Pyrophosphate fluorescent probe, preparation method and application thereof
By designing the fluorescent pyrophosphate probe FL-TPY-Zn2+, the problem of distinguishing between pyrophosphate and nucleoside triphosphate in existing technologies has been solved, realizing a rapid and selective method for detecting pyrophosphate and screening pyrophosphate hydrolases, thus improving detection efficiency and selectivity.
Patent Information
- Application Number
- CN202411147510.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-21
- Publication Date
- 2026-02-13
- Estimated Expiration
- 2044-08-21
AI Technical Summary
Existing fluorescent probes have poor selectivity in distinguishing between pyrophosphate (PPi) and nucleoside triphosphates (NTPs), making it impossible to effectively detect the activity of pyrophosphate hydrolases and the optimal amount added for the reaction. Furthermore, traditional detection methods are inefficient and complex to operate.
A fluorescent pyrophosphate probe (FL-TPY-Zn2+) was developed to generate an enhanced fluorescence response by selectively binding to pyrophosphate and to monitor fluorescence changes during the hydrolysis process of pyrophosphatase, thereby enabling the screening of pyrophosphatases.
It enables rapid and selective detection of pyrophosphate with high sensitivity and specificity, effectively screening pyrophosphate hydrolases, reducing costs and simplifying operation.
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Figure CN119039214B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a fluorescent probe, a preparation method and an application thereof, in particular to a pyrophosphate fluorescent probe, a preparation method and an application thereof. BACKGROUND
[0002] Pyrophosphate (PPi, P2O7 4- ) detection has been extensively studied due to its important significance in biology. PPi is a common metabolic byproduct, which is produced in the biosynthesis of DNA, RNA, protein, peptidoglycan, cellulose, starch and other biological polymers. Because PPi plays an important role in many biological processes by participating in enzymatic reactions. Especially in polymerase chain reaction (PCR), when nucleotide triphosphates (NTPs) are incorporated into the amplified DNA or RNA, PPi is released, but the accumulation of product PPi will inhibit the PCR process.
[0003] Inorganic pyrophosphatase (IPP) is a ubiquitous enzyme in nature, which is involved in the hydrolysis of PPi generated in the synthesis of various metabolites such as carbohydrates, nucleic acids and proteins, generating two molecules of orthophosphate ion (Pi). This process is a high-energy process, which hydrolyzes PPi generated in various metabolic processes to prevent its excessive accumulation, thereby promoting the thermodynamic equilibrium to proceed in the direction of biosynthesis.
[0004] So far, few fluorescent probes that can effectively distinguish PPi and NTPs have been reported, although many PPi fluorescent probes are available, but the probes that bind closely to PPi have poor selectivity for PPi (between PPi and NTPs). Therefore, in order to determine the accumulation of PPi in the PCR process, the optimal amount of IPP activity in the reaction process, it is necessary to develop a high-sensitivity PPi sensor to distinguish PPi and NTPs.
[0005] The current method for detecting IPP activity mainly relies on the amount of Pi, the hydrolysis product of PPi, to evaluate the activity of the enzyme. The main defect of this method is that the analysis and detection are slow, the operation is complex and the efficiency is low. Therefore, it is of great significance to establish a rapid, specific and sensitive analysis and detection method. Fluorescent probes have the advantages of high sensitivity, good selectivity, real-time monitoring and the like.
[0006] In recent years, there are also many reports on the method of screening PPi by designing fluorescent probes, but due to its strong response to NTPs, it cannot effectively distinguish PPi from NTPs, so it cannot selectively detect PPi in the PCR process (there are a large number of NTPs), and thus cannot effectively determine the IPP enzyme activity and the optimal amount of addition. Adding excessive IPP in the reaction will increase the cost. Therefore, it is necessary to develop a fluorescent probe that can quickly and selectively detect pyrophosphate and a method for analyzing and detecting inorganic pyrophosphatase activity. SUMMARY
[0007] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a pyrophosphate fluorescent probe which produces a selective fluorescence response when combined with pyrophosphate and has activity in screening pyrophosphatase.
[0008] The present application also provides a preparation method of the pyrophosphate fluorescent probe, and its application in detecting pyrophosphate and screening pyrophosphatase.
[0009] The technical concept and principle of the present application are as follows: through research, it is found that the pyrophosphate fluorescent probe of the present application can selectively bind to free pyrophosphate and produce enhanced fluorescence, while the fluorescence response of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (dTTP) and phosphate (Pi) is weak, and has good selectivity and specificity, which can be used for selective detection of PPi.
