oven; the post-treatment method in step S3 is adding dilute hydrochloric acid, water washing and separation multiple times, adding dilute sodium hydroxide to neutralize and rotary drying, and then column chromatography and drying.

[0021] Another aspect of the present invention provides the application of the above-mentioned dual-lock fluorescent probe for detecting H2S and H2O2, for the detection of H2S and H2O2.

[0022] Specifically, when H2S is added alone, the disulfide bond of the probe breaks, releasing the ferrocene group, but the fluorescence of the parent compound remains off due to the inhibition of the phenylboronic ester. When H2O2 is added alone, the phenylboronic ester is oxidized to a hydroxyl group, but the fluorescence of the parent compound also fails to activate under the PET effect of ferrocene. Probe P1's fluorescence remains off in the presence of either H2S or H2O2 alone; its green fluorescence is only activated and significantly enhanced when both H2S and H2O2 are present simultaneously, or when H2S is added before H2O2 (or vice versa).

[0023] The present invention has the following advantages:

[0024] The probe P1 of this invention exhibits a short response time and excellent recognition effect for both H2S and H2O2. The fluorescence is in a closed state due to the presence of phenylboronic acid ester and ferrocene groups. In the presence of H2S alone, the phenylboronic acid ester is oxidized, but the ferrocene remains, resulting in weak fluorescence. In the presence of H2O2 alone, the ferrocene is removed, but the presence of phenylboronic acid ester still inhibits fluorescence. Only when H2S and H2O2 are present simultaneously, or when H2S is added before H2O2 (or vice versa), is the phenylboronic acid ester oxidized and the ferrocene removed, thus unlocking both "locks" and significantly enhancing the green fluorescence of the probe at 553 nm. Attached Figure Description

[0025] Figure 1 The 1H NMR spectrum (CDCl3) of compound NA-SS-Boc in Example 1;

[0026] Figure 2 The 1H NMR spectrum (CDCl3) of compound NA-SS-NH2 in Example 1;

[0027] Figure 3 The 1H NMR spectrum (CDCl3) of compound P1 in Example 1;

[0028] Figure 4 The fluorescence spectra of probe P1 in Example 2 are for NaHS, H2O2, NaHS added first and then H2O2, H2O2 added first and then NaHS, and NaHS and H2O2 added simultaneously.

[0029] Figure 5 The graph shows the change in fluorescence intensity of P1 / H2O2 with the addition of NaHS and the change in fluorescence intensity of P1 / NaHS with the addition of H2O2 over time in Example 3.

[0030] Figure 6 The images show the fluorescence droplets of P1 / H2O2 on NaHS and P1 / NaHS on H2O2 in Example 4.

[0031] Figure 7 This is a schematic diagram of the recognition of H2S and H2O2 by a dual-lock fluorescent probe. The yellow lock represents phenylboronic acid ester, the blue lock represents ferrocene linked by disulfide bonds, the yellow key represents H2O2, and the blue key represents H2S. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0033] This invention provides a dual-locked fluorescent probe for detecting H2S and H2O2. The dual-locked fluorescent probe for detecting H2S and H2O2 is P1, wherein P1 uses 1,8-naphthalenediamide as the fluorescent parent compound, and the chemical structural formula of P1 is as follows:

[0034]

[0035] Furthermore, on one side of the fluorescent matrix, a disulfide bond is selected as the H2S response site, and the ferrocene bonded to the disulfide bond is selected as the fluorescence quenching group. On the other side of the fluorescent matrix, a phenylboronic acid ester is selected as the H2O2 reaction site and the fluorescence activity inhibiting group.

[0036] In another aspect, this invention provides a method for preparing the aforementioned dual-lock fluorescent probe for detecting H2S and H2O2. The method involves amidation of 4-borate pinacol ester-1,8-naphthalenedicarboxylic anhydride with t-Boc-cystamine to obtain a first intermediate NA-SS-Boc, followed by deBoc reaction with trifluoroacetic acid to obtain a second intermediate NA-SS-NH2. Finally, NA-SS-NH2 and ferrocene carboxylic acid are amidated to obtain the target probe P1. The structures of NA-SS-Boc and NA-SS-NH2 are as follows:

[0037]

[0038] Furthermore, it includes the following steps:

[0039] S1: Synthesis of the first intermediate NA-SS-Boc: The compound 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride and t-Boc-cystamine were dissolved in the first solvent, and the temperature was gradually increased to reflux under an inert atmosphere. After the reaction was completed, the mixture was cooled, washed with water, extracted and dried by rotary evaporation, and separated by column chromatography to obtain the light white solid NA-SS-Boc.

