Squarylium cyanine fluorescent probe based on methimazole and its preparation method and application

Abstract

The present invention belongs to the field of chemical analysis and testing, and in particular to a kind of methimazole-based squaryl cyanide fluorescent probe and its preparation method and application.Methimazole and 2 bromoethylamine hydrobromide are dissolved in a toluene solution, and the reaction solution is heated at 60 DEG C for 8 hours under tetrabutylammonium bromide as a phase inversion agent and 40% sodium hydroxide solution as an alkali condition, and the obtained crude product is recrystallized by methanol to obtain intermediate 1.Then intermediate 1 and asymmetric squaryl cyanide dye SQ01 are reacted with 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate and N, N-diisopropylethylamine at room temperature, and the obtained crude product is separated by thin layer chromatography to obtain final product SQM-1.The squaryl cyanide fluorescent probe SQM-1 obtained by the present invention can quickly identify deoxythymidine triphosphate (dTTP) in a hexadecyltrimethylammonium bromide surfactant aqueous solution.

Description

Technical Field

[0001] The invention belongs to the field of chemical analysis and testing, and particularly relates to a methimazole-based squaraine fluorescent probe and a preparation method and application thereof. Background Art

[0002] Deoxythymidine triphosphate (dTTP) is one of the four deoxynucleotides that carries the thymine base and can pair with adenine to form the double helix structure of DNA. Depleting dTTP by inhibiting thymidine synthase leads to the accumulation of deoxyuridine triphosphate (dUTP) and an increase in the ratio of dUTP to dTTP. DNA polymerase cannot distinguish between dUTP and dTTP, which will lead to large-scale misincorporation of uracil and extensive DNA damage response, resulting in "thymic death". Therefore, deoxythymidine triphosphate plays a very important role in biology. It is the basis for maintaining the transmission of genetic information and the maintenance of cell structure and function.

[0003] Currently, nucleotide detection methods primarily rely on high-performance liquid chromatography, gel electrophoresis, polymerase chain reaction, and nuclear magnetic resonance. These methods typically require sample pretreatment and have lengthy detection cycles. Fluorescence analysis is a sensitive and rapid method. Therefore, developing a simple and rapid assay for intracellular thymidine triphosphate levels is of vital importance to human health. Summary of the Invention

[0004] The present invention provides a methimazole-based squaraine fluorescent probe SQM-1, the structural formula of which is:

[0005]

[0006] The present invention also provides a method for preparing a fluorescent probe based on a methimazole structure. The chemical reaction formula for preparing the fluorescent probe is:

[0007]

[0008] The specific preparation method is as follows: methimazole and 2-bromoethylamine hydrobromide are dissolved in a toluene solution at a molar equivalent ratio of 1:1.2-1.5, and then 0.1 molar equivalent of tetrabutylammonium bromide (TBAB) and 1.5 molar equivalent of 40% sodium hydroxide solution are added. The mixture is heated at 60°C for 8 hours. After the reaction is completed, the solvent is removed, and the crude product is extracted with deionized water and ethyl acetate. The aqueous phase is freeze-dried to obtain a solid, which is washed with methanol and the methanol is removed under reduced pressure to obtain a white solid intermediate 1.

[0009] The freeze-drying conditions of the aqueous phase are as follows: the aqueous phase is frozen at -78°C, and the intermediate 1 is obtained by vacuum freeze-drying.

[0010] Subsequently, intermediate 1 and asymmetric cyanine dye SQ01 were dissolved in a dichloromethane (DCM) solution at a 1:1 molar equivalent, and then 1 molar equivalent of 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP) and 2.5 molar equivalents of N,N-diisopropylethylamine (DIPEA) were added. The reaction was carried out at room temperature for 3-6 hours. The resulting crude product was separated by thin-layer chromatography using a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1 to obtain the final pure blue solid.

[0011] The present invention also provides an application of the fluorescent probe: the prepared methimazole-based squaraine fluorescent probe SQM-1 can quickly identify dTTP in a CTAB surfactant aqueous solution.

[0012] The concentration of the CTAB surfactant aqueous solution was 0.4-1.0 mM.

