CN108675989B - Fe3+ molecular fluorescence test agent and preparation method thereof - Google Patents

Abstract

The present invention provides a Fe ³⁺ molecular fluorescence test agent and a preparation method thereof. The Fe ³⁺ molecular fluorescent test agent has the structure shown in formula I. The Fe ³⁺ molecular fluorescence test reagent provided by the present invention uses 1,8-naphthalimide as the fluorescent group, so that it has good photostability, the excitation and emission light are both long-wavelength visible light, and the Stokes shift is large. High quantum yield. At the same time, the Fe ³⁺ molecular fluorescence test agent in The sensitivity of the group to Fe ³⁺ is high, and the nitrogen in its pyridine ring and the amino nitrogen at the 4-position can be complexed with Fe ³⁺ to form an ion complex, which can induce electron transfer or energy transfer, so that the test agent Different fluorescence responses at different Fe ³⁺ concentrations. The above reasons also make the Fe ³⁺ molecular fluorescence test agent provided by the present invention have better test accuracy and sensitivity.

Description

Fe3+ molecular fluorescence test agent and preparation method thereof

technical field

The invention relates to the technical field of organic synthesis and elemental analysis, in particular to a Fe ³⁺ molecular fluorescence test agent and a preparation method thereof.

Background technique

Iron is one of the most abundant trace elements in the body. It is an important component of hemoglobin, myoglobin and various enzymes, and participates in processes such as oxygen uptake, oxygen metabolism, and electron transfer. If there is a lack of iron in the body, it can affect the synthesis of hemoglobin and reduce the activity of enzymes such as cytochrome c, ribonucleotide reductase, and succinate dehydrogenase, resulting in serious body dysfunction. The change of Fe ³⁺ content in the human body is related to many diseases, such as iron deficiency can lead to anemia, cancer, diabetes and organ dysfunction, etc., while iron excess can generate reactive oxygen species through the Fenton reaction to induce Alzheimer's disease, Huntington's disease, etc. disease and Parkinson's disease. Therefore, the rapid and accurate detection of iron ion content in the environment and human body is of great significance for environmental safety and human health.

At present, there are many analytical techniques for detecting trace Fe ³⁺ , including atomic absorption spectrometry, plasma emission spectrometry, plasma mass spectrometry, electrochemical method, titration method, etc. Most of these methods require the use of expensive large-scale instruments, are complicated to operate, have poor portability and are not suitable for online real-time monitoring. Due to the simple equipment required for fluorescence analysis, and the advantages of fast response, high sensitivity, and easy operation, the use of fluorescent probes for qualitative and quantitative detection of Fe ³⁺ has become a research hotspot.

In recent years, there have been few reports on the study of Fe ³⁺ fluorescent probes. Most of them are iron ion fluorescent probes with rhodamine as the fluorescent group, such as Chinese patents CN107011351A, CN 105884788A, Synthesis and evaluation of a novel rhodamine B-based'off-on'fluorescent chemosensor for the selective determination of Fe ³⁺ ions , Sensors and Actuators B: Chemicals, 2017, 242, 921-931. The detection principle of rhodamine-based fluorescent probes is as follows: by interacting with metal ions, the self-lactam spiro-ring structure is opened, which can cause changes in ultraviolet-visible absorption and fluorescence spectrum, and finally achieve the purpose of detecting different metal ion concentrations.

However, because rhodamine participates in the ion coupling reaction, its excitation wavelength and emission wavelength will change due to the change of the detected ions, which leads to the fact that rhodamine-based fluorescence sensors with different ions cannot use the same excitation and emission wavelengths for detection. Moreover, the Fe ³⁺ fluorescence sensor reported above has an excitation wavelength of about 560 nm and an emission wavelength of about 580 nm, and its Stockes shift is only about 20 nm. It is difficult for ordinary optical spectroscopic lenses to distinguish the excitation light from the fluorescence, which affects the detection results. Generally, spectral detection equipment with high spectral resolution is used for fluorescence signal acquisition, which will greatly increase the difficulty and cost of detection.

For the above reasons, it is necessary to use a fluorophore with a larger Stokes shift for the design of Fe ³⁺ molecular fluorescence sensor.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide a Fe ³⁺ molecular fluorescent test agent and a preparation method thereof, so as to solve the problem of the Fe ³⁺ molecular fluorescent test agent being too small in Stokes shift in the prior art.

In order to achieve the above object, according to one aspect of the present invention, a kind of Fe ³⁺ molecular fluorescence test agent is provided, which has the structure shown in formula I:

According to another aspect of the present invention, there is also provided a preparation method of Fe ³⁺ molecular fluorescence test agent, which comprises the following steps:

In step S1, compound A and compound B are subjected to a condensation reaction to form compound C; the structures of compound A, compound B and compound C are as follows, wherein X in compound A is halogen:

In step S2, compound C is subjected to hydrolysis reaction to obtain Fe ³⁺ molecular fluorescence test agent:

Further, before step S1, the preparation method further includes the step of preparing compound A, which includes: performing an esterification reaction with 4-cyanobenzoic acid and tert-butanol to obtain tert-butyl 4-cyanobenzoate; - tert-butyl cyanobenzoate, catalyst and methanol are mixed to form a mixed system, and hydrogen is introduced into the mixed system to carry out hydrogenation reaction to obtain tert-butyl 4-aminomethylbenzoate E; wherein the catalyst is a nickel catalyst, a palladium-carbon catalyst and one or more of the cobalt catalysts; 4-aminomethyl benzoic acid tert-butyl ester E and compound F are reacted to obtain compound A; wherein 4-amino methyl benzoic acid tert-butyl ester E, compound F have the following structure:

wherein X in compound F is halogen.