[0040] S2: Synthesis of the second intermediate NA-SS-NH2: Dissolve NA-SS-Boc from step S1 in the second solvent, add trifluoroacetic acid, and react at room temperature under an inert atmosphere. After the reaction is complete, wash with water, extract, evaporate to dryness, and then dry directly in an oven to obtain white solid NA-SS-NH2.

[0041] S3: Synthesis of dual-lock fluorescent probe P1: NA-SS-NH2 and ferrocene carboxylic acid from step S2 were dissolved in the second solvent. Dicyclohexylcarbodiimide was added as a dehydrating agent, and 4-dimethylaminopyridine was added as a catalyst. After the reaction was carried out completely at room temperature under an inert atmosphere, the mixture was washed with dilute hydrochloric acid and separated. After neutralization with alkali, the mixture was evaporated to dryness and separated by column chromatography to obtain a light brown solid P1.

[0042] The overall synthesis route is as follows:

[0043]

[0044] Furthermore, the first solvent is acetonitrile, and the second solvent is dichloromethane.

[0045] Further, in step S1, the molar ratio of the raw material compound 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride to t-Boc-cystamine is 1:1-1.5; in step S2, the molar ratio of NA-SS-Boc to trifluoroacetic acid is 1:1-3; and in step S3, the molar ratio of NA-SS-NH2 to ferrocene carboxylic acid is 1:1-2. The equivalents of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to NA-SS-NH2 are 1-1.8 and 0.5-1, respectively. The inert atmosphere in steps S1, S2, and S3 is a nitrogen atmosphere.

[0046] Further, the post-treatment method in step S1 is water washing, extraction and separation, rotary drying, column chromatography and drying; the post-treatment method in step S2 is water washing, extraction multiple times and direct rotary drying, and drying in an oven; the post-treatment method in step S3 is adding dilute hydrochloric acid, water washing and separation multiple times, adding dilute sodium hydroxide to neutralize and rotary drying, and then column chromatography and drying.

[0047] Another aspect of the present invention provides the application of the above-mentioned dual-lock fluorescent probe for detecting H2S and H2O2, for the detection of H2S and H2O2.

[0048] Example 1: The synthesis of a dual-locked fluorescent probe P1 for detecting H2S and H2O2 involved in this example includes the following steps:

[0049] (1) Synthesis of compound NA-SS-Boc: First, 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride (0.52 g, 1.60 mmol) was dissolved in 15 mL of ethanol and stirred for 10 minutes to ensure complete dispersion. Then, t-Boc-cystamine (0.41 g, 1.61 mmol) was slowly added, and the mixture was refluxed under a nitrogen atmosphere for 12 hours. After the reaction was complete, the mixture was washed three times with water, extracted, and evaporated to dryness to obtain the crude product. Using petroleum ether / CH2Cl2 (5:1, v / v) as the eluent, 0.78 g of the light white solid compound NA-SS-Boc was obtained by column chromatography, with a yield of 87%.

[0050] The light-colored solid NA-SS-Boc obtained above was measured using a nuclear magnetic resonance instrument (Varian instrument 400MHz), as shown above. Figure 1 As shown, the data is as follows:

[0051] 1H NMR (400MHz, CDCl3) δ (ppm): δ9.10 (t, J = 8.6 Hz, 1H), 8.55 (tt, J = 14.4, 7.1 Hz, 2H), 8.27 (t, J = 7.5 Hz, 1H), 7.75 (q, J = 8.4 Hz, 1H), 4 .53(dt,J=14.9,7.4Hz,2H),3.43(t,J=8.4Hz,2H),3.15-2.99(m,2H),2.78(d,J=6.7Hz,2H),1.40(s,12H),1.25(t,J=5.7Hz,9H).

[0052] (2) Synthesis of compound NA-SS-NH2: Trifluoroacetic acid (0.06 g, 0.53 mmol) was dissolved in 5 mL of anhydrous dichloromethane, and then compound NA-SS-Boc (0.29 g, 0.52 mmol) was slowly added dropwise. After the addition was complete, the reaction was carried out for 5 hours. The mixture was washed three times with pure water, extracted, and the solvent was concentrated. After drying, 0.22 g of white solid compound NA-SS-NH2 was obtained, with a yield of 93%.

[0053] The white solid compound NA-SS-NH2 obtained above was measured by nuclear magnetic resonance (NMR) instrument (Varian instrument 400MHz), as follows: Figure 2 As shown, the data is as follows:

[0054] 1 H NMR (400MHz, CDCl3) δ (ppm): δ9.07 (dd, J=19.9, 8.4Hz, 1H), 8.53 (dd, J=16.0, 7.5Hz, 1H), 8.45 (dd, J=17.2, 7.0Hz, 1H), 8.23 ​​(dd, J=24. 8,7.2Hz,1H),7.72(dt,J=17.0,7.4Hz,1H),4.50(d,J=41.1Hz,2H),3.13(dd,J=16.3,8.5Hz,2H),2.72(s,4H),1.43(d,J=2.1Hz,12H).