[0013] The specific application method is: add 2 μL of 1×10 -2 mol / L nucleotide and analogue solutions (the nucleotides in each well are guanosine triphosphate (GTP), adenosine triphosphate

[0014] ATP, cyclic adenosine monophosphate (cAMP), guanosine monophosphate (GMP), guanine (G), O-6-benzylguanine (O6-BG), deoxyguanosine (dG), adenine (A), cytosine (C), thymidine acetate (TAA), guanosine, β-nicotinamide mononucleotide (NMN), sodium monododecyl phosphate (SMP), cytidine triphosphate (CTP), deoxythymidine triphosphate (dTTP), oxidized coenzyme I (NAD), sodium alendronate (AST), blank), 196 μL of 0.5 mM hexadecyltrimethylammonium bromide (CTAB surfactant) solution and 2 μL of squaraine fluorescent probe (concentration of 0.5 × 10 -3 mol / L), and a solution containing a squaraine fluorescent probe without nucleotides (Blank) was used as a comparison. The solutions in each well were mixed evenly, and the fluorescence intensity of the solution in each well was measured using a microplate reader.

[0015] The results showed that the fluorescence intensity of the mixed solution containing deoxythymidine triphosphate at 655nm was 50 times that of the solution without nucleotides; and the fluorescent probe did not show any particularly obvious fluorescence intensity changes for other nucleotides and analogs, thus demonstrating the selective recognition effect of the fluorescent probe on dTTP in cetyltrimethylammonium bromide (CTAB surfactant) solution.

[0016] The squaraine dye used in this fluorescent probe is a near-infrared dye with excellent optical properties and photostability. It produces strong fluorescence when excited by a specific wavelength. When methimazole and the squaraine dye SQ01 are linked via an amide bond, the resulting squaraine fluorescent probe significantly enhances fluorescence intensity upon recognition of dTTP, while other nucleotide analogs exhibit no similar effect, thus achieving the desired detection of dTTP.

[0017] In the structure of the probe prepared by the present invention, methimazole and squaraine dye form a complex with dTTP through multiple hydrogen bond interactions and / or electrostatic attraction, resulting in fluorescence enhancement, thereby achieving the function of identifying dTTP.

[0018] Beneficial effects

[0019] The raw materials of the present invention are readily available, the synthesis method is relatively simple, the reaction conditions are easily controlled, and a pure product can be obtained through simple post-processing. Fluorescence phenomena show that the fluorescence intensity of the well plate probe SQM-1 with dTTP added at 655 nm is significantly enhanced after the addition of different nucleotides, demonstrating the recognition effect of the probe SQM-1 on dTTP in this system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The probe prepared in Example 1 was 0.5×10 -3 Fluorescence spectra of 5-nitro-1,1-diol-1 in 0.5 mM CTAB aqueous solution after interaction with different nucleotides and analogs.

[0021] Figure 2 The probe prepared in Example 1 was 0.5×10 -3 mol / L in 0.5 mM CTAB aqueous solution after reacting with different concentrations of dTTP.

[0022] Figure 3 The fluorescent probe prepared in Example 1 was 0.5×10 -3 mol / L concentration of dTTP in 0.5mM CTAB surfactant aqueous solution after the addition of five times the interfering nucleotides and analogs at 655nm.

[0023] Figure 4 This is the hydrogen spectrum of the probe prepared in Example 1.

[0024] Figure 5 This is a graph showing the recognition ability of the probe prepared in Example 1 for dTTP in aqueous CTAB surfactant solutions with different concentrations. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments:

[0026] Example 1

[0027] (1) Methimazole (100 mg, 0.88 mmol) and 2-bromoethylamine hydrobromide (216 mg, 1.05 mmol) were dissolved in a round-bottom flask with 4 mL of toluene as solvent. Tetrabutylammonium bromide (28 mg, 0.088 mmol) and 132 μL of 40% sodium hydroxide solution were then added and heated at 60°C for 8 hours. After the reaction, the solvent was removed and the crude product was extracted with deionized water and ethyl acetate. The aqueous phase was lyophilized to obtain a solid which was washed with methanol. The methanol was removed under reduced pressure to obtain 248 mg of intermediate 1 as a white solid.

[0028] (2) The intermediate 1 (10 mg, 0.064 mmol) obtained in step (1) and the asymmetric cyanine dye SQ01 (30 mg, 0.053 mmol) were dissolved in 3 mL of dichloromethane solution, followed by the addition of 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate (30 mg, 0.058 mmol) and N,N-diisopropylethylamine (220 μL, 0.135 mmol). The reaction was carried out at room temperature for 3 hours. The solvent was removed under reduced pressure to obtain a blue solid. After separation and purification by thin layer chromatography (developing solvent volume ratio: dichloromethane: methanol = 10:1), 20 mg of the final product was obtained with a yield of 54%.