[0010] In addition, the enzymatic hydrolysis kinetics of pyrophosphate can be characterized by fluorescence, and the fluorescence response of IPP can be analyzed. The present application finds a method for screening pyrophosphatase based on the hydrolysis of PPi to produce free Pi, thereby causing the fluorescence change of the probe. The substrate and probe preparation process of this strategy is simple and suitable for large-scale screening of enzymes.
[0011] The technical scheme of the present application is as follows:
[0012] The pyrophosphate fluorescent probe of the present application has a structure as shown in formula I, and the name is: 4'-(3,5-dimethoxyphenyl)-2,2':6',2"-terpyridine zinc (II) ligand, which is abbreviated as FL-TPY-Zn. 2+
[0013]
[0014] The preparation method of the pyrophosphate fluorescent probe provided by the application comprises the following steps: dispersing 4'-(3,5-dimethoxyphenyl)-2,2':6',2"-terpyridine and zinc nitrate (II) in an organic solvent, and evaporating and concentrating to obtain yellow solid FL-TPY-Zn 2+ ; wherein 4'-(3,5-dimethoxyphenyl)-2,2':6',2"-terpyridine is abbreviated as FL-TPY.
[0015] The preparation method of the pyrophosphate fluorescent probe provided by the application further comprises the following steps: dispersing 4'-(3,5-dimethoxyphenyl)-2,2':6',2"-terpyridine in an organic solvent, then adding an organic solvent solution of zinc nitrate (II), evaporating and removing the organic solvent, and then dissolving with an organic solvent for three times to obtain yellow solid FL-TPY-Zn 2+ ; wherein the molar ratio of the amount of FL-TPY to the amount of zinc nitrate (II) is 1:1. In order to ensure that one terpyridyl group is coordinated with one zinc ion, the amount of FL-TPY and the amount of zinc nitrate (II) need to be molarly matched at 1:1. Because the precursor and the zinc ion exist in a 2:1 coordination form, recrystallization may lose zinc ions in the solution, so the 1:1 molar ratio of the amount of FL-TPY to the amount of zinc nitrate (II) is used for feeding, and then the solid product obtained by feeding is directly dissolved in a buffer to prepare a probe mother liquor.
[0016] The preparation method of the pyrophosphate fluorescent probe provided by the application further comprises the following steps: dispersing 4'-(3,5-dimethoxyphenyl)-2,2':6',2"-terpyridine in an organic solvent, then adding an organic solvent solution of zinc nitrate (II), evaporating and removing the organic solvent, and then dissolving with an organic solvent for three times to obtain yellow solid FL-TPY-Zn
[0017] The preparation method of the pyrophosphate fluorescent probe provided by the application further comprises the following steps: dispersing 4'-(3,5-dimethoxyphenyl)-2,2':6',2"-terpyridine in an organic solvent, then adding an organic solvent solution of zinc nitrate (II), evaporating and removing the organic solvent, and then dissolving with an organic solvent for three times to obtain yellow solid FL-TPY-Zn
[0018] The pyrophosphate fluorescent probe provided by the application can be applied to the detection of pyrophosphate.
[0019] The pyrophosphate fluorescent probe provided by the application can be applied to the screening of pyrophosphate hydrolysis effective enzymes. The application comprises the following steps: using a saturated fluorescent solution of FL-TPY-Zn 2+ and pyrophosphate with a molar ratio of 3:1 as a substrate to screen pyrophosphate hydrolysis effective enzymes or monitor the kinetics of pyrophosphatase hydrolysis; FL-TPY-Zn 2+It selectively binds to PPi, exhibiting an enhanced fluorescence response. By screening pyrophosphatases that catalyze the conversion of PPi to two molecules of phosphate through fluorescence quenching rate, and which have no obvious fluorescence response to phosphate, it is possible to rapidly screen effective enzymes for pyrophosphate hydrolysis.
[0020] The application of the above-mentioned fluorescent pyrophosphate probe in screening for effective pyrophosphate hydrolysates, and its further technical solution is the FL-TPY-Zn described above. 2+ The molecular structure II, which coordinates with pyrophosphate in a 3:1 molar ratio, is shown below:
[0021]
[0022] The reaction process or principle of the present invention is as follows: Figure 14 As shown.