Further, step S1 includes: mixing compound A, compound B and acid binding agent with a first solvent to obtain a first mixture; subjecting the first mixture to a condensation reaction at a temperature of 80-100° C. to obtain a first product system; The first product system is cooled and poured into water, filtered to obtain a precipitate, which is compound C; preferably, the first solvent is N-methylpyrrolidone, N,N-dimethylacetamide, dimethyl sulfoxide, dimethyl sulfoxide One or more of acetamide and tetrahydrofuran; preferably, the acid binding agent is N,N-diisopropylethylamine, N,N-dimethylformamide, 4-dimethylaminopyridine and triethylamine One or more of amines; preferably, the molar ratio between compound A, compound B and acid binding agent is 1-6:1-5:1-15.

Further, after the filtering step, step S1 further includes the step of washing the precipitate, and the washing step includes: dissolving the precipitate in chloroform, adding water to it for washing, and separating liquids to obtain an organic phase and an aqueous phase; The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give compound C.

Further, step S2 includes: mixing and reacting the compound C, the carbonyl removal reagent and the second solvent to obtain a second product system; using a chloroform/methanol mixed solution with a volume ratio of 1:1 to dilute the second product system, Then the solvent is evaporated to obtain ^Fe molecular fluorescence test reagent; preferably, the carbonyl removal reagent is trifluoroacetic acid, a mixed reagent of hydrochloric acid and ethyl acetate with a volume ratio of 1:2, or silica gel; preferably, the second solvent is Dichloromethane and/or chloroform; preferably, the mole number of compound C is 1.8-2.0% of the mole number of the carbonyl removal reagent, preferably 1.9%.

Further, the step of performing the esterification reaction between 4-cyanobenzoic acid and tert-butanol includes: mixing and reacting 4-cyanobenzoic acid and the fourth solvent of the acylating reagent to obtain an intermediate; removing the solvent in the intermediate Then, it is mixed and reacted with an esterification catalyst and tert-butanol to obtain a fourth product system; the fourth product system is purified to obtain tert-butyl 4-cyanobenzoate; preferably, the acylating reagents are oxalyl chloride, chlorine One or more of sulfoxide, phosphorus trichloride and phosphorus pentachloride; preferably, the esterification catalyst is pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine and triethylamine one or more of; preferably, the molar ratio between the 4-cyanobenzoic acid and the acylating agent is 2～3:3～5; the volume ratio between the esterification catalyst and tert-butanol is 1～3 1.1:1.

Further, in the step of preparing tert-butyl 4-aminomethylbenzoate E, the weight ratio between tert-butyl 4-cyanobenzoate and the catalyst is 10-20:1-3; 4-cyanobenzoic acid The weight ratio between tert-butyl ester and methanol is 1-3:8-20; preferably, after hydrogen is introduced, the pressure of the reaction system is 0.8-1.2 MPa.

Further, the step of reacting tert-butyl 4-aminomethylbenzoate E with compound F includes: mixing and reacting tert-butyl 4-aminomethylbenzoate E, compound F and the fifth solvent to obtain the fifth solvent. product system; filter the fifth product system, and dry the obtained precipitate to obtain compound A; preferably, the fifth solvent is one or more of ethanol, methanol, propanol and isopropanol; preferably, 4- The molar ratio between tert-butyl aminomethylbenzoate E and compound F was 1:1.

The Fe ³⁺ molecular fluorescence test reagent provided by the present invention uses 1,8-naphthalimide as the fluorescent group, so that it has good photostability, and the excitation (449nm) and emission light (533nm) are both long-wavelength visible light. The Tox shift is large (84 nm) and the quantum yield is high. At the same time, the Fe ³⁺ molecular fluorescence test agent in The group (test group) has high sensitivity to Fe ₃₊ , and the nitrogen in the pyridine ring and the 4-amino nitrogen can be complexed with Fe ³⁺ to form an ionic complex, which can induce electron transfer or energy transfer , so that the test agent has different fluorescence responses at different Fe ³⁺ concentrations. The above reasons also make the Fe ³⁺ molecular fluorescence test agent provided by the present invention have better test accuracy and sensitivity.

Description of drawings

The accompanying drawings forming a part of the present application are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the attached image:

Fig. 1 shows the hydrogen nuclear magnetic resonance spectrum of tert-butyl-4-aminomethylpyridine-1,8-naphthalimide methylbenzoate prepared in Example 1 of the present invention;

Fig. 2 shows the hydrogen nuclear magnetic resonance spectrum of 4-aminomethylpyridine-1,8-naphthalimidomethylbenzoic acid prepared in Example 1 of the present invention;

Fig. 3 shows the change diagram of the fluorescence spectrum of the Fe ³⁺ fluorescent test solution prepared in Example 1 of the present invention as the volume of the Fe ³⁺ solution to be tested increases;

Fig. 4 shows the variation diagram of the fluorescence intensity of Fe ³⁺ fluorescent test solution prepared in Example 1 of the present invention as the Fe ³⁺ concentration increases;

5 shows a schematic diagram of the selective identification of Fe ³⁺ fluorescence by the Fe ³⁺ fluorescent test solution prepared in Example 1 of the present invention;

6 shows a schematic diagram of the anti-interference identification of Fe ³⁺ detected by the Fe ³⁺ fluorescent test solution prepared in Example 1 of the present invention;

Detailed ways

It should be noted that the embodiments in the present application and the features of the embodiments may be combined with each other in the case of no conflict. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

The present application will be described in further detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present application.