[0055] (3) Synthesis of probe P1: Compound NA-SS-NH2 (0.20 g, 0.43 mmol) and 4-dimethylaminopyridine (0.03 g, 0.25 mmol) were dispersed in 10 mL of anhydrous dichloromethane. Then, ferrocene carboxylic acid (0.10 g, 0.43 mol) and dicyclohexylcarbodiimide (0.11 g, 0.53 mmol) were added to the mixture. After reacting for 24 hours, the mixture was washed with dilute hydrochloric acid and water several times, neutralized with dilute sodium hydroxide, and then evaporated to dryness. The crude product was purified by column chromatography using CH2Cl2 / methanol (25:1, v / v) as the eluent, and 0.24 g of brown solid P1 was successfully obtained, with a yield of 83%.

[0056] The brown solid compound P1 obtained above was measured by a Varian instrument (400MHz), as shown above. Figure 3 As shown, the data is as follows:

[0057] 1 H NMR (400MHz, CDCl3) δ (ppm): 9.14 (d, J = 8.5Hz, 1H), 8.62 (d, J = 7.3Hz, 1H), 8.58 (d, J = 7.4Hz, 1H), 8.31 (d, J = 6.9Hz, 1H), 7.79 (t, J = 8.0Hz, 1H ),6.94(s,1H),4.87(s,2H),4.59(t,J=7.8Hz,3H),4.33(s,2H),4.21(d,J=1.2Hz,4H),3.75-3.67(m,2H),3.15-3.03(m,4H),1.44(s,12H).

[0058] Example 2:

[0059] For the dual-lock fluorescent probe P1 obtained in Example 1, we used NaHS instead of the analyte H2S, first as follows: Figure 4As shown, the black line represents probe P1 without any analyte, the red line represents P1 with NaHS alone, the green line represents P1 with H2O2 alone, the purple line represents P1 with both NaHS and H2O2, the orange line represents P1 with NaHS first and then H2O2, and the orange line represents P1 with H2O2 first and then NaHS. It can be observed that when probe P1 (10 μM) is added alone with 0.2 mM NaHS, even though the disulfide bonds are cleaved, causing the ferrocene group to fall from the fluorescent parent compound and interrupting the PET process, the fluorescence of the parent compound remains very weak due to the inhibition effect of phenylboronic acid esters. Similarly, when probe P1 is added alone with 0.5 mM H2O2, although the phenylboronic acid ester is oxidized to hydroxyl groups, the fluorescence of the parent compound is still limited under the PET effect of ferrocene. Only when NaHS (0.2mM) and H2O2 (0.5mM) are present simultaneously (NaHS is added before H2O2, H2O2 is added before NaHS, or both NaHS and H2O2 are added at the same time), phenylboronic acid ester and ferrocene leave the parent compound at the same time, and both "locks" are opened, resulting in a significant enhancement of the green fluorescence of the probe at 553nm.

[0060] Example 3:

[0061] The response times of the dual-locked fluorescent probe P1 obtained in Example 1 to NaHS and H2O2 were tested. Figure 5 Figure a shows a dotted line graph showing the fluorescence intensity of probe P1 after reacting with H2O2 for a sufficient time (2h) and then adding NaHS. It can be seen that the fluorescence of P1 (10μM) / H2O2 (0.5mM) at 553nm gradually increases with time after the addition of 0.2mM NaHS, and reaches its maximum at about 10 minutes and then does not change with time. Figure 5 The graph shows the fluorescence intensity of probe P1 after reacting with NaHS for a sufficient time (2h) and then adding H2O2. It was found that the fluorescence intensity of P1 (10μM) / NaHS (0.2mM) stabilized and reached saturation after about 30 minutes with the addition of 0.5mM H2O2.

[0062] Example 4:

[0063] The fluorescence titration responses of the dual-locked fluorescent probe P1 obtained in Example 1 to NaHS and H2O2 were tested. Figure 6Figure a shows the fluorescence spectra of probe P1 after the reaction with 0.5 mM H2O2 reached saturation time, followed by the addition of 0, 0.02 mM, 0.04 mM, 0.08 mM, 0.12 mM, 0.16 mM, and 0.2 mM NaHS, respectively. It can be observed that the fluorescence emission peak of P1 (10 μM) / H2O2 (0.5 mM) at 553 nm gradually increases with the continuous addition of NaHS, and reaches saturation at 0.2 mM, with the fluorescence intensity increasing by 7.7 times compared to the previous concentration. Figure 6 b shows the fluorescence spectra of probe P1 after the reaction with 0.2 mM NaHS reached saturation time, followed by the addition of 0, 0.02 mM, 0.04 mM, 0.08 mM, 0.14 mM, 0.2 mM, 0.28 mM, 0.36 mM, 0.42 mM, and 0.5 mM H2O2, respectively. It was found that the fluorescence intensity of P1 (10 μM) / NaHS (0.2 mM) also increased continuously with the addition of H2O2, reaching a peak at the addition of 0.5 mM H2O2, with an enhancement of 13.4 times.