[0023] The present invention has the following beneficial effects:
[0024] 1) In this invention, FL-TPY and zinc nitrate (II) are coordinated in a 1:1 ratio to obtain the fluorescent probe FL-TPY-Zn. 2+ Dispersed in HEPES buffer (10 mM, pH 7.4), the probe molecules are selectively induced to aggregate by pyrophosphate (PPi), forming fluorescent nanoparticles and resulting in enhanced fluorescence emission.
[0025] 2) The fluorescence response method of the pyrophosphate fluorescent probe of the present invention, wherein the fluorescent probe of the present invention is FL-TPY-Zn. 2+ The pyrophosphate complex exhibits a significant fluorescence response to pyrophosphatase, with a coordination ratio of 3:1.
[0026] 3) The pyrophosphate fluorescent probe of the present invention has a strong fluorescence response to pyrophosphate, which is 212 times stronger. After sonication for 10 minutes, the detection limit is 37 nM. It has a weak fluorescence response to adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (dTTP), and phosphate (Pi), etc., and has good selectivity and specificity.
[0027] 4) The fluorescent probe FL-TPY-Zn of this invention 2+ When combined with pyrophosphate at a 3:1 ratio, it can be used for kinetic monitoring of pyrophosphatase hydrolysis of pyrophosphate substrates. The fluorescent probe FL-TPY-Zn of this invention... 2+ It can also be used for selective detection of pyrophosphate during PCR amplification. Attached Figure Description
[0028] Figure 1 The 1H NMR spectrum of FL-TPY in deuterated DMSO is shown in Example 1 of this invention.
[0029] Figure 2 NMR spectrum of Example 1 FL-TPY in deuterated DMSO.
[0030] Figure 3 High resolution mass spectrum of Example 2 FL-TPY-Zn 2+
[0031] Figure 4 Fluorescence response curve of Example 2 FL-TPY-Zn 2+
[0032] Figure 5 Linear relationship of Example 2 FL-TPY-Zn 2+
[0033] Figure 6 Jobs curve of Example 2 FL-TPY-Zn 2+
[0034] Figure 7 High resolution mass spectrum of Example 2 FL-TPY-Zn 2+
[0035] Figure 8 Fluorescence response comparison of Example 3 FL-TPY-Zn 2+
[0036] Figure 9 Fluorescence quenching process diagram of Example 3 FL-TPY-Zn 2+
[0037] Figure 10 Fluorescence kinetics curve of Example 3 FL-TPY-Zn 2+
[0038] Figure 11 PCR amplification process and product RNA and PPi relationship diagram of Example 4
[0039] Figure 12 Fluorescence response comparison of Example 4 FL-TPY-Zn 2+
[0040] Figure 13 Fluorescence response comparison of Example 4 FL-TPY-Zn 2+ Comparison of fluorescence response of reaction solution (without IPP) to PCR amplification process.
[0041] Figure 14 Reaction process or schematic diagram of the present application.
[0042] Figure 15 Process route diagram of Example 1 of the present application.
[0043] Figure 16 Process route diagram of Example 2 of the present application.
[0044] Figure 17 Process route diagram of Example 3 of the present application. DETAILED DESCRIPTION
[0045] The present application will be further described in detail below with reference to the accompanying drawings and in conjunction with the examples, but the present application is not limited to the examples.
[0046] Example 1 Preparation of fluorescent probe (FL-TPY-Zn) for selective recognition of pyrophosphate 2+ )
[0047] 1.96 g of 3,4,5-trimethoxybenzaldehyde and 2.42 g of 2-acetylpyridine were added to 80 mL of ethanol, after dissolution, 1.2 g of KOH particles were added, stirred at room temperature for 0.5 h, 80 mL of 25% ammonia water was added, and the reaction was stirred at room temperature for 6 h. The reaction solution was reduced pressure filtered, washed with ice ethanol, the precipitate was collected, and vacuum dried to obtain white flaky FL-TPY (1H NMR spectrum is shown in Figure 1 , and the carbon spectrum is shown in Figure 2 ).
[0048] 79.8 mg of FL-TPY and 59.5 mg of zinc nitrate hexahydrate (II) were dispersed in a methanol solution, heated to reflux, and the solvent was removed under vacuum for three times. After vacuum drying, yellow solid FL-TPY-Zn 2+ (mass spectrum is shown in Figure 3 ) was obtained.
[0049] The process route of this example is shown in Figure 15 .
