A Fluorescent Probe Based on Naphthalimide for the Detection of Nerve Agent Simulants

Abstract

The present invention discloses a fluorescence probe based on naphthalimide for detecting nerve agent simulants, which relates to the technical field of fluorescence probes. To further expand the detection means of fluorescence probes based on naphthalimide for nerve agent simulants; the structural formula of the organic probe 1 of the present invention is synthesized with naphthalimide as the fluorophore and piperazine as the recognition site; the synthesis method of the organic probe 1 of the present invention is simple, and the organic probe 1 has good fluorescence responses to DCP and DCNP, can visually detect DCP and DCNP, can well recognize DCP and DCNP, and has a low detection limit; it provides a new detection means for fluorescence probes based on naphthalimide for nerve agent simulants, which is conducive to the further development of nerve agent detection technology based on fluorescence probes.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and specifically to a fluorescent probe based on naphthalimide for the detection of nerve agent simulants. Background Art

[0002] Nerve agents are a class of highly toxic organophosphorus compounds, and ingestion of a small amount within a short time can cause death. Sarin (GB) is a highly toxic and lethal nerve agent that can disrupt the human nervous system by inhibiting the activity of acetylcholinesterase (AChE), resulting in hundreds of deaths in the past few decades. Therefore, there is an urgent need to develop a rapid and sensitive detection method. Many techniques have been used for the detection of nerve agents, including mass spectrometry, electrochemical sensors, biosensors, semiconductor sensors, and nuclear magnetic resonance analysis. These techniques have at least one of the following disadvantages, such as high cost, poor portability, complex operation, and low sensitivity. In contrast, the detection technique based on fluorescent probes has attracted increasing attention due to its low cost, high sensitivity, and fast response speed.

[0003] As an excellent fluorophore, naphthalimide has optical properties such as high photo / thermal stability, large Stokes shift, and high fluorescence quantum yield, and is widely used for the detection of metal ions, reactive nitrogen, reactive sulfur, etc. After the recognition group of naphthalimide reacts with the analyte, it usually causes changes in the ICT or PET effect, altering the luminescence characteristics of the probe to achieve the purpose of detecting the analyte. In the article "A Fluorescence-Enhanced Probe Based on Naphthalimide for Nerve Agent Simulants" (Journal of Shanghai Normal University (Natural Science Edition). 2020, 49(02), written by Zhao Lei, etc.), a rapid-response fluorescent probe (PND) with naphthalimide as the fluorophore and hydrazino group as the active group was designed and synthesized; however, in order to meet the needs of nerve agent detection, gradually enrich the detection means, and further explore the characteristics of fluorescent probes based on naphthalimide for the detection of nerve agent simulants, the existing naphthalimide-based fluorescent probes still need to be further developed. Summary of the Invention

[0004] The purpose of the present invention is to provide a fluorescent probe based on naphthalimide for the detection of nerve agent simulants, so as to further expand the detection means of naphthalimide-based fluorescent probes for nerve agent simulants.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A fluorescent probe based on naphthalimide for the detection of nerve agent simulants, and its structural formula is:

[0006]

[0007] Preferably, the synthesis method of the above fluorescent probe includes:

[0008] (1) Synthesis of Compound 3

[0009] 4-bromo-1,8-naphthalic anhydride, the β-alanine tert-butyl ester hydrochloride after the first step of dehydrochlorination, and 4-dimethylaminopyridine were added to ethanol; heated under reflux and stirred at 80-90 °C for 9-10 h, and then cooled to room temperature; the solid product was collected by suction filtration through a Buchner funnel, the solid product was dissolved in DCM, and then the solution was extracted with DCM and saturated brine; the organic phases were combined, dried with anhydrous magnesium sulfate for 2-4 h, the magnesium sulfate was removed by suction filtration under reduced pressure, and the compound 3 was obtained by rotary evaporation under reduced pressure. Its structural formula is

[0010] (2) Synthesis of compound 2

[0011] Compound 3 and piperazine were added to 2-methoxyethanol, heated under reflux and stirred for 5-6 h. After the reaction was completed, it was cooled to room temperature, and the reaction solution was extracted with DCM and saturated brine. The product was purified by column chromatography, using DCM:MeOH = 20:1 (v / v) as the eluent, and compound 2 was obtained. Its structural formula is

[0012]

[0013] (3) Synthesis of organic probe 1

[0014] Compound 2 was dissolved in DCM, stirred in an ice bath for 25-35 min, trifluoroacetic acid was quickly added to the solution, and then stirred for 2-3 h; TCL analysis was carried out using DCM:MeOH = 15:1 (v / v) as the developing agent, and column chromatography purification was carried out by wet loading, using DCM:MeOH = 10:1 (v / v) as the eluent, and organic probe 1, that is, the above-mentioned fluorescent probe, was obtained by rotary evaporation under reduced pressure.