[0029] Figure 1 The methimazole-based squaraine fluorescent probe prepared in Example 1 was reacted with different nucleotides and analogs (guanosine triphosphate (GTP), adenosine triphosphate (ATP)) in a 0.5 mM CTAB surfactant aqueous solution.

[0030] Fluorescence spectra of the probe SQM-1 after the addition of various 10 mM nucleotide and analog solutions. The figure shows the fluorescence intensity changes of the probe SQM-1 after the addition of dTTP, while the fluorescence intensity of the probe remains unchanged for other nucleotides. This demonstrates the probe's selective recognition of dTTP in this system.

[0031] Figure 2The fluorescent probe prepared in Example 1 was 0.5×10 -3 Fluorescence spectra of the probe after interaction with various concentrations of dTTP in aqueous CTAB solution at 10 μM concentration. The figure shows that the fluorescence intensity of the probe solution gradually increases with increasing dTTP concentration. At the lowest concentration of 10 μM, the fluorescence peak at 655 nm is still distinguishable from the background fluorescence curve without nucleotide addition, demonstrating that the probe has a low detection limit and high sensitivity for dTTP.

[0032] Figure 3 The fluorescent probe prepared in Example 1 was 0.5×10 -3 The fluorescence intensity diagram of the probe solution at 655nm after the addition of five times the interfering nucleotides and analogues at a concentration of mol / L in CTAB aqueous solution and dTTP. The figure shows that when only dTTP solution is added, the fluorescence intensity of the probe solution at 655nm is significantly increased relative to the blank, while the probe has no significant effect on other nucleotides (guanosine triphosphate (GTP), adenosine triphosphate (ATP), cyclic adenosine monophosphate (cAMP), guanosine monophosphate (GMP), guanine (G), O-6 benzylguanine (O6-BG), deoxyguanosine (dG), adenine (A), cytosine (C), thymine acetate (TAA), guanosine nucleoside (Guanosine), β-nicotinamide mononucleotide (NMN), There was no particularly obvious fluorescence change in sodium monododecyl phosphate (SMP), cytidine triphosphate (CTP), deoxythymidine triphosphate (dTTP), oxidized coenzyme I (NAD), sodium alendronate (AST), and blank; subsequently, after adding 5 times the amount of other nucleotide and analog solutions to the detection system, the fluorescence intensity change at 655nm of the probe showed that the addition of other proteins and redox substances did not lead to a decrease in the detection results of the probe for dTTP, indicating that the fluorescent probe has strong anti-interference ability during the detection process.

[0033] Figure 4This is the hydrogen spectrum of the methimazole-based squaraine fluorescent probe prepared in Example 1. 1H NMR (400 MHz, Methanol) δ7.88 (s, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.30 (m, J = 22.0 Hz, 2H), 7.25 (d, J = 4.0 Hz, 1H), 7.22 (s, 1H), 7.19 (d, J = 1.4 Hz, 1H), 7.04 (d, J = 1.4 Hz, 1H), 6.05(s,1H),5.95(s,1H),4.23(q,J=6.8Hz,2H),4.08(s,2H),3.71(s,3H),3.58(m,J=44.4Hz, 4H), 1.75 (s, 12H), 1.41 (td, J = 7.2, 2.5Hz, 6H), 1.29 (m, J = 25.4Hz, 9H), 0.87 (t, J = 6.9Hz, 3H).

[0034] Example 2

[0035] (1) Methimazole (200 mg, 1.76 mmol) and 2-bromoethylamine hydrobromide (493 mg, 2.64 mmol) were dissolved in a round-bottom flask with 8 mL of toluene as solvent. Tetrabutylammonium bromide (55 mg, 0.176 mmol) and 250 μL of 40% sodium hydroxide solution were then added and heated at 60°C for 8 hours. After the reaction, the solvent was removed and the crude product was extracted with deionized water and ethyl acetate. The aqueous phase was lyophilized to obtain a solid which was washed with methanol. The methanol was removed under reduced pressure to obtain 506 mg of intermediate 1 as a white solid.

[0036] (2) The intermediate 1 (20 mg, 0.064 mmol) obtained in step (1) and the asymmetric cyanine dye SQ01 (60 mg, 0.106 mmol) were dissolved in 5 mL of dichloromethane solution, followed by the addition of 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate (30 mg, 0.058 mmol) and N,N-diisopropylethylamine (220 μL, 0.135 mmol). The reaction was carried out at 0°C for 6 hours. The solvent was removed under reduced pressure to obtain a blue solid. The final product (44 mg) was separated and purified by thin layer chromatography (developing solvent volume ratio: dichloromethane: methanol = 10:1) with a yield of 59%.

[0037] Example 3

[0038] 2 μL of 1×10 -2mol / L deoxythymidine triphosphate solution, 13 groups of parallel, 13 groups of different concentrations of hexadecyltrimethylammonium bromide solution (0mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 2.0mM, 3.0mM) and 2μL of squaraine fluorescent probe (concentration of 0.5×10 -3 mol / L), the solution in each well was mixed evenly, and the fluorescence intensity of the solution in each well was detected by a microplate reader. The results showed that the fluorescent probe can effectively identify dTTP in a 0.4-1.0mM CTAB aqueous solution system.

[0039] The specific recognition ability of the probe to dTTP was studied in aqueous solutions of CTAB surfactant with different concentrations. Figure 5 The horizontal axis represents the concentration of the CTAB surfactant solution, and the vertical axis represents the difference in fluorescence intensity of the probe before and after the addition of dTTP, represented by I-I0. The figure shows that the probe has a clear recognition ability for dTTP when the CTAB concentration is between 0.4mM and 1.0mM.

Claims

1. A methimazole-based squaraine fluorescent probe, characterized in that: The structural formula of the probe is: 。 2. A method for preparing a methimazole-based squaraine fluorescent probe according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Using toluene as solvent, methimazole and 2-bromoethylamine hydrobromide were dissolved in a round-bottom flask, and then tetrabutylammonium bromide and 40% sodium hydroxide solution were added in sequence. The mixture was heated at 60°C for 8 hours. After the reaction, the solvent was removed, and the crude product was extracted with deionized water and ethyl acetate. The solid obtained after freeze-drying the aqueous phase was washed with methanol, and the methanol was removed under reduced pressure to obtain a white solid intermediate 1. ; (2) The intermediate 1 obtained in step (1) and the asymmetric cyanine dye SQ01 are reacted The product was dissolved in dichloromethane solution, and then 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) were added. The product was stirred at room temperature for 3-6 hours, and the solvent was removed to obtain a blue crude product. The product was extracted with ethyl acetate and water, and the organic solvent was removed under reduced pressure. Finally, the product was purified by thin-layer chromatography using dichloromethane and methanol as developing solvents to obtain the methimazole-based cyanine fluorescent probe SQM-1. .

3. The method for preparing a methimazole-based squaraine fluorescent probe according to claim 2, wherein: In step (1), the molar equivalent ratio of methimazole, 2-bromoethylamine hydrobromide, tetrabutylammonium bromide and sodium hydroxide is: 1:1.2-1.5:0.1:1.

5.

4. The method for preparing a methimazole-based squaraine fluorescent probe according to claim 2, wherein: The freeze-drying conditions of the aqueous phase in step (1) are as follows: the aqueous phase is frozen at -78°C, and the intermediate 1 is obtained by vacuum freeze-drying.

5. The method for preparing a methimazole-based squaraine fluorescent probe according to claim 2, wherein: In step (2), the molar equivalent ratio of intermediate 1, asymmetric cyanine dye SQ01, 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate and N,N-diisopropylethylamine is 1:1:1:2.

5.

6. The method for preparing a methimazole-based squaraine fluorescent probe according to claim 2, wherein: In step (2), the developing solvent is a mixed solvent of dichloromethane and methanol in a volume ratio of 10:

1.

7. A use of the methimazole-based squaraine fluorescent probe according to claim 1, characterized in that: The methimazole-based squaraine fluorescent probe is used to detect deoxythymidine triphosphate in a hexadecyltrimethylammonium bromide surfactant aqueous solution.

8. The use of the methimazole-based squaraine fluorescent probe according to claim 7, characterized in that: The concentration of the cetyltrimethylammonium bromide surfactant aqueous solution is: 0.4-1.0mM.

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