A regenerative formaldehyde fluorescent probe and a preparation method and use method thereof

Abstract

The application discloses a regenerative formaldehyde fluorescent probe and a preparation method and use method thereof, and the probe is (E)-2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-3-methylpyrrolidine-3-carboxylate (IPF2). The probe is based on an analyte regeneration mechanism: after formaldehyde reacts with a pyrrolidine group, intramolecular lactone hydrolysis is triggered, a fluorophore is released, and formaldehyde is regenerated, so that the "on" type sensitive detection of formaldehyde is realized. The probe has simple synthesis steps, a large stokes shift, high selectivity and a low detection limit, can be used for quantitative analysis of formaldehyde in aqueous solution and biological samples, and has application prospects in the fields of environmental monitoring and biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to a method for preparing and using (E)-2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-3-methylpyrrolidine-3-carboxylic acid ester as a formaldehyde fluorescent probe. Background Technology

[0002] Formaldehyde (FA) is the simplest endogenous carbonyl molecule in living organisms, a colorless gas with a pungent odor. It participates in various biosynthetic processes and metabolisms, including nucleotides and adenosine triphosphate (ATP). While the concentration of FA in a normal healthy human body is sub-millimolecular, it can significantly increase in many pathological conditions. FA is also widely used in various fields such as plastics, cosmetics, food and pharmaceuticals, wood processing, and medicine. However, FA has attracted considerable attention as an exogenous carcinogen. Studies have shown that FA is not only present in foods such as spinach, meat, fruits, and seafood, but is also commonly used as a preservative in hospitals in the form of formalin solution. With the acceleration of urbanization, FA generated during home renovations has become one of the major indoor pollutants, posing a significant risk to human health. Long-term exposure to high levels of FA is associated with various diseases, including respiratory diseases, diabetes, Alzheimer's disease, and cancer. Therefore, the detection of FA is crucial for environmental monitoring, human health, and the monitoring of disease diagnosis and rehabilitation.

[0003] Fluorescent probes offer advantages such as ease of operation, rapid response, high sensitivity, low signal-to-noise ratio, and good biocompatibility, accurately reflecting the spatiotemporal distribution of fatty acids (FAs) in organisms. Therefore, fluorescent probes for FA detection have been developed year by year and are used as a practical tool in physiological and pathological studies within organisms.

[0004] Several fluorescent probes with different response modes have been used to identify formaldehyde (FA). These can be broadly categorized as follows: 1) Based on a homoallylamine responsive group, involving a 2-aza-Cope rearrangement mechanism: FA reacts with homoallylamine to form an imine, undergoes a [3,3]-σ migration rearrangement, and subsequently hydrolyzes to form an aldehyde. 2) Based on a hydrazine responsive group: FA reacts with a hydrazine group to form methylene hydrazine, thereby altering the photoinduced electron transfer (PET) process and causing a change in fluorescence. 3) Based on an amino responsive group: The amino group on the fluorophore condenses with the carbonyl group of FA to form a simple Schiff base, causing a change in the electronic structure of the probe molecule and achieving a change in fluorescence. 4) Based on an o-methylaminobenzoate responsive group: The secondary amine in this fluorophore first nucleophilically attacks formaldehyde, the double bond of formaldehyde breaks to form a hydroxyl group, and then the hydroxyl group undergoes intramolecular nucleophilic attack on the ester group, resulting in ester group cleavage and the release of the fluorophore.

[0005] However, most of the fluorescent probes reported so far are formaldehyde-consuming, which usually require a large amount of formaldehyde to cause a change in the fluorescence of the probe during the test, leaving a gap in the determination of low concentrations of formaldehyde (see review article: Pengfei Wang; Minggui Gong; Rui Zhao; Zhaozhou Li; Huaibin Kang; Ying Hou; Wenfen Zhang; Huawei Niu; Shusheng Zhang. Food Chemistry, 2025, 481, 144041). https: / / doi.org / 10.1016 / j.foodchem.2025.144041 Fluorescent probes for analyte regeneration mechanisms have unique advantages, as they can regenerate formaldehyde while simultaneously enhancing the fluorescence signal, thereby reducing interference with formaldehyde concentration during the detection process and providing a new tool for studying the physiological, pathological processes and signaling pathways of formaldehyde. Summary of the Invention

[0006] The present invention aims to provide an "on" type formaldehyde fluorescent probe based on the analyte regeneration mechanism, which has the characteristics of high sensitivity, large Stokes shift and formaldehyde regeneration.

[0007] In a first aspect, the present invention provides a fluorescent probe for detecting formaldehyde, which is (E)-2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-3-methylpyrrolidine-3-carboxylic acid ester (IPF2 for short), with the structure shown in formula (I):

[0008]

[0009] The working principle of the probe of this invention is as follows: the probe molecule uses a 3-esterified pyrrolidine derivative as the formaldehyde recognition group. When formaldehyde is present, the nitrogen atom on the pyrrolidine nucleophilically attacks the formaldehyde, forming an acetal intermediate. Subsequently, an intramolecular esterification reaction occurs, releasing a strongly fluorescent dicyanoisophorone fluorophore. Simultaneously, the acetal intermediate releases one molecule of formaldehyde, thus regenerating the formaldehyde. Therefore, the probe of this invention does not consume formaldehyde during detection and is a "regenerative" fluorescent probe, suitable for the sensitive detection of low concentrations of formaldehyde.

[0010] In a second aspect, the present invention provides a method for preparing the fluorescent probe, the synthetic route of which is as follows:

[0011]

[0012] The specific steps are as described in claim 3.

[0013] A third aspect of the present invention provides a method for using the fluorescent probe to detect formaldehyde content in samples for non-diagnostic purposes, wherein quantitative detection is achieved by establishing a standard curve of fluorescence intensity versus formaldehyde concentration.

[0014] Beneficial effects

[0015] 1) The fluorescent probe provided by this invention is a yellow-green solid powder with good optical stability.

[0016] 2) The fluorescent probe provided by this invention has a solution that is sensitive to the concentration of formaldehyde. As the concentration of formaldehyde increases, the color of its aqueous solution changes from colorless to light red under sunlight, and the fluorescence of its aqueous solution changes from colorless to red under a 365 nm ultraviolet lamp.

[0017] 3) The fluorescent probe provided by this invention has no fluorescence when the probe solution is alone, but emits fluorescence at a wavelength of 650 nm after reacting with formaldehyde, exhibiting an on-type fluorescence response. This probe has the ability to detect analyte regeneration, enabling specific detection of low doses of analyte.

[0018] 4) The fluorescent probe provided by this invention has a linear relationship with formaldehyde concentration and can be used for accurate measurement of formaldehyde concentration.

[0019] The formaldehyde fluorescent probe based on DCI dye, which is an analyte regeneration method, provided by this invention has a specific response to formaldehyde solution and can achieve sensitive quantitative detection of formaldehyde in samples. It has the advantages of simple operation, low cost, sensitive response, and easy promotion and application. Attached Figure Description

[0020] Figure 1 : 1H NMR spectrum of fluorescent probe IPF2 (400 MHz, DMSO-d6).

[0021] Figure 2 Color comparison of the fluorescent probe IPF2 (10 μM) under sunlight before and after the addition of 200 μM formaldehyde in phosphate buffer (pH 7.4, 10 mM, 10% DMSO).

[0022] Figure 3 Fluorescence comparison of the fluorescent probe IPF2 (10 μM) under UV lamp (365 nm) before and after the addition of 200 μM formaldehyde under the same conditions.

[0023] Figure 4 UV-Vis absorption spectra of fluorescent probe IPF2 (10 μM) reacted with formaldehyde of different concentrations (0–300 μM) in phosphate buffer (pH 7.4, 10 mM, 10% DMSO).

[0024] Figure 5Fluorescence emission spectra (λ) of fluorescent probe IPF2 (10 μM) reacting with different concentrations of formaldehyde (0–300 μM) under the same conditions. ex = 525 nm), and the linear relationship between fluorescence intensity and formaldehyde concentration at 650 nm.

[0025] Figure 6 : A bar chart of the fluorescence response of the fluorescent probe IPF2 (10 μM) to common aldehydes and metal ions (200 μM each) under the same conditions (λ) ex = 525 nm, λ em = 650 nm). Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] The compound numbers in the examples correspond to the numbers in the compounds described above.

[0029] Example 1: Synthesis of fluorescent probe IPF2

[0030] (1) Synthesis of intermediate (E)-1-(tert-butyl)3-(2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)-3-methylpyrrolidine-1,3-dicarboxylic acid ester (5)

[0031] Under nitrogen protection, 100.0 mg (307.8 μmol) (E)-2-(3-(3-chloro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile (1) and 84.7 mg (369.5 μmol) 1-(tert-butoxycarbonyl)-3-methylpyrrolidine-3-carboxylic acid (2) were dissolved in 10 mL of anhydrous dichloromethane. 7.5 mg (61.6 μmol) 4-dimethylaminopyridine (3) and 70.8 mg (369.5 μmol) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4) were added in an ice bath at 0 °C. The reaction was stirred at room temperature for 2 hours. After the reaction was monitored by thin-layer chromatography to ensure complete reaction, the solvent was removed under reduced pressure, and the product was purified by column chromatography to give 147.0 mg of a pale yellow solid intermediate (5), with a yield of 94%.

[0032] like Figure 1 As shown, 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.95 (s, 2H), 6.87 (s, 1H), 4.02 (dd, J =22.0, 11.2 Hz, 1H), 3.54 (dq, J = 20.8, 12.7, 10.2 Hz, 2H), 3.34 (dd, J =30.5, 11.2 Hz, 1H), 2.61 (s, 2H), 2.58–2.51 (m, 1H), 2.45 (s, 2H), 1.93 (dt,J = 13.5, 7.3 Hz, 1H), 1.55 (s, 3H), 1.47 (s, 9H), 1.09 (s, 6H).

[0033] (2) Synthesis of fluorescent probe IPF2 (7)

[0034] 100.0 mg (186.5 μmol) of intermediate (5) was dissolved in 5 mL of anhydrous dichloromethane, and 47.2 μL (205.2 μmol) of tert-butyldimethylsilyltrifluoromethanesulfonate (6) was added. The reaction was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was filtered, the precipitate was collected, dried, and 70.7 mg of yellow-green solid product IPF2 (7) was obtained, with a yield of 87%. 1 H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 16.2 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 16.2 Hz, 1H), 6.93 (s, 1H), 3.78 (d, J= 12.2 Hz, 1H), 3.43 (dt, J = 11.8, 7.4 Hz, 1H), 3.34–3.28 (m, 1H), 3.22 (d,J = 12.2 Hz, 1H), 2.63 (s, 2H), 2.61–2.56 (m, 1H), 2.53 (s, 2H), 2.07 (dt, J= 13.5, 7.7 Hz, 1H), 1.56 (s, 3H), 1.02 (s, 6H).

[0035] Example 2: Color and fluorescence response of compound IPF2 to formaldehyde

[0036] Prepare a 1 mM IPF2 dimethyl sulfoxide (DMSO) stock solution. Add 50 μL of this stock solution to 5 mL of phosphate buffer solution (pH 7.4, 10 mM, containing 10% DMSO) to bring the final probe concentration to 10 μM. Then add formaldehyde to bring the final concentration to 200 μM. Figure 2 As shown, the solution changes color from colorless to pale red under sunlight; as Figure 3 As shown, the solution changed from no fluorescence to red fluorescence under a 365 nm UV lamp. This indicates that the probe has a direct "on" response to formaldehyde.

[0037] Example 3: UV titration detection of IPF2 by different concentrations of formaldehyde

[0038] A series of samples were prepared with a probe concentration of 10 μM phosphate buffer solution (pH 7.4, 10 mM, 10% DMSO). Formaldehyde was added to each sample to achieve final concentrations of 0, 30, 60, 90, 120, 150, 180, 210, 240, and 300 μM. The UV-Vis absorption spectra of each system were measured. Figure 4 As shown, with increasing formaldehyde concentration, the absorbance at 385 nm gradually decreases, while the absorbance at 482 nm gradually increases, and A... 482 / A 385 The ratio showed a good linear relationship with formaldehyde concentration (0–90 μM).

[0039] Example 4: Fluorescent titration detection of IPF2 by different concentrations of formaldehyde

[0040] Using the same series of solutions as in Example 3, fluorescence emission spectra were measured at an excitation wavelength of 525 nm. Figure 5 As shown, the fluorescence intensity at 650 nm gradually increases with increasing formaldehyde concentration, reaching a plateau at 200 μM formaldehyde. Fluorescence intensity I 650 It showed a good linear relationship with formaldehyde concentration (0–90 μM) (R 2 > 0.99).

[0041] Example 5: Selectivity of compound IPF2 against common interfering substances

[0042] Prepare a phosphate buffer solution (pH 7.4, 10 mM, 10% DMSO) sample with a probe concentration of 10 μM, and add the following interfering substances (final concentration 200 μM): acetaldehyde, glyoxal, methylglyoxal, salicylaldehyde, glutathione, hydrogen sulfide, sodium thiosulfate, and Cu. 2+ Fe 3+ Zn 2+ Mg 2+ K +Cl - ,Br - I - Hydrogen peroxide. The fluorescence intensity of each sample at 650 nm was measured at an excitation wavelength of 525 nm. For example... Figure 6 As shown, only formaldehyde caused a significant fluorescence enhancement, while other interfering substances showed almost no response, indicating that the probe has excellent selectivity for formaldehyde.

[0043] Example 6:

[0044] Intermediate synthesis: Under nitrogen protection, (E)-2-(3-(3-chloro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile (100.0 mg, 307.8 μmol, 1.0 equivalent), 1-(tert-butoxycarbonyl)-3-methylpyrrolidine-3-carboxylic acid (70.6 mg, 307.8 μmol, 1.0 equivalent), and anhydrous dichloromethane (8 mL) were added to a 50 mL round-bottom flask. After cooling to 0°C in an ice bath, 4-dimethylaminopyridine (3.8 mg, 30.8 μmol, 0.1 equivalent) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (58.9 mg, 307.8 μmol, 1.0 equivalent) were added sequentially. The ice bath was removed, and the mixture was allowed to return to room temperature (25°C) and stirred for 4 hours. TLC monitoring showed that the reaction was essentially complete. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) to give 132.5 mg of a pale yellow solid intermediate (5), in 85% yield. ¹H NMR characterization confirmed that the structure was consistent with the intermediate in Example 1.

[0045] Synthesis of probe IPF2: The above intermediate (5) (100.0 mg, 186.5 μmol) was dissolved in anhydrous dichloromethane (5 mL), and tert-butyldimethylsilyltrifluoromethanesulfonate (42.9 μL, 186.5 μmol, 1.0 equivalent) was added. The reaction was stirred at room temperature (25 °C) for 18 hours. After the reaction was completed, the mixture was filtered, the precipitate was washed with cold dichloromethane, and dried under vacuum to obtain a yellow-green solid powder product IPF2-L 64.1 mg, with a yield of 80%. The ¹H NMR (400 MHz, DMSO-d6) spectrum was consistent with that of the product IPF2 in Example 1, confirming the correct structure.

[0046] Example 7:

[0047] Intermediate synthesis: Under nitrogen protection, (E)-2-(3-(3-chloro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonadionitrile (100.0 mg, 307.8 μmol, 1.0 equivalent), 1-(tert-butoxycarbonyl)-3-methylpyrrolidine-3-carboxylic acid (105.9 mg, 461.7 μmol, 1.5 equivalent), and anhydrous dichloromethane (12 mL) were added to a 100 mL round-bottom flask. After cooling to 0 °C in an ice bath, 4-dimethylaminopyridine (11.3 mg, 92.3 μmol, 0.3 equivalent) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (88.4 mg, 461.7 μmol, 1.5 equivalent) were added sequentially. The ice bath was removed, and the mixture was stirred at room temperature (25°C) for 1.5 hours. TLC monitoring showed that the starting material had been completely converted. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1, v / v) to give 155.8 mg of a pale yellow solid intermediate (5), yield ~ quantitative (>99%). The structure was confirmed by ¹H NMR characterization.

[0048] Synthesis of probe IPF2: The above intermediate (5) (100.0 mg, 186.5 μmol) was dissolved in anhydrous dichloromethane (5 mL), and tert-butyldimethylsilyltrifluoromethanesulfonate (64.3 μL, 279.8 μmol, 1.5 equivalents) was added. The reaction was stirred at room temperature (25 °C) for 6 hours. After the reaction was completed, the mixture was filtered, the precipitate was washed with cold dichloromethane, and dried under vacuum to obtain 73.4 mg of the yellow-green solid powder product IPF2-H, with a yield of 91%. The ¹H NMR spectrum was consistent with that of the product IPF2 in Example 1.

Claims

1. A regenerable formaldehyde fluorescent probe, characterized in that, The fluorescent probe is (E)-2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-3-methylpyrrolidine-3-carboxylic acid ester, with the molecular formula C2 25 H 26 ClN3O2, abbreviated as IPF2, has the structure shown in formula (I): 。 2. The regenerable formaldehyde fluorescent probe according to claim 1, characterized in that, The fluorescent probe is in the form of a solid powder.

3. A method for preparing a regenerative formaldehyde fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: (1) Under nitrogen protection, (E)-2-(3-(3-chloro-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile and 1-(tert-butoxycarbonyl)-3-methylpyrrolidine-3-carboxylic acid were dissolved in anhydrous dichloromethane. 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added in an ice bath. The reaction was allowed to proceed to room temperature for 1-5 hours to obtain (E)-1-(tert-butyl)3-(2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl)-3-methylpyrrolidine-1,3-dicarboxylic acid ester; (2) Dissolve the intermediate obtained in step (1) in anhydrous dichloromethane, add tert-butyldimethylsilyltrifluoromethanesulfonate, and react at room temperature for 4 to 20 hours to obtain (E)-2-chloro-4-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-3-methylpyrrolidine-3-carboxylic acid ester, which is the fluorescent probe.

4. The method for preparing a regenerable formaldehyde fluorescent probe according to claim 3, characterized in that, In step (1), the molar ratio of (E)-2-(3-(3-chloro-4-hydroxystyryl)-5,5-dimethylcyclohexyl-2-en-1-yl)malonitrile, 1-(tert-butoxycarbonyl)-3-methylpyrrolidine-3-carboxylic acid, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1 : (1.0~1.5) : (0.1~0.3) : (1.0~1.5); the reaction temperature is 0~30℃ and the reaction time is 1~5 hours.

5. The preparation method according to claim 3, characterized in that, In step (2), the molar ratio of the intermediate obtained in step (1) to tert-butyldimethylsilyltrifluoromethanesulfonate is 1:1 to 1.5; the reaction temperature is 10 to 35°C; and the reaction time is 4 to 20 hours.

6. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio is 1:1.2:0.2:1.2; the amount of anhydrous dichloromethane is 10 ml; the reaction temperature is 25℃; and the reaction time is 2 hours.

7. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio is 1:1.1; the amount of anhydrous dichloromethane is 5 ml; the reaction temperature is 25°C; and the reaction time is 12 hours.

8. A method of using the regenerable formaldehyde fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: (a) Prepare at least 5 standard solutions with different formaldehyde concentrations by adding the same concentration of the compound shown in formula (I) to phosphate buffer solutions containing different concentrations of formaldehyde; wherein the concentration of the compound shown in formula (I) in the standard solutions is 100 nM to 10 μM; and the concentration of formaldehyde in the standard solutions is 0.1 nM to 1 mM. (b) The fluorescence emission spectra of the standard solutions were measured respectively, with an excitation wavelength of 525 nm. The formaldehyde concentration was plotted on the x-axis, and the fluorescence emission peak intensity value at 650 nm was plotted on the y-axis. 650 Establish a standard curve with the vertical axis as the ordinate; (c) Add the compound of formula (I) to the sample to be tested, so that its concentration is equal to that of the compound of formula (I) in the standard solution, measure the fluorescence emission spectrum of the sample to be tested at an excitation wavelength of 525 nm, and calculate the formaldehyde content in the sample to be tested according to the standard curve.

9. The method according to claim 8, characterized in that, The phosphate buffer solution has a pH of 7.4, a concentration of 10 mM, and contains 10% dimethyl sulfoxide by volume.

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