[0015] Preferably, in step (1), the β-alanine tert-butyl ester hydrochloride after the first step of dehydrochlorination refers to that β-alanine tert-butyl ester hydrochloride is added to K2CO3 / ACN and reacted for 3-4 h to remove hydrochloric acid, and the liquid part is obtained by cooling and filtration; the ratio of 4-bromo-1,8-naphthalic anhydride to the β-alanine tert-butyl ester hydrochloride after the first step of dehydrochlorination is 1 mmol: (1-1.2 mmol); 4-dimethylaminopyridine is used as a catalyst and its molar amount is at least 10% of 4-bromo-1,8-naphthalic anhydride; the ratio of 4-bromo-1,8-naphthalic anhydride to ethanol is 14 mmol: (15-25) ml;

[0016] Preferably, in step (2), the ratio of compound 3, piperazine, and 2-methoxyethanol is 1 mmol: 2 mmol: (15-25) ml;

[0017] Preferably, in step (3), the ratio of compound 2, DCM, and trifluoroacetic acid is 1 mmol: (4-10 ml): (1-5 ml);

[0018] Preferably, in step (3), two drops of TEA are added during column packing for column chromatography purification to neutralize the acidity of a small amount of trifluoroacetic acid.

[0019] Preferably, the above-mentioned fluorescent probe is used for detecting and identifying DCP and DCNP.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. For the fluorescent probe based on naphthalimide for detecting nerve agent simulants, the synthesis method of organic probe 1 is simple and has high selectivity. Organic probe 1 has good fluorescence responses to DCP and DCNP. When responding, the fluorescence of the probe changes significantly from colorless to green, enabling visual detection of DCP and DCNP, being able to well identify DCP and DCNP, and having a low detection limit and a short response time.

[0022] 2. For the fluorescent probe based on naphthalimide for detecting nerve agent simulants, it provides a new detection means for the naphthalimide-based fluorescent probe for nerve agent simulants, which is conducive to the further development of the nerve agent detection technology based on fluorescent probes. Description of the Drawings

[0023] Figure 1 1H NMR spectrum of organic probe 1 of the present invention.

[0024] Figure 2 13C NMR spectrum of organic probe 1 of the present invention.

[0025] Figure 3 Infrared spectrum of organic probe 1 of the present invention.

[0026] Figure 4 UV absorption spectrum of organic probe 1 of the present invention for DCP in DMF.

[0027] Figure 5 Fluorescence spectrum (a) and linear relationship diagram (b) of organic probe 1 of the present invention after adding DCP.

[0028] Figure 6 Fluorescence responses of organic probe 1 of the present invention to several interfering substances.

[0029] Figure 7 Time-dependent fluorescence diagram of organic probe 1 of the present invention under 10eq DCP.

[0030] Figure 8 Fluorescence change diagram of adding 0 - 10eq DCP solution to 10 μM organic probe 1 solution of the present invention.

[0031] Figure 9This is the UV absorption spectrum of the organic probe 1 of the present invention to DCNP in DMF.

[0032] Figure 10 The fluorescence spectrum (a) and linear relationship diagram (b) of the organic probe 1 of the present invention after adding DCNP.

[0033] Figure 11 This is a time-dependent fluorescence graph of the organic probe 1 of the present invention under 10 eqDCNP. DETAILED DESCRIPTION

[0034] Example 1:

[0035] In this example, an organic probe 1 was synthesized using naphthaleneimide as a fluorophore and piperazine as a recognition site. The synthesis route is as follows:

[0036]

[0037] The specific synthesis steps are:

[0038] (1) Synthesis of compound 3

[0039] In a 50 mL single-necked flask, 4-bromo-1,8-naphthalic anhydride (3.88 g, 14.01 mmol), β-alanine tert-butyl ester hydrochloride (2.23 g, 15.41 mmol) after the first step of dehydrochlorination, 4-dimethylaminopyridine (2.23 g, 18.25 mmol) and 20 mL of ethanol were added. The mixture was heated under reflux at 85°C with stirring for 10 hours and then cooled to room temperature. The solid product collected by vacuum filtration on a Buchner funnel was dissolved in DCM, and the solution was then extracted with DCM and saturated brine. The organic phases were combined and dried over anhydrous magnesium sulfate for 2 hours. The magnesium sulfate was removed by vacuum filtration and vacuum rotary evaporation to obtain compound 3 (3.31 g, yield 55.03%) as a yellow solid.

[0040] The H NMR spectrum of the obtained compound 3 is as follows: 1 H NMR (400MHz, DMSO-d6) δ8.43-8.21(d,J=25.7Hz,2H),8.16-7.96(d,J=28.0Hz,2 H),7.89-7.77(s,1H),4.24-4.11(s,2H),2.63-2.53(s,2H),1.45-1.22(s,9H).

[0041] The C NMR spectrum of the obtained compound 3 is as follows: 13C NMR(101MHz,DMSO-d6)δ170.61-170.43,163.18-162.95,163.10-162.92, 133.20-132.89,132.10-131.85,131.85-131.59,131.45-131.16,130.15- 129.94,129.76-129.55,129.31-129.01,128.58-128.35,123.00-122.79 ,122.22-122.01,80.71-80.53,36.46-36.10,33.84-33.53,28.16-27.94.

[0042] (2) Synthesis of Compound 2

[0043] Compound 3 (3.11 g, 7.71 mmol) and piperazine (1.33 g, 15.41 mmol) were added to a 50 mL single-necked flask, followed by the addition of 20 mL of 2-methoxyethanol. The mixture was heated under reflux and stirred for 6 hours. After the reaction was completed, the mixture was cooled to room temperature and extracted with DCM and saturated brine. The product was purified by column chromatography using DCM:MeOH = 20:1 (v / v) as the eluent to obtain compound 2 (502 mg, yield 15.82%) as a yellow solid.

[0044] The H NMR spectrum of the obtained compound 2 is as follows: 1 H NMR (400MHz, CDCl3) δ8.63-8.56(d,J=7.3Hz,1H),8.56-8.50(d,J=8.1Hz,1H),8.48-8.41(s,1H),7.75-7.66(m,1H),7.26-7.21(d ,J=8.1Hz,1H),4.51-4.41(m,2H),3.39-3.27(s,4H),3.11-2.80(s,4H),2.75-2.63(m,2H),1.81-1.62(s,1H),1.48-1.40(s,9H).

[0045] The C NMR spectrum of the obtained compound 2 is as follows: 1313C NMR (101 MHz, CDCl3) δ 170.63 - 170.57, 164.32 - 164.27, 163.84 - 163.77, 156.23 - 156.15, 129.95 - 129.90, 126.22 - 126.16, 125.69 - 125.54, 123.15 - 123.09, 116.51 - 116.44, 115.05 - 114.77, 53.24 - 53.01, 51.71 - 51.44, 36.18 - 35.99, 34.04 - 33.81, 28.13 - 27.95.

[0046] (3) Synthesis of Organic Probe 1

[0047] Dissolve compound 2 (502 mg, 1.22 mmol) in 10 mL of DCM and stir in an ice bath for 30 minutes. Quickly add 5 mL of trifluoroacetic acid to the solution and then stir for 3 hours. Perform TCL analysis using a developing solvent of DCM:MeOH = 15:1. Purify by column chromatography using wet loading (add two drops of TEA when packing the column to neutralize the acidity of a small amount of trifluoroacetic acid), and use DCM:MeOH = 10:1 as the eluent. Rotate and evaporate under reduced pressure to obtain yellow solid organic probe 1 (211 mg, yield 49.18%).

[0048] Refer to Figure 1 , and the 1H NMR spectrum of the obtained organic probe 1 is as follows: 1 1H NMR (400 MHz, DMSO-d6) δ 8.50 - 8.43 (d, J = 8.5 Hz, 1H), 8.42 - 8.36 (d, J = 7.2 Hz, 1H), 8.36 - 8.26 (d, J = 8.1 Hz, 1H), 7.83 - 7.70 (m, 1H), 7.41 - 7.32 (d, J = 8.1 Hz, 1H), 4.34 - 4.06 (m, 2H), 3.49 - 3.36 (m, 8H), 2.60 - 2.52 (t, J = 7.7 Hz, 2H), 1.26 - 1.15 (m, 1H).