As described in the background section, the Stokes shift of the Fe ³⁺ molecular fluorescent test agent in the prior art is too small.

In order to solve the above problems, the present invention provides a kind of Fe ³⁺ molecular fluorescence test agent, which has the structure shown in formula I:

The Fe ³⁺ molecular fluorescence test reagent provided by the present invention uses 1,8-naphthalimide as the fluorescent group, so that it has good photostability, and the excitation (449nm) and emission light (533nm) are both long-wavelength visible light. The Tox shift is large (84 nm) and the quantum yield is high. At the same time, the Fe ₃₊ molecular fluorescence test agent in The group (test group) has high sensitivity to Fe ₃₊ , and the nitrogen in the pyridine ring and the 4-amino nitrogen can be complexed with Fe ³⁺ to form an ionic complex, which can induce electron transfer or energy transfer , so that the test agent has different fluorescence responses at different Fe ³⁺ concentrations. The above reasons also make the Fe ³⁺ molecular fluorescence test agent provided by the present invention have better test accuracy and sensitivity.

According to another aspect of the present invention, there is also provided a preparation method of Fe ³⁺ molecular fluorescence test agent, which comprises the following steps:

In step S1, compound A and compound B are subjected to a condensation reaction to form compound C; the structures of compound A, compound B and compound C are as follows, wherein X in compound A is halogen:

In step S2, compound C is subjected to hydrolysis reaction to obtain Fe ³⁺ molecular fluorescence test agent:

As mentioned above, the Fe ³⁺ molecular fluorescence test agent prepared by the modified preparation method of the present invention has both better test accuracy and sensitivity. In addition, in the preparation method, compound C is prepared by condensation reaction of compound A and compound B, and then the Fe ³⁺ molecular fluorescence test agent is prepared by hydrolysis of compound C. The reaction route is simple, the cost is low, and the process conditions are mild, which is suitable for industrialization. mass production.

The above-mentioned compound A can be selected from commercially available products. In a preferred embodiment, before step S1, the preparation method further includes a step of preparing compound A, which includes: esterifying 4-cyanobenzoic acid with tert-butanol chemical reaction to obtain tert-butyl 4-cyanobenzoate; mix tert-butyl 4-cyanobenzoate, a catalyst (one or more of a nickel catalyst, a palladium-carbon catalyst and a cobalt catalyst) and methanol to form a mixed system , hydrogen is introduced into the mixed system to carry out hydrogenation reaction to obtain tert-butyl 4-aminomethylbenzoate E; tert-butyl 4-aminomethylbenzoate E is reacted with compound F to obtain compound A; wherein 4- Aminomethylbenzoic acid tert-butyl ester E, compound F have the following structures:

wherein X in compound F is halogen.

Using the above method to prepare compound A, the route is short, the process is simple, the cost is lower, and the yield is relatively high.

The specific process conditions in each of the above synthesis steps can be adjusted, and the details are as follows:

In a preferred embodiment, the above step S1 includes: mixing compound A, compound B and acid binding agent with a first solvent to obtain a first mixture; and heating the first mixture at 80-100°C, more preferably at 90°C The condensation reaction is carried out at the temperature to obtain the first product system; the first product system is cooled, poured into water, and filtered to obtain a precipitate, which is compound C.

Preferably, the first solvent includes, but is not limited to, one or more of N-methylpyrrolidone, N,N-dimethylacetamide, dimethylsulfoxide, dimethylacetamide and tetrahydrofuran.

Preferably, the above acid binding agent is one or more of N-methylpyrrolidone, N,N-dimethylacetamide, dimethyl sulfoxide, dimethylacetamide and tetrahydrofuran. The use of these acid binding agents is beneficial to further improve the reaction efficiency between compound A and compound B, and to improve the reaction conversion rate.

Preferably, the molar ratio between compound A, compound B and the acid binding agent is 1-6:1-5:1-15; preferably, after the filtering step, step S1 further includes the step of washing the precipitate, and the washing The steps include: dissolving the precipitate in chloroform, adding water for washing, and separating liquids to obtain an organic phase and an aqueous phase; drying the organic phase with anhydrous sodium sulfate, filtering and evaporating to obtain compound C.

Examples are as follows: tert-butyl-4-chloro-1,8-naphthalimidomethylbenzoate (compound A), 2-aminomethylpyridine (compound B) and N,N-dicarboxylate Isopropylethylamine was suspended in N-methylpyrrolidone (NMP) and heated at 90°C for 18 hours. Cool the mixture and pour into water. The precipitate was obtained by filtration, then dissolved in _CHCl3 and washed with water, and the layers were separated. The organic layer was dried _{over Na2SO4} , filtered and evaporated to give the crude product, which was hot slurried with hot methanol at 55-60°C, filtered and washed with cold methanol at 0-10°C. The obtained solid (solid after washing with cold methanol) was recrystallized with hot _CHCl3 at 50-55°C to obtain yellow crystals of tert-butyl-4-aminomethylpyridine-1,8-naphthalimidomethylbenzene Formate (Compound C).