[0064] In summary, this invention chemically synthesizes a dual-locked fluorescent probe P1 for detecting H2S and H2O2. Initially, probe P1 exhibits almost no fluorescence due to the dual fluorescence quenching effect of phenylboronic acid ester and ferrocene. Only when H2S and H2O2 are present simultaneously, causing the phenylboronic acid ester to be oxidized by H2O2, and the disulfide bond to be cleaved by H2S, resulting in the removal of ferrocene, does its green fluorescence at 553 nm significantly activate. The reaction times of probe P1 / H2O2 to H2S and P1 / H2S to H2O2 are 10 and 30 minutes, respectively. Furthermore, the saturation response concentrations of probe P1 / H2O2 to H2S and P1 / H2S to H2O2 are 0.2 mM and 0.5 mM, respectively. It can be observed that probe P1 exhibits a short response time and excellent recognition effect for both H2S and H2O2.

[0065] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A dual-lock fluorescent probe for detecting H2S and H2O2, characterized in that: The dual-lock fluorescent probe for detecting H2S and H2O2 is P1, which uses 1,8-naphthalenediamide as the fluorescent precursor. The chemical structure of P1 is as follows: 。 2. A method for preparing the dual-locked fluorescent probe for detecting H2S and H2O2 as described in claim 1, characterized in that: The first intermediate NA-SS-Boc was obtained by amidation of 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride with t-Boc-cystamine. Then, it was subjected to de-Boc reaction with trifluoroacetic acid to obtain the second intermediate NA-SS-NH2. Finally, NA-SS-NH2 and ferrocene carboxylic acid were amidated to obtain the target probe P1. The structures of NA-SS-Boc and NA-SS-NH2 are as follows: 。 3. The method for preparing a dual-locked fluorescent probe for detecting H2S and H2O2 according to claim 2, characterized in that, Includes the following steps: S1: Synthesis of the first intermediate NA-SS-Boc: The compound 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride and t-Boc-cystamine were dissolved in the first solvent, and the temperature was gradually increased to reflux under an inert atmosphere. After the reaction was completed, the mixture was cooled, washed with water, extracted and dried by rotary evaporation, and separated by column chromatography to obtain the light white solid NA-SS-Boc. S2: Synthesis of the second intermediate NA-SS-NH2: Dissolve NA-SS-Boc from step S1 in the second solvent, add trifluoroacetic acid, and react at room temperature under an inert atmosphere. After the reaction is complete, wash with water, extract, evaporate to dryness, and then dry directly in an oven to obtain white solid NA-SS-NH2. S3: Synthesis of dual-lock fluorescent probe P1: NA-SS-NH2 and ferrocene carboxylic acid from step S2 were dissolved in the second solvent. Dicyclohexylcarbodiimide was added as a dehydrating agent, and 4-dimethylaminopyridine was added as a catalyst. After the reaction was carried out completely at room temperature under an inert atmosphere, the mixture was washed with dilute hydrochloric acid and separated. After neutralization with alkali, the mixture was evaporated to dryness and separated by column chromatography to obtain a light brown solid P1. The overall synthesis route is as follows: 。 4. The method for preparing the dual-locked fluorescent probe for detecting H2S and H2O2 according to claim 3, characterized in that: The first solvent is ethanol, and the second solvent is dichloromethane.

5. The method for preparing a dual-locked fluorescent probe for detecting H2S and H2O2 according to claim 4, characterized in that: In step S1, the molar ratio of the raw material compound 4-boronic acid pinacol ester-1,8-naphthalenedicarboxylic anhydride to t-Boc-cystamine is 1:1-1.

5. In step S2, the molar ratio of NA-SS-Boc to trifluoroacetic acid is 1:1-3. In step S3, the molar ratio of NA-SS-NH2 to ferrocene carboxylic acid is 1:1-2. The equivalents of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to NA-SS-NH2 are 1-1.8 and 0.5-1, respectively.

6. The application of the dual-lock fluorescent probe for detecting H2S and H2O2 as described in claim 1, characterized in that: Reagents for the preparation of H2S and H2O2 detection.

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