[0049] Refer to Figure 2 , and the 13C NMR spectrum of the obtained organic probe 1 is as follows: 1313C NMR (101 MHz, DMSO-d6) δ 173.00 - 172.85, 163.81 - 163.65, 163.29 - 163.12, 159.28 - 159.12, 158.98 - 158.80, 154.93 - 154.74, 132.58 - 132.22, 131.36 - 131.04, 131.04 - 130.67, 129.41 - 129.24, 126.99 - 126.67, 125.83 - 125.64, 122.95 - 122.79, 119.21 - 118.99, 117.06 - 116.89, 116.25 - 115.83, 50.20 - 49.78, 43.68 - 43.25, 36.26 - 35.80, 32.91 - 32.47.

[0050] The high-resolution mass spectrum of the obtained organic probe 1 is as follows: HRMS (ESI) calcd for C 19 H 19 N3O4 [M + H] + 353.14483, found 354.14401.

[0051] Example 2:

[0052] The detection experiment was carried out using the organic probe 1 prepared in Example 1:

[0053] As Figure 4 shown, in DMF, the ultraviolet absorption of the organic probe 1 itself is at 405 nm. After adding 100 μM of DCP, the absorption peak blue-shifts from 405 nm to about 400 nm.

[0054] As Figure 5 shown in a of [reference], a fluorescence titration experiment was carried out with 10 μM of the organic probe 1 in 2 mL of DMF. After adding DCP to the solution, the fluorescence at 510 nm gradually increased. When 200 μM of DCP was added, the organic probe 1 completely reacted with DCP and the fluorescence intensity basically no longer changed.

[0055] The determination of fluorescence linearity and detection limit, as Figure 5 shown in b of [reference], within the concentration range of 0 - 30 μM, the organic probe 1 has a linear fluorescence response to DCP. Using the formula the detection limit was calculated to be 12 nM.

[0056] As Figure 6As shown, DCNP (100 μM) can also cause fluorescence changes in organic probe 1. Different from DCP, the emission peak of DCNP is at 520 nm. Other interfering substances such as acetyl chloride (AC), acetic acid (HAc), triethyl phosphate (TEP), and diethyl cyanomethylphosphonate (DCMP) do not have obvious interference on the detection results. This indicates that the selectivity of organic probe 1 is relatively good, and it can respond to both mimics of two nerve agents.

[0057] As Figure 7 shown, after adding 50 μM of DCP to the organic probe 1 solution, a fluorescence response signal can be given after 5 minutes, with a short response time.

[0058] As Figure 8 shown, when 0 - 5 eq of DCP solution was added to 10 μM of the organic probe 1 DMF solution, it was found that the green fluorescence of the solution gradually increased.

[0059] As Figure 9 shown, in DMF, the ultraviolet absorption of organic probe 1 itself is at 405 nm. After adding 100 μM of DCNP, the absorption peak blue-shifts from 405 nm to about 395 nm.

[0060] As Figure 10 shown in a of , a fluorescence titration experiment was carried out with 10 μM of organic probe 1 in 2 mL of DMF. After adding DCNP to the solution, the fluorescence at 520 nm gradually increased. When 100 μM of DCNP was added, organic probe 1 completely reacted with DCNP, and the fluorescence intensity basically no longer changed.

[0061] Determination of fluorescence linearity and detection limit. As Figure 10 shown in b of , in the concentration range of 0 - 90 μM, organic probe 1 has a linear fluorescence response to DCNP. Using the formula the detection limit was calculated to be 22 nM.

[0062] As Figure 11 shown, after adding 100 μM of DCNP to the organic probe 1 DMF solution, it reached stability at 60 s, and the intensity basically did not fluctuate much within the subsequent 3 min, with a short response time.

[0063] It can be seen from this example that organic probe 1 has a good fluorescence response to DCP and DCNP, can visually detect DCP and DCNP, can well identify DCP and DCNP, and has a low detection limit and a short response time.

[0064] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection defined by the claims.

[0065] Any matters not described in detail in the present invention are well-known technologies to those skilled in the art.

Claims

1. Use of a naphthalimide-based fluorescent probe for detecting nerve agent simulants in detecting and identifying DCP and DCNP for non-disease diagnosis or treatment purposes, characterized in that: The structural formula of the fluorescent probe is: The application is to detect and identify DCNP, or to detect and identify DCP and DCNP simultaneously.

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