In a preferred embodiment, the above step S2 includes: mixing and reacting compound C, the carbonyl removal reagent and the second solvent to obtain a second product system; using a chloroform/methanol mixture with a volume ratio of 1:1 The solution dilutes the second product system, and then evaporates the solvent to obtain the Fe ³⁺ molecular fluorescence test agent. The tert-butoxycarbonyl (BOC) group of reactant C was removed using a carbonyl removal reagent. The chloroform/methanol mixed solution with a volume ratio of 1:1 can be used as a solvent to dilute the product, thereby removing excess carbonyl removal reagent.

Preferably, the above-mentioned carbonyl removal reagent is trifluoroacetic acid, a mixed reagent of hydrochloric acid and ethyl acetate in a volume ratio of 1:2, or silica gel. For the purpose of reducing side reactions and simplifying the post-treatment process, trifluoroacetic acid is more preferably used as the carbonyl removal reagent. Preferably, the second solvent is dichloromethane and/or chloroform, wherein using dichloromethane is less toxic and more environmentally friendly. Preferably, the mole number of compound C is 1.8-2.0% of the mole number of the carbonyl removal reagent, more preferably 1.9%.

An example is as follows: trifluoroacetic acid (TFA) is added to _CH2Cl2 where tert-butyl- ₄ -aminomethylpyridine-1,8-naphthalimidomethylbenzoate (compound C) is located in solution. The resulting solution was stirred at room temperature for about 1 hour until thin layer chromatography (TLC) showed that most of the tert-butyl-4-aminomethylpyridine-1,8-naphthalimidomethylbenzoate disappeared. The mixture was then diluted with 1:1 by volume _CHCI3 :MeOH and the solvent was evaporated. Repeat 4 to 8 times to remove TFA, then put it on the pump for 30 minutes to completely dry to give yellow crystals of 4-aminomethylpyridine-1,8-naphthalimidomethylbenzoic acid (Fe ³⁺ Molecular Fluorescence Test Agent).

In a preferred embodiment, the step of esterifying 4-cyanobenzoic acid with tert-butanol includes: mixing and reacting 4-cyanobenzoic acid, an acylating reagent and a fourth solvent to obtain an intermediate ; After removing the solvent in the intermediate, mix and react with the esterification catalyst and tert-butanol mixture to obtain the fourth product system; purify the fourth product system to obtain tert-butyl 4-cyanobenzoate. The acylation reagent is used for acylation reaction with 4-cyanobenzoic acid to form an intermediate product, and then it can react with tert-butanol under the action of an esterification catalyst to form tert-butyl 4-cyanobenzoate. Compared with the direct reaction between acid and pure, using the route provided by the present invention to prepare tert-butyl 4-cyanobenzoate, the raw material conversion rate is higher. Preferably, the acylating reagent is one or more of oxalyl chloride, thionyl chloride, phosphorus trichloride and phosphorus pentachloride; preferably, the esterification catalyst is pyridine, N,N-dimethylformamide One or more of (DMF), 4-dimethylaminopyridine (DMAP) and triethylamine.

Preferably, the molar ratio between 4-cyanobenzoic acid and the acylating agent is 2-3:3-5, more preferably 2:3; the total weight of the esterification catalyst and tert-butanol is denoted as n, and the removal of The weight of the solvent intermediate is denoted as m, and m:n is 1-3:10-20, more preferably 3:20; the volume ratio between the esterification catalyst and tert-butanol is 1-1.1:1, preferably 1:1.

An example is as follows: 4-cyanobenzoic acid is dissolved in dry _CH2Cl2 , oxalyl _chloride and dimethylformamide (DMF) are added. The resulting mixture was stirred at room temperature for 1 hour until gas evolution ceased. The solvent was removed and the resulting residue was treated with a pyridine/tert-butanol mixture and stirred at room temperature for 6 hours. The solvent was evaporated under reduced pressure, and the green residue was suspended in water, followed by extraction with ethyl acetate. The combined organic layers were washed, dried _{over Na2SO4} and evaporated. The crude product was purified by an ethyl acetate/petroleum ether column to give tert-butyl 4-cyanobenzoate as a white solid.

In a preferred embodiment, in the step of preparing tert-butyl 4-aminomethylbenzoate E, the weight ratio between tert-butyl 4-cyanobenzoate and the catalyst is 10-20:1-3, More preferably, it is 10:1; the weight ratio between tert-butyl 4-cyanobenzoate and methanol is 1～3:8～20, more preferably 3:20; The pressure is 0.8 to 1.2 MPa, more preferably 1 MPa. Utilizing the above-mentioned process to prepare tert-butyl 4-aminomethylbenzoate E, the reaction efficiency is higher, and the above-mentioned catalyst can specifically adopt one or more of a nickel catalyst, a palladium-carbon catalyst and a cobalt catalyst.

An example is as follows: tert-butyl 4-cyanobenzoate and nickel catalyst were mixed in methanol and hydrogenated at a pressure of 1 MPa for 16 hours. The catalyst was removed by filtration and then the solvent was removed under reduced pressure to yield tert-butyl 4-aminomethylbenzoate as a white solid.

In a preferred embodiment, the step of reacting tert-butyl 4-aminomethylbenzoate E with compound F comprises: mixing tert-butyl 4-aminomethylbenzoate E, compound F and a fifth solvent and react to obtain the fifth product system; filter the fifth product system, and dry the obtained precipitate to obtain compound A.

Preferably, the fifth solvent is one or more of ethanol, methanol, propanol and isopropanol; Preferably, the mol ratio between tert-butyl 4-aminomethylbenzoate E and compound F is 1: 1.

An example is as follows: 4-chloro-1,8-naphthalenedicarboxylic anhydride and tert-butyl 4-aminomethylbenzoate are put into ethanol to form a suspension. Stir at room temperature for 16 hours. The obtained precipitate was filtered and dried at 50°C for 8 hours to obtain tert-butyl-4-chloro-1,8-naphthalimidomethylbenzoate as a white powder.

According to another aspect of the present invention, there is also provided a Fe ³⁺ molecular fluorescence test solution, which comprises the above-mentioned Fe ³⁺ molecular fluorescence test agent and a solvent for dissolving the Fe ³⁺ molecular fluorescence test agent; the preferred solvent is ethanol , one or more of acetonitrile, dimethyl sulfoxide and N,N-dimethylformamide, and the concentration of the Fe ³⁺ molecular fluorescence test agent in the Fe ³⁺ molecular fluorescence test solution is 1 to 10 μmol/L . Based on the excellent performance of the Fe ³⁺ molecular fluorescence test agent, the above-mentioned Fe ³⁺ molecular fluorescence test solution also has good test sensitivity and accuracy.

The beneficial effects of the present invention are further described below by examples:

Example 1

Synthesis of Compound 1

4-Cyanobenzoic acid (80 g, 544 mmol) was dissolved in dry _{CH2Cl2 (} 1000 mL). Oxalyl chloride (104 mL, 816 mmol) and dimethylformamide (DMF) 10 mL were added. The resulting reaction mixture was stirred at room temperature for 1 hour until gas evolution ceased. Remove the solvent. The resulting residue was treated with 600 mL of a pyridine/tert-butanol mixture (1:1) and stirred at room temperature for 6 hours. The solvent was evaporated under reduced pressure and the green residue was suspended in _H2O . The aqueous suspension was extracted with ethyl acetate (3 x 500 mL). The combined organic layers were washed with 10% _KHSO4 (2 x 500 mL), _H2O (500 mL), _NaHCO3 (500 mL), _H2O (500 mL) and brine (500 mL). The solvent was dried _{over Na2SO4} and evaporated. The crude product was purified by column chromatography using ethyl acetate/petroleum ether (1:4) to give a white solid 58g (52% yield).

Synthesis of Compound 2

tert-Butyl 4-cyanobenzoate (58 g, 37 mol) and nickel catalyst (5.8 g) were mixed in methanol (500 mL) and hydrogenated under a pressure of 10 kg for 16 hours. The catalyst was removed by filtration, and then the solvent was removed under reduced pressure to obtain 47 g of a white solid (80% yield).

Synthesis of Compound 3

46.4 g (200 mmol) of 4-chloro-1,8-naphthalenedicarboxylic anhydride and 41.4 g (200 mmol) of tert-butyl 4-aminomethylbenzoate were placed in 250 mL of EtOH to form a suspension. Stir at RT for 16 hours. The obtained precipitate was filtered and dried at 50°C for 8 hours to obtain 48 g of white powder (yield 57%).

Synthesis of compound 4

Combine 24.33 g (225 mmol) 2-aminomethylpyridine and 31.3 g (74.3 mmol) tert-butyl-4-chloro-1,8-naphthalimidomethylbenzoate and 13 mL (74.6 mmol) N,N-Diisopropylethylamine was suspended in 110 mL of N-methylpyrrolidone (NMP) and heated at 90°C for 18 hours. The mixture was cooled and poured into water (2 L). The precipitate was obtained by filtration, then dissolved in _CHCl3 (800 mL) and washed with water (5 x 800 mL). The organic layer was dried _{over Na2SO4} , filtered and evaporated to give 63.55 g of crude product. The residue was triturated with hot methanol (600 mL), filtered and washed with cold methanol (600 mL). _The obtained solid was recrystallized with hot CHCl to obtain 32.0 g of yellow crystals (87% yield), tert-butyl-4-aminomethylpyridine-1,8-naphthalimidomethylbenzoate, Its H NMR spectrum is shown in Figure 1.

Synthesis of compound 5

87.5 mL (1.14 mol) of trifluoroacetic acid (TFA) was added to 10.68 g (21.64 mmol) of tert-butyl-4-aminomethylpyridine-1,8-naphthalimidomethylbenzoate in in _{CH2Cl2 (} 160 mL). The resulting solution was stirred at room temperature for about 1 hour until thin layer chromatography (TLC) showed that most of the tert-butyl-4-aminomethylpyridine-1,8-naphthalimidomethylbenzoate disappeared. The mixture was then diluted with 1:1 _CHCl3 :MeOH (1.2 L) and the solvent was evaporated. This was repeated 6 times to remove TFA, then placed on the pump for 30 minutes to completely dry to obtain 9.35 g of yellow crystals (yield 99%), 4-aminomethylpyridine-1,8-naphthalimidomethyl Benzoic acid, its H NMR spectrum is shown in Figure 2.

Preparation of Fe ³⁺ Molecular Fluorescence Test Solution

Weigh 0.0022 g of 4-aminomethylpyridine-1,8-naphthalimidomethylbenzoic acid (compound 5) into 100 ml of solution (volume ratio water: ethanol: acetonitrile = 1:9:10) to obtain 5 ×10 ^-2 mol/L Fe ³⁺ molecular fluorescence test stock solution.

Fluorescence performance test of Fe ³⁺ molecular fluorescence test solution

(1) Spectral characteristics of Fe ³⁺ fluorescent test solution to different concentrations of Fe ³⁺

Dissolve 0.1622g of ferric chloride in 0.05mol/L Tris-HCl buffer at pH 6.0, and use a 100mL volumetric flask to make a 1.0×10 ^-2 mol/L Fe ³⁺ solution to be tested. Pipette 300μL of Fe ³⁺ fluorescent test solution and add it to the fluorescence cuvette containing 2700μl 0.05mol/L Tris-HCl (pH 6.0) buffer solution, pipette evenly with a pipette tip to get 5μmol/L Fe ³⁺ Fluorescent test solution. Under the condition that the voltage of the photomultiplier tube is 700V, and the excitation wavelength and emission wavelength are 449nm and 533nm, respectively, the fluorescence spectrophotometer is used to measure the above-mentioned series of different volumes (0μl, 30μl, 60μl, 90μl, 120μl, 150μl, 180μl, 210 μl, 240 μl, 270 μl and 300 μl) changes in the fluorescence intensity of emitted light in the Fe ³⁺ solution to be tested. Fig. 3 is a graph showing the change of the fluorescence spectrum of the Fe ³⁺ fluorescent test solution prepared by the present invention as the volume of the Fe ³⁺ solution to be tested increases, and it can be seen that the Fe ³⁺ fluorescent reagent exhibits a strong effect in the presence of Fe ³⁺ Fluorescence quenching. The fluorescence intensity of the sensor at 533 nm gradually decreased with the increase of Fe ³⁺ addition amount. Figure 4 is a graph showing the change of the fluorescence intensity of the Fe ³⁺ fluorescent test solution prepared by the present invention as the Fe ³⁺ concentration increases. From this figure, it can be seen that when the Fe ³⁺ concentration is within the range of 99 μmol/L-909 μmol/L When , the fluorescence intensity has a good linear relationship with the Fe ³⁺ concentration, and the fitted linear equation can quantitatively detect Fe ³⁺ .

(2) Spectral characteristics of Fe ³⁺ fluorescent test solution to other heavy metal ions

16 kinds of metal ion salts [NaCl, KCl, CH ₃ COOLi·2H ₂ O, Al(NO ₃ ) ₃ , CaCl ₂ , MgCl ₂ ·6H ₂ O, ZnCl ₂ , FeSO ₄ ·7H ₂ O, CoCl ₂ ·6H For ₂ O, MnCl ₂ 4H ₂ O, CrCl ₃ 6H ₂ O, BaCl ₂ 2H ₂ O, NiCl ₂ 6H ₂ O, CuCl ₂ 2H ₂ O, CdCl ₂ 2.5H ₂ O, HgCl ₂ ] 0.05 mol/L Tris-HCl buffer pH 6.0 was precisely formulated into stock solutions with respective concentrations of 10 mmol/L. At the same time, standard solutions in which each metal ion and Fe ³⁺ coexist were also prepared, so that the concentration of a single metal ion and Fe ³⁺ in each standard solution was 10 mmol/L. The fluorescence selectivity experiment is shown in Figure 6. The concentrations of Na ⁺ , K ⁺ , Li ⁺ , Al ³⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Fe ²⁺ , Co ²⁺ are all 10 mmol/L. , Mn ²⁺ , Cr ³⁺ , Ba ²⁺ , Ni ²⁺ , Cu ²⁺ , Cd ²⁺ , Hg ²⁺ and Fe ³⁺ ion standard solutions were respectively taken 150μl and added with 5μmol/L Fe ³⁺ fluorescent reagent 3ml In the fluorescence cuvette, after the fluorescence is stabilized, the change of the fluorescence intensity of the emitted light is detected by the excitation light with a wavelength of 449 nm (the voltage of the photomultiplier tube is 650V). Observing Figure 5, it can be seen that the Fe ³⁺ fluorescent test solution has an obvious response effect to Fe ³⁺ , and the fluorescence intensity decreases to the lowest value at 533 nm, which shows that the fluorescent reagent has good selectivity to Fe ³⁺ . After the addition of Cr ³⁺ , the fluorescence intensity of the sensor also decreased slightly, but other equivalent metal ions had no effect on the fluorescence emission intensity of the reagent. In order to further study the interference effect of other common metal cations on the determination of Fe ³⁺ , we carried out a competition experiment, that is, 150ul of 10mmol/LFe ³⁺ solution containing other ions (concentrations of 10mmol/L) was added to the solution containing 5μmol/L The measurement was carried out in a cuvette containing 3 ml of Fe ³⁺ fluorescent reagent (the results are shown in the white histogram in Figure 6). The competition experiment showed that except for Cr ³⁺ , the fluorescence emission intensity at 533 nm did not change significantly when other ions and Fe ³⁺ coexisted. Therefore, in addition to Cr ³⁺ , other common metal cations and anions (different metal cations are added as chloride, nitrate, sulfate or acetate) do not interfere with the determination of Fe ³⁺ . When the concentration of Cr ³⁺ was reduced to 50 μmol/L, the detection of Fe ³⁺ at 500 μmol/L no longer interfered. Therefore, it is feasible for this test fluid to be used in practical applications.