[0050] Example 2 FL-TPY-Zn 2+ for selective detection of pyrophosphate
[0051] The FL-TPY-Zn 2+ prepared in Example 1 was dissolved with HEPES buffer (10 mM, pH 7.4), and made up to 200 mL in a volumetric flask to prepare a suspension with a concentration of 1.0 mM as a probe mother liquor. It was stored in a refrigerator at 0-2°C, and was taken out and placed at room temperature before use.
[0052] At 25℃, 2925 μL HEPES buffer (10 mM, pH 7.4) was placed in a quartz cuvette, 75 μL of the mother liquor was added and mixed thoroughly to obtain a 25 μM probe solution. Different concentrations of pyrophosphate solution were added, and after ultrasonic treatment for 10 minutes, the fluorescence spectra of different concentrations of pyrophosphate aqueous solution were measured using a fluorescence spectrometer, with the same volume of HEPES buffer added as a blank control Figure 4 ,5). The FL-TPY-Zn 2+ complex was prepared and the job's curve was obtained by coordination experiment with pyrophosphate Figure 6 ). The molecular weight of the complex was tested by high-resolution mass spectrometry when the fluorescence of the FL-TPY-Zn 2+ complex was saturated by coordination with pyrophosphate Figure 7 ). To the 25 μM probe solution, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (dTTP), phosphate (Pi), F - , Cl - , Br - , CH3COO - , CO3 2- , HCO3 - , SO4 2- , NO2 - , C2O4 - were added to a final concentration of 10 μM, and after ultrasonic treatment for 10 minutes, the fluorescence spectra were recorded using a fluorescence spectrometer Figure 8 . The excitation wavelength was selected to be 365 nm, the emission wavelength range was 400-700 nm, the excitation and emission slit widths were 10 / 10 nm, and the voltage was 400 V.
[0053] The process route of this example is shown in Figure 16 .
[0054] The above results show that:
[0055] The fluorescence probe FL-TPY-Zn 2+ prepared in Example 1 has a weak fluorescence response to ATP, ADP, AMP, GTP, CTP, dTTP, Pi, F - , Cl - , Br - , CH3COO - , CO3 2- , HCO3 - , SO4 2- , NO2 - , C2O4 - , etc., and the fluorescence emission of the system at 538 nm is enhanced by about 212 times after the addition of PPi, showing good selectivity and specificity.
[0056] Example 3 FL-TPY-Zn 2+ Pyrophosphatase hydrolysis kinetics monitoring with PPi complex
[0057] To the quartz cell containing the fluorescent probe FL-TPY-Zn 2+ (25 μM) in HEPES buffer (10 mM, pH 7.4), PPi solution with final concentration of 8 μM was added respectively. After 10 min sonication, pyrophosphatase (IPP) with final enzyme activity of 0, 0.2, 0.4, 0.6 U / μL was added respectively, and the same volume of HEPES buffer was added as blank control. The fluorescence quenching process of hydrolysis was shown in Figure 9 The excitation wavelength was selected as 365 nm, the emission wavelength as 527 nm, the excitation and emission slit width as 10 / 10 nm, the voltage as 400 V, and the test time as 8000 s, and the fluorescence kinetics curve was obtained. The obtained fluorescence kinetics curves were shown in Figure 10
[0058] The process route of this example was shown in Figure 17
[0059] The above results show that:
[0060] Example 2 FL-TPY-Zn 2+ The saturated fluorescence solution of 3:1 coordination with PPi as substrate can be used for monitoring the kinetics process of IPP hydrolyzing PPi.
[0061] Example 4 FL-TPY-Zn 2+ Selective detection of pyrophosphate for mRNA amplification process
[0062] At 25 °C, inorganic pyrophosphatase (IPP, 0.002 U / μL) was introduced before mRNA amplification reaction, and the terminator (RNase Inhibitor) was added after 3 h reaction. The PCR process was shown in Figure 11 , and the components of the reaction system were shown in the following table.
