Double-lock fluorescent probe for detecting H2S and H2O2 as well as preparation method and application of double-lock fluorescent probe
By designing a double-locked fluorescent probe based on 1,8-naphthalene diformimide, the synchronous detection of H2S and H2O2 is achieved by using disulfide bonds and phenylborate to combine ferrocene groups, the problem of spectral overlapping crosstalk in the prior art is solved, and the accuracy and time resolution of the detection are improved.
Patent Information
- Application Number
- CN202510709169.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-09-02
AI Technical Summary
Existing fluorescent probes cannot efficiently detect H2S and H2O2 at the same time under the same test environment, and are prone to spectral overlapping crosstalk and uneven fluorescence intensity distribution, affecting the time resolution.
A double-locked fluorescent probe was designed, using 1,8-naphthalene diformimide as the fluorescence parent, combining disulfide bonds and phenylborate as the response sites of H2S and H2O2, and ferrocene as the fluorescence quenching group, and fluorescence was turned on only when H2S and H2O2 exist simultaneously.
The synchronous response to H2S and H2O2 in the same test environment is achieved, which improves the accuracy and accuracy of detection, avoids false positive/false negative results, and has a short response time and excellent recognition effect.
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Figure CN120574265A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of organic chemical synthesis and fluorescence detection, and particularly relates to a double-locked fluorescent probe for detecting H2S and H2O2, and a preparation method and application thereof. Background Art
[0002] Normal physiological activities of living organisms require a certain supply of oxygen. Hypoxia can lead to cellular dysfunction, abnormal accumulation of intracellular reactive oxygen species (ROS), or imbalance in antioxidant metabolism, causing cells to be in a state of oxidative stress. Long-term exposure to this state can cause cellular dysfunction and tissue damage, leading to a range of diseases such as inflammation, neurological diseases, and cancer. Hydrogen sulfide (H2S) and hydrogen peroxide (H2O2), as important redox signaling molecules, are involved in the complex redox regulation of oxidative stress associated with various biological processes and diseases. Studies have shown that intracellular H2S and H2O2 are closely linked to maintain cellular homeostasis, and their levels directly reflect the degree of oxidative stress and disease status. Therefore, it is of great significance to accurately and sensitively monitor the real-time fluctuations in H2S and H2O2 concentrations in cells to reflect their redox state.
[0003] In recent years, fluorescent probes have attracted extensive research interest from researchers due to their superior sensitivity, selectivity, and biocompatibility compared to traditional detection methods. Currently, a large number of fluorescent probes for detecting H2S or H2O2 have been developed. However, these fluorescent probes are mainly designed based on a strategy of one sensor for each analyte, and therefore cannot meet the requirements for simultaneous detection of hydrogen peroxide and hydrogen sulfide. If two independent fluorescent probes are used to simultaneously detect these two analytes in a single system, the instrument setup will be complicated, resulting in spectral overlap, crosstalk, and uneven fluorescence intensity distribution, making simultaneous sensing difficult and potentially reducing the 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 test environment to better elucidate the interactions between these two biological species in signal transduction and redox homeostasis regulation.
[0004] The "Dual Lock, Dual Key" probe was developed based on a synergistic dual-factor stimulation design. In this "Dual Lock, Dual Key" fluorescent probe system, the presence of a single stimulus alone cannot cause the probe to activate fluorescence. Only when two stimuli (double keys) are present simultaneously can the two "locks" be opened, ultimately allowing the fluorescence signal to be observed. The multi-signal "Dual Lock, Dual Key" probe demonstrates high accuracy and precision during the identification process and avoids "false positive / false negative" results. These advantages perfectly compensate for the shortcomings of de-superimposed fluorescent probes and enable real-time monitoring of concentration fluctuations of hydrogen sulfide and hydrogen peroxide. Summary of the Invention
[0005] The present invention uses 1,8-naphthalimide as a fluorescent matrix, selects phenyl borate as a H2O2 reaction site and fluorescence activity inhibition group on one side of the matrix, introduces a disulfide bond at the amine position as an H2S action site, and connects ferrocene on the other side of the disulfide bond as a fluorescence quenching group to obtain a dual-locked fluorescent probe that can simultaneously detect H2S and H2O2. To this end, the present invention provides a dual-locked fluorescent probe for detecting H2S and H2O2, as well as a preparation method and application thereof.
[0006] The technical solution adopted in the present invention is as follows:
[0007] In one aspect, the present invention provides a dual-lock fluorescent probe for detecting H2S and H2O2. The dual-lock fluorescent probe for detecting H2S and H2O2 is P1, which uses 1,8-naphthalene diimide as a fluorescent matrix. The chemical structure 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, ferrocene connected to the disulfide bond is used as the fluorescence quenching group, and phenylboronic acid ester is selected as the H2O2 reaction site and fluorescence activity inhibition group on the other side of the fluorescent matrix.
[0010] Another aspect of the present invention provides a method for preparing the above-mentioned double-locked fluorescent probe for detecting H2S and H2O2, wherein 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride is subjected to an amidation reaction with t-Boc-cystamine to obtain a first intermediate NA-SS-Boc, which is then subjected to a de-Boc reaction with trifluoroacetic acid to obtain a second intermediate NA-SS-NH2, and finally NA-SS-NH2 is subjected to an amidation reaction with ferrocenecarboxylic acid to obtain the target probe P1. The structures of NA-SS-Boc and NA-SS-NH2 are as follows:
[0011]
[0012] Preferably, the steps include:
[0013] S1: Synthesis of the first intermediate NA-SS-Boc: Dissolve the compound 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride and t-Boc-cystamine in a first solvent, gradually heat to reflux under an inert atmosphere, cool after the reaction, wash with water, extract and dry, and separate by column chromatography to obtain a light white solid NA-SS-Boc;
[0014] S2: Synthesis of the second intermediate NA-SS-NH2: Dissolve the NA-SS-Boc prepared in step S1 in a second solvent, add trifluoroacetic acid, and react at room temperature under an inert atmosphere. After the reaction is completed, wash with water, extract and spin-dry, and then directly place in an oven to dry to obtain a white solid NA-SS-NH2;
[0015] S3: Synthesis of the double-locked fluorescent probe P1: NA-SS-NH2 and ferrocenecarboxylic acid prepared in step S2 were dissolved in a second solvent, dicyclohexylcarbodiimide was added as a dehydrating agent, and 4-dimethylaminopyridine was added as a catalyst. After thorough reaction at room temperature under an inert atmosphere, the separated liquid was washed with dilute hydrochloric acid, neutralized with alkali, and then dried by spin drying. The light brown solid P1 was obtained by column chromatography.
[0016] The overall synthetic 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 compounds 4-boronic acid pinacol ester-1,8-naphthalic anhydride to t-Boc-cystamine is 1:1-1.5, the molar ratio of NA-SS-Boc to trifluoroacetic acid in step S2 is 1:1-3, the molar ratio of NA-SS-NH2 to ferrocenecarboxylic acid in step S3 is 1:1-2, and 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 nitrogen atmosphere.
[0020] Preferably, the post-treatment method in step S1 is water washing, extraction and separation, spin drying, column chromatography and drying; the post-treatment method in step S2 is water washing, extraction multiple times and then direct spin drying, and drying in an oven; the post-treatment method in step S3 is adding dilute hydrochloric acid to wash and separate the liquid multiple times, adding dilute sodium hydroxide to neutralize and then spin drying, column chromatography and drying.
[0021] Another aspect of the present invention provides the use of the above-mentioned double-locked fluorescent probe for detecting H2S and H2O2, which is used for the detection of H2S and H2O2.
[0022] Specifically, when H2S alone is added to the probe, the disulfide bond cleaves, releasing the ferrocene group. However, the fluorescence of the parent is turned off due to the inhibition of phenylboronate. When H2O2 is added to the probe alone, the phenylboronate is oxidized to a hydroxyl group, but the parent fails to turn on fluorescence under the action of ferrocene's PET. Probe P1's fluorescence is turned off in the presence of either H2S or H2O2 alone. Only when H2S and H2O2 are present simultaneously, or when H2S is added first followed by H2O2 (or H2O2 first followed by H2S), does its green fluorescence turn on, significantly enhancing its fluorescence.
[0023] The present invention has the following advantages:
[0024] Probe P1 of the present invention has a short response time and excellent recognition effect for H2S and H2O2. The presence of phenylboronate and ferrocene groups causes fluorescence to be in an off state. In the presence of H2S alone, phenylboronate is oxidized, but ferrocene is still present, and the fluorescence is still very weak. In the presence of H2O2 alone, ferrocene is removed, but the presence of phenylboronate still inhibits fluorescence. Only when H2S and H2O2 are present simultaneously, or when H2S is added first and H2O2 second (H2O2 added first and H2S second), phenylboronate is oxidized and ferrocene is removed, and both "locks" are unlocked, allowing the probe to turn on green fluorescence at 553nm, significantly enhancing it. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 is the H NMR spectrum (CDCl3) of the compound NA-SS-Boc in Example 1;
[0026] Figure 2 is the H NMR spectrum (CDCl3) of the compound NA-SS-NH2 in Example 1;
[0027] Figure 3 is the H NMR spectrum (CDCl3) of compound P1 in Example 1;
[0028] Figure 4 The fluorescence spectra of probe P1 in Example 2 to NaHS, H2O2, adding NaHS first and then H2O2, adding H2O2 first and then NaHS, and adding NaHS and H2O2 simultaneously;
[0029] Figure 5 The fluorescence intensity changes over time after the addition of P1 / H2O2 and the addition of P1 / NaHS and H2O2 in Example 3;
[0030] Figure 6 The fluorescence droplet images of P1 / H2O2 to NaHS and P1 / NaHS to H2O2 in Example 4 are shown;
[0031] Figure 7 Schematic diagram of the double-lock fluorescent probe for the recognition of H2S and H2O2. The yellow lock represents phenylboronic acid ester, the blue lock represents disulfide-linked ferrocene, the yellow key represents H2O2, and the blue key represents H2S. DETAILED DESCRIPTION
[0032] The present invention will be further described below with reference to specific examples, but the present invention is not limited to the following examples.
[0033] In one aspect, the present invention provides a dual-lock fluorescent probe for detecting H2S and H2O2. The dual-lock fluorescent probe for detecting H2S and H2O2 is P1, which uses 1,8-naphthalene diimide as a fluorescent matrix. The chemical structure of P1 is as follows:
[0034]
[0035] Furthermore, a disulfide bond is selected as the H2S response site on one side of the fluorescent matrix, ferrocene connected to the disulfide bond is used as the fluorescence quenching group, and phenylboronic acid ester is selected as the H2O2 reaction site and fluorescence activity inhibition group on the other side of the fluorescent matrix.
[0036] Another aspect of the present invention provides a method for preparing the above-mentioned double-locked fluorescent probe for detecting H2S and H2O2, wherein 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride is subjected to an amidation reaction with t-Boc-cystamine to obtain a first intermediate NA-SS-Boc, which is then subjected to a de-Boc reaction with trifluoroacetic acid to obtain a second intermediate NA-SS-NH2, and finally NA-SS-NH2 is subjected to an amidation reaction with ferrocenecarboxylic acid to obtain the target probe P1. The structures of NA-SS-Boc and NA-SS-NH2 are as follows:
[0037]
[0038] Further, the following steps are included:
[0039] S1: Synthesis of the first intermediate NA-SS-Boc: Dissolve the compound 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride and t-Boc-cystamine in a first solvent, gradually heat to reflux under an inert atmosphere, cool after the reaction, wash with water, extract and dry, and separate by column chromatography to obtain a light white solid NA-SS-Boc;
[0040] S2: Synthesis of the second intermediate NA-SS-NH2: Dissolve the NA-SS-Boc prepared in step S1 in a second solvent, add trifluoroacetic acid, and react at room temperature under an inert atmosphere. After the reaction is completed, wash with water, extract and spin-dry, and then directly place in an oven to dry to obtain a white solid NA-SS-NH2;
[0041] S3: Synthesis of the double-locked fluorescent probe P1: NA-SS-NH2 and ferrocenecarboxylic acid prepared in step S2 were dissolved in a second solvent, dicyclohexylcarbodiimide was added as a dehydrating agent, and 4-dimethylaminopyridine was added as a catalyst. After thorough reaction at room temperature under an inert atmosphere, the separated liquid was washed with dilute hydrochloric acid, neutralized with alkali, and then dried by spin drying. The light brown solid P1 was obtained by column chromatography.
[0042] The overall synthetic route is as follows:
[0043]
[0044] Furthermore, the first solvent is acetonitrile, and the second solvent is dichloromethane.
[0045] Furthermore, in step S1, the molar ratio of the raw material compounds 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride to t-Boc-cystamine is 1:1-1.5, the molar ratio of NA-SS-Boc to trifluoroacetic acid in step S2 is 1:1-3, the molar ratio of NA-SS-NH2 to ferrocenecarboxylic acid in step S3 is 1:1-2, and 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 nitrogen atmosphere.
[0046] Furthermore, the post-processing method in step S1 is water washing, extraction and separation, spin drying, column chromatography and drying; the post-processing method in step S2 is water washing, extraction multiple times and then direct spin drying, and drying in an oven; the post-processing method in step S3 is adding dilute hydrochloric acid to wash and separate the liquid multiple times, adding dilute sodium hydroxide to neutralize and then spin drying, column chromatography and drying.
[0047] Another aspect of the present invention provides the use of the above-mentioned double-locked fluorescent probe for detecting H2S and H2O2, which is used for the detection of H2S and H2O2.
[0048] Example 1: The synthesis of a double-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-naphthalene dicarboxylic anhydride (0.52 g, 1.60 mmol) was dissolved in 15 mL of ethanol and stirred for 10 minutes to completely disperse it. 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 with water three times, extracted and separated, and the crude product was dried to obtain a crude product. 0.78 g of light white solid compound NA-SS-Boc was obtained by column chromatography using petroleum ether / CH2Cl2 (5:1, v / v) as eluent, with a yield of 87%.
[0050] The above obtained light white solid NA-SS-Boc was measured by nuclear magnetic resonance instrument (Varian instrument 400MHz), as shown in FIG. 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 NA-SS-NH2: Trifluoroacetic acid (0.06 g, 0.53 mmol) was dissolved in 5 mL of anhydrous dichloromethane, and NA-SS-Boc (0.29 g, 0.52 mmol) was slowly added dropwise. The mixture was allowed to react for 5 hours. The mixture was washed three times with pure water, extracted, and the solvent was concentrated. After drying, 0.22 g of NA-SS-NH2 was obtained as a white solid (yield: 93%).
[0053] The white solid compound NA-SS-NH2 obtained above was measured by nuclear magnetic resonance instrument (Varian instrument 400MHz), as shown in FIG. 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, and then ferrocenecarboxylic acid (0.10 g, 0.43 mol) and dicyclohexylcarbodiimide (0.11 g, 0.53 mmol) were added to the above mixture. After reacting for 24 hours, the separated liquid was washed with dilute hydrochloric acid several times, neutralized with dilute sodium hydroxide, and then dried by spin drying. The crude product was purified by column chromatography using CH2Cl2 / methanol (25:1, v / v) as the eluent to successfully obtain 0.24 g of brown solid P1 with a yield of 83%.
[0056] The brown solid compound P1 obtained above was measured by a nuclear magnetic resonance instrument (Varian instrument 400MHz), as shown in FIG. 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 double-lock fluorescent probe P1 obtained in Example 1, we replaced the analyte H2S with NaHS. Figure 4As shown, the black line represents probe P1 without any analyte added, the red line represents P1 with NaHS alone, the red line represents P1 with H2O2 alone, the green line represents P1 with both NaHS and H2O2, the purple line represents P1 with NaHS added first followed by H2O2, and the orange line represents P1 with H2O2 added first followed by NaHS. It can be seen that when probe P1 (10 μM) is treated with 0.2 mM NaHS alone, even though the disulfide bond is cleaved, causing the ferrocene group to fall from the fluorescent matrix and interrupting the PET process, the fluorescence of the matrix remains weak due to the inhibition of phenylboronic acid ester. Furthermore, when the probe is treated with 0.5 mM H2O2 alone, although the phenylboronic acid ester is oxidized to a hydroxyl group, the fluorescence of the matrix is still limited by the PET action of ferrocene. Only when NaHS (0.2 mM) and H2O2 (0.5 mM) are present at the same time (add NaHS first and then H2O2, add H2O2 first and then NaHS, or add NaHS and H2O2 at the same time), phenylboronic acid ester and ferrocene leave the matrix at the same time, both "locks" are opened, resulting in a significant enhancement of the green fluorescence of the probe at 553 nm.
[0060] Example 3:
[0061] The double-locked fluorescent probe P1 obtained in Example 1 was tested for its response time to NaHS and H2O2, respectively. Figure 5 a shows a dotted line graph of the fluorescence intensity of probe P1 after adding H2O2 for a sufficient reaction time (2h) and then adding NaHS. It can be found that after the addition of 0.2mM NaHS, the fluorescence of P1 (10μM) / H2O2 (0.5mM) at 553nm gradually increases with time, reaches the maximum at about 10 minutes, and does not change with time. Figure 5 a shows a dotted line graph showing the change in fluorescence intensity of probe P1 over time after adding NaHS for a sufficient reaction time (2 h) and then adding H2O2. It was found that after the addition of 0.5 mM H2O2, the fluorescence intensity of P1 (10 μM) / NaHS (0.2 mM) reached saturation after about 30 minutes.
[0062] Example 4:
[0063] The double-lock fluorescent probe P1 obtained in Example 1 was tested for its fluorescence titration response to NaHS and H2O2, respectively. Figure 6Figure a shows the fluorescence spectra of probe P1 after adding 0.5 mM H2O2 for a 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. It can be seen that the fluorescence emission peak of P1 (10 μM) / H2O2 (0.5 mM) at 553 nm gradually increases with the addition of NaHS, reaching saturation at 0.2 mM, with a 7.7-fold year-on-year increase in fluorescence intensity. Figure 6 b shows the fluorescence spectra of probe P1 after adding 0.2 mM NaHS for a saturation reaction 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. The fluorescence intensity of P1 (10 μM) / NaHS (0.2 mM) also increased with the addition of H2O2, reaching a peak at 0.5 mM H2O2, an increase of 13.4 times.
[0064] In summary, the present invention has obtained the double-locked fluorescent probe P1 for detecting H2S and H2O2 by chemical synthesis. At the beginning, the fluorescence of probe P1 was almost non-existent due to the dual fluorescence quenching effect of phenylboronic acid ester and ferrocene. When P1 only has H2S and H2O2 in the presence of both, phenylboronic acid ester is oxidized by H2O2, and the disulfide bond is cut by H2S, causing ferrocene to be removed, and the green fluorescence at 553nm is significantly turned on. The reaction times of probe P1 / H2O2 to H2S and P1 / H2S to H2O2 are 10 and 30 minutes, respectively. At the same time, the saturation response concentrations of probe P1 / H2O2 to H2S and P1 / H2S to H2O2 are 0.2mM and 0.5mM, respectively. It can be found that probe P1 has a shorter response time and excellent recognition effect for H2S and H2O2.
[0065] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.
Claims
1. A dual-lock fluorescent probe for detecting H2S and H2O2, characterized by: The dual-lock fluorescent probe for detecting H2S and H2O2 is P1, which uses 1,8-naphthalene diimide as the fluorescent matrix. The chemical structure of P1 is as follows:
2. The dual-lock fluorescent probe for detecting H2S and H2O2 according to claim 1, characterized in that: On one side of the fluorescent matrix, a disulfide bond is selected as the H2S response site, and ferrocene connected to the disulfide bond is used as a fluorescence quenching group. On the other side of the fluorescent matrix, phenyl borate is selected as the H2O2 reaction site and fluorescence activity inhibition group.
3. A method for preparing a double-locked fluorescent probe for detecting H2S and H2O2 according to claim 1, characterized in that: 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride is subjected to an amidation reaction with t-Boc-cystamine to obtain the first intermediate NA-SS-Boc, which is then subjected to a de-Boc reaction with trifluoroacetic acid to obtain the second intermediate NA-SS-NH2. Finally, NA-SS-NH2 and ferrocenecarboxylic acid are subjected to an amidation reaction to obtain the target probe P1. The structures of NA-SS-Boc and NA-SS-NH2 are as follows:
4. The method for preparing a double-locked fluorescent probe for detecting H2S and H2O2 according to claim 3, characterized in that: The following steps are involved: S1: Synthesis of the first intermediate NA-SS-Boc: Dissolve the compound 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride and t-Boc-cystamine in a first solvent, gradually heat to reflux under an inert atmosphere, cool after the reaction, wash with water, extract and dry, and separate by column chromatography to obtain a light white solid NA-SS-Boc; S2: Synthesis of the second intermediate NA-SS-NH2: Dissolve the NA-SS-Boc prepared in step S1 in a second solvent, add trifluoroacetic acid, and react at room temperature under an inert atmosphere. After the reaction is completed, wash with water, extract and spin-dry, and then directly place in an oven to dry to obtain a white solid NA-SS-NH2; S3: Synthesis of the double-locked fluorescent probe P1: NA-SS-NH2 and ferrocenecarboxylic acid prepared in step S2 were dissolved in a second solvent, dicyclohexylcarbodiimide was added as a dehydrating agent, and 4-dimethylaminopyridine was added as a catalyst. After thorough reaction at room temperature under an inert atmosphere, the separated liquid was washed with dilute hydrochloric acid, neutralized with alkali, and then dried by spin drying. The light brown solid P1 was obtained by column chromatography. The overall synthetic route is as follows:
5. The method for preparing a double-locked fluorescent probe for detecting H2S and H2O2 according to claim 4, characterized in that: The first solvent is acetonitrile, and the second solvent is dichloromethane.
6. The method for preparing a double-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 compounds 4-boronic acid pinacol ester-1,8-naphthalene dicarboxylic anhydride and t-Boc-cystamine is 1:1-1.5, the molar ratio of NA-SS-Boc and trifluoroacetic acid in step S2 is 1:1-3, and the molar ratio of NA-SS-NH2 and ferrocenecarboxylic acid in step S3 is 1:1-2. The equivalents of dicyclohexylcarbodiimide and 4-dimethylaminopyridine to NA-SS-NH2 are 1-1.8 and 0.5-1, respectively.
7. Use of the dual-lock fluorescent probe for detecting H2S and H2O2 according to claim 1, characterized in that: Used for the detection of H2S and H2O2.