Example 2

The technique of each step in this embodiment is the same as that of Example 1, except that in the synthesis process of compound 1, the consumption of oxalyl chloride is adjusted so that the mol ratio of 4-cyanobenzoic acid and oxalyl chloride is 2:3, The volume ratio between pyridine and tert-butanol was 1.1:1. The yield of final compound 1 was 49%.

Example 3

The process of each step in this embodiment is the same as that of embodiment 1, except that: in the synthesis process of compound 1, the consumption of oxalyl chloride is adjusted so that the molar ratio of 4-cyanobenzoic acid and oxalyl chloride is 3:5. The yield of final compound 1 was 53%.

Example 4

The process of each step in this embodiment is the same as that of embodiment 1, except that: in the synthesis process of compound 1, oxalyl chloride is replaced with thionyl chloride, and its consumption is adjusted to make 4-cyanobenzoic acid and chlorinated The molar ratio of sulfoxide was 3:2. The pyridine was replaced with 4-dimethylaminopyridine, and its amount was adjusted so that the volume ratio between 4-dimethylaminopyridine and tert-butanol was 1.2:1, and the final yield of compound 1 was 45%.

Example 5

The technique of each step in this embodiment is the same as that of Embodiment 1, except that in the synthesis process of compound 2, the consumption of the catalyst is adjusted so that the weight ratio between tert-butyl 4-cyanobenzoate and the nickel catalyst is 20:3, adjust the amount of methanol so that the weight ratio between tert-butyl 4-cyanobenzoate and methanol is 1:8. The yield of compound 2 was 82%.

Example 6

The technique of each step in this embodiment is the same as that of Embodiment 1, except that in the synthesis process of compound 2, the consumption of the catalyst is adjusted so that the weight ratio between tert-butyl 4-cyanobenzoate and the nickel catalyst is 20:3, adjust the amount of methanol so that the weight ratio between tert-butyl 4-cyanobenzoate and methanol is 3:20. The yield of compound 2 was 84%.

Example 7

The technique of each step in this embodiment is the same as that of Embodiment 1, except that in the synthesis process of compound 2, the nickel catalyst is replaced with a palladium-carbon catalyst, and the consumption of the catalyst is adjusted to make tert-butyl 4-cyanobenzoate. The weight ratio between the ester and the palladium-carbon catalyst was 15:1, and the amount of methanol was adjusted so that the weight ratio between tert-butyl 4-cyanobenzoate and methanol was 1:6. The yield of compound 2 was 74%.

Example 8

The process of each step in this embodiment is the same as that of embodiment 1, except that: in the synthesis process of compound 4, the consumption of 2-aminomethylpyridine and N,N-diisopropylethylamine is adjusted so that tert-butyl 1:1 molar ratio between yl-4-chloro-1,8-naphthalimidomethylbenzoate, 2-aminomethylpyridine and N,N-diisopropylethylamine :1. The yield of compound 4 was 85%.

Example 9

The process of each step in this embodiment is the same as that of embodiment 1, except that: in the synthesis process of compound 4, the consumption of 2-aminomethylpyridine and N,N-diisopropylethylamine is adjusted so that tert-butyl The molar ratio between yl-4-chloro-1,8-naphthalimidomethylbenzoate, 2-aminomethylpyridine and N,N-diisopropylethylamine is 6:5 :15. The yield of compound 4 was 82%.

Example 10

The process of each step in this example is the same as that of Example 1, except that in the synthesis process of compound 4, N, N-diisopropylethylamine was replaced with triethylamine, and 2-aminomethyl was adjusted. The amount of pyridine and triethylamine used to make the molar ratio between tert-butyl-4-chloro-1,8-naphthalimidomethylbenzoate, 2-aminomethylpyridine and triethylamine 8:5:5. The yield of compound 4 was 73%.

Example 11

The process of each step in this example is the same as that of Example 1, except that: in the synthesis process of compound 5, the consumption of trifluoroacetic acid was adjusted so that tert-butyl-4-aminomethylpyridine-1,8-naphthalene was The moles of dicarboximidomethylbenzoate were 1.8% of the moles of trifluoroacetic acid. The yield of compound 5 was 97%.

Example 12

The process of each step in this example is the same as that of Example 1, except that: in the synthesis process of compound 5, the consumption of trifluoroacetic acid was adjusted so that tert-butyl-4-aminomethylpyridine-1,8-naphthalene was The moles of dicarboximidomethylbenzoate were 2.0% of the moles of trifluoroacetic acid. The yield of compound 5 was 98%.

Example 13

The process of each step in this embodiment is the same as that of embodiment 1, except that: in the synthesis process of compound 5, trifluoroacetic acid is replaced with a mixed reagent of hydrochloric acid and ethyl acetate with a volume ratio of 1:2, and the To be used, the mole number of tert-butyl-4-aminomethylpyridine-1,8-naphthalimidomethyl benzoate is 3.0% of the mole number of the mixed reagent. The yield of compound 5 was 75%.

It can be seen from the above examples that the Fe ³⁺ molecular fluorescence test agent provided by the present invention has the advantages of high measurement efficiency, high sensitivity and good accuracy when measuring the Fe ³⁺ concentration in the sample.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (15)

1. a Fe ³⁺ molecular fluorescence testing agent, is characterized in that, described Fe ³⁺ molecular fluorescence testing agent has the structure shown in formula I: 2. the preparation method of the Fe ³⁺ molecular fluorescence test agent of claim 1, is characterized in that, described preparation method comprises the following steps: In step S1, compound A and compound B are subjected to a condensation reaction to form compound C; the structures of compound A, compound B and compound C are as follows, wherein X in compound A is halogen: In step S2, the compound C is subjected to a hydrolysis reaction to obtain the Fe ³⁺ molecular fluorescence test agent: 3. preparation method according to claim 2 is characterized in that, before described step S1, described preparation method also comprises the step of preparing described compound A, it comprises: 4-cyanobenzoic acid and tert-butanol are carried out esterification to obtain tert-butyl 4-cyanobenzoate; The tert-butyl 4-cyanobenzoate, the catalyst and methanol are mixed to form a mixed system, and hydrogen is introduced into the mixed system for hydrogenation to obtain tert-butyl 4-aminomethylbenzoate E; wherein the The catalyst is one or more of nickel catalyst, palladium carbon catalyst and cobalt catalyst; The tert-butyl 4-aminomethylbenzoate E and the compound F are reacted to obtain the compound A; wherein the tert-butyl 4-aminomethylbenzoate E and the compound F have the following structures: wherein X in the compound F is halogen. 4. preparation method according to claim 2 or 3, is characterized in that, described step S1 comprises: Mixing the compound A, the compound B and the acid binding agent with a first solvent to obtain a first mixture; performing the condensation reaction on the first mixture at a temperature of 80-100° C. to obtain a first product system; The first product system is cooled, poured into water, filtered to obtain a precipitate, which is the compound C. 5. preparation method according to claim 4, is characterized in that, The first solvent is one or more of N-methylpyrrolidone, N,N-dimethylacetamide, dimethyl sulfoxide and tetrahydrofuran; The acid binding agent is one or more of N,N-diisopropylethylamine, N,N-dimethylformamide, 4-dimethylaminopyridine and triethylamine; The molar ratio between the compound A, the compound B and the acid binding agent is 1-6:1-5:1-15. 6. The preparation method according to claim 4, characterized in that, after the filtering step, the step S1 further comprises a step of washing the precipitate, and the washing step comprises: Dissolving the precipitate in chloroform, adding water to it for washing, and separating the liquids to obtain an organic phase and an aqueous phase; The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to give the compound C. 7. preparation method according to claim 2 or 3, is characterized in that, described step S2 comprises: Mixing and reacting the compound C, the carbonyl removal reagent and the second solvent to obtain the second product system; The second product system was diluted with a chloroform/methanol mixed solution with a volume ratio of 1:1, and then the solvent was evaporated to obtain the Fe ³⁺ molecular fluorescence test agent. 8. preparation method according to claim 7, is characterized in that, The decarbonylation reagent is trifluoroacetic acid, a mixed reagent of hydrochloric acid and ethyl acetate with a volume ratio of 1:2, or silica gel; The second solvent is dichloromethane and/or chloroform; The mole number of the compound C is 1.8-2.0% of the mole number of the carbonyl removal reagent. 9. preparation method according to claim 8, is characterized in that, The moles of compound C were 1.9% of the moles of the decarbonylation reagent. 10. preparation method according to claim 3 is characterized in that, the step of carrying out esterification reaction of described 4-cyanobenzoic acid and described tert-butanol comprises: Mixing and reacting the 4-cyanobenzoic acid, the acylating reagent and the fourth solvent to obtain an intermediate; After removing the solvent in the intermediate, it is mixed and reacted with an esterification catalyst and tert-butanol to obtain a fourth product system; The fourth product system is purified to obtain the tert-butyl 4-cyanobenzoate. 11. The preparation method according to claim 10, wherein the step S1 comprises: The acylating reagent is one or more of oxalyl chloride, thionyl chloride, phosphorus trichloride and phosphorus pentachloride; The esterification catalyst is one or more of pyridine, N,N-dimethylformamide, 4-dimethylaminopyridine and triethylamine; The molar ratio between the 4-cyanobenzoic acid and the acylating agent is 2-3:3-5; the volume ratio between the esterification catalyst and the tert-butanol is 1-1.1:1 . 12. The preparation method according to claim 3 or 10, wherein in the step of preparing the tert-butyl 4-aminomethylbenzoate E, the tert-butyl 4-cyanobenzoate and the The weight ratio between the catalysts is 10-20:1-3; the weight ratio between the tert-butyl 4-cyanobenzoate and the methanol is 1-3:8-20. 13 . The preparation method according to claim 12 , wherein after the hydrogen gas is introduced, the pressure of the reaction system is 0.8-1.2 MPa. 14 . 14. The preparation method according to claim 3 or 10, wherein the step of reacting the tert-butyl 4-aminomethylbenzoate E with the compound F comprises: The tert-butyl 4-aminomethylbenzoate E, the compound F and the fifth solvent are mixed and reacted to obtain the fifth product system; The fifth product system is filtered, and the obtained precipitate is dried to obtain the compound A. 15. preparation method according to claim 14, is characterized in that, The fifth solvent is one or more of ethanol, methanol, propanol and isopropanol; The molar ratio between the tert-butyl 4-aminomethylbenzoate E and the compound F is 1:1.