[0063] The table of component concentration of PCR amplification process (containing IPP) of this example
[0064] Component Name Final Concentration of Reaction System Tris-HCl pH 8.0 4 mM MgCl2 2 mM TCEP 0.25 mM Spermidine 0.2 mM NTPs (mix) 40 mM T7 RNA Polymerase 12.5 U / μl Inorganic Pyrophosphatase 0.002 U / μl RNase Inhibitor 2 U / μl DNA Template 50 ng / μl
[0065] The sample after complete reaction was diluted ten times with HEPES buffer (10 mM, pH 7.4), and different volumes of the diluted reaction solution were added to the quartz cell containing the fluorescent probe FL-TPY-Zn 2+ The mRNA sample (1.078 mg / μL) was tested for fluorescence spectrum. The same volume of HEPES buffer was added as blank control. The excitation wavelength was 365 nm, the emission wavelength range was 400-700 nm, the excitation and emission slit width was 10 / 10 nm, the voltage was 400 V, and the obtained fluorescence spectrum is shown in Fig. 1. Figure 12
[0066] As a control, the mRNA amplification reaction solution (without pyrophosphatase) was tested for fluorescence spectrum for 3 h, and the rest of the components and test conditions were unchanged. The obtained fluorescence spectrum is shown in Fig. 2. Figure 13
[0067] The above results show that:
[0068] The fluorescent probe FL-TPY-Zn was prepared according to Example 1 2+ The selective detection of pyrophosphate can be achieved in the mRNA amplification process, and there is no obvious fluorescence peak at 527 nm after the introduction of IPP, which has good selectivity and specificity. In addition, the probe can bind to mRNA to induce weak fluorescence enhancement at 628 nm.
[0069] In addition to the above examples, the present application can have other implementation manners. Any technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope of the present application.
Claims
1. A pyrophosphate fluorescent probe, characterized in that, The probe has the structure shown in Formula I and is named 4'-(3,5-dimethoxyphenyl)-2,2':6',2”-terpyridine zinc(II) ligand, abbreviated as FL-TPY-Zn. 2+ 2. A method for preparing the pyrophosphate fluorescent probe as described in claim 1, characterized in that, The process includes the following steps: dispersing 4'-(3,5-dimethoxyphenyl)-2,2':6',2”-terpyridine and zinc(II) nitrate in an organic solvent, followed by evaporation and concentration to obtain a yellow solid FL-TPY-Zn. 2+ ; among which 4'-(3,5-dimethoxyphenyl)-2,2':6',2”-terpyridine is abbreviated as FL-TPY.
3. The method for preparing the pyrophosphate fluorescent probe according to claim 2, characterized in that, The process includes the following steps: dispersing 4'-(3,5-dimethoxyphenyl)-2,2':6',2”-terpyridine in an organic solvent, adding an organic solvent solution of zinc nitrate (II), evaporating to remove the organic solvent, and then dissolving it in an organic solvent. This process is repeated three times to obtain a yellow solid FL-TPY-Zn. 2+ The molar ratio of FL-TPY to zinc nitrate (II) is 1:
1.
4. The method for preparing the pyrophosphate fluorescent probe according to claim 2, characterized in that, The organic solvent is methanol or ethanol.
5. The method for preparing the pyrophosphate fluorescent probe according to claim 2, characterized in that, The preparation method of FL-TPY includes the following steps: 3,5-dimethoxybenzaldehyde, 2-acetylpyridine and flake KOH are added to ethanol, then concentrated ammonia is added, and the mixture is stirred and reacted at room temperature for more than 8 hours. The reaction solution is filtered under reduced pressure, washed with water and ice-cold ethanol, the precipitate is collected, and the precipitate is dried under vacuum overnight to obtain white flake FL-TPY.
6. The application of the pyrophosphate fluorescent probe as described in claim 1 in the detection of pyrophosphate.
7. The application of the pyrophosphate fluorescent probe as described in claim 1 in screening effective enzymes for pyrophosphate hydrolysis.
8. The application of the pyrophosphate fluorescent probe according to claim 7 in screening effective enzymes for pyrophosphate hydrolysis, characterized in that, Includes the following steps: FL-TPY-Zn 2+ A saturated fluorescent solution coordinated with pyrophosphate at a molar ratio of 3:1 was used as a substrate for screening pyrophosphate-hydrolyzing enzymes or for monitoring the kinetics of pyrophosphate-hydrolyzing enzymes; FL-TPY-Zn 2+ It selectively binds to PPi, exhibiting an enhanced fluorescence response. By screening pyrophosphatases that catalyze the conversion of PPi to two molecules of phosphate through fluorescence quenching rate, and which have no obvious fluorescence response to phosphate, it is possible to rapidly screen effective enzymes for pyrophosphate hydrolysis.
9. The application of the pyrophosphate fluorescent probe according to claim 8 in screening effective enzymes for pyrophosphate hydrolysis, characterized in that, The FL-TPY-Zn 2+ The molecular structure II, which coordinates with pyrophosphate in a 3:1 molar ratio, is shown below: