Uridine analogue modified by fluorescent chromophore DPP derivative, and synthetic method and application thereof

Abstract

The invention discloses a fluorescent chromophore DPP derivative modified uridine analogue as well as a synthesis method and application thereof, and belongs to the field of biological medicine and detection. The invention provides the uridine analogue modified by the fluorescent chromophore DPP derivative, and the uridine analogue modified by the fluorescent chromophore DPP derivative has an excellent fluorescent property, can be used as a fluorescent probe, and is widely applied to the fields of biochemistry, medicine and the like. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and detection, and specifically relates to a uridine analog modified by a fluorescent chromophore DPP derivative, and a synthesis method and application thereof. Background Art

[0002] In the field of nucleic acid research, a variety of technical means play a key role, such as fluorescence spectroscopy, electrophoresis, nuclear magnetic resonance and X-ray single crystal diffraction, which jointly promote the progress of research in this field. Among them, fluorescence spectroscopy technology is highly respected in the nucleic acid research community due to its significant advantages such as ease of operation, high sensitivity and strong selectivity. It has shown a wide range of application value in exploring the process of nucleic acid conformational transition and its dynamic mechanism, and can accurately capture the conformational change information of nucleic acid molecules in different states.

[0003] Fluorescent nucleoside analogs have unique photophysical properties, which can change regularly according to the changes in the microenvironmental conditions. This property makes them a powerful tool for the study of nucleic acid structure and function, and they are widely used in this research field. Isostructural fluorescent nucleoside analogs have a high degree of similarity in structure to natural nucleosides. After being inserted into the nucleic acid sequence, they cause minimal disturbance to the structural integrity and functional activity of the parent nucleic acid, so they can effectively replace natural nucleosides to perform normal cellular functions. Based on the specific requirements for ideal fluorescent nucleosides, this study successfully constructed this type of isostructural fluorescent nucleoside analogs through careful design and synthetic operations, and deeply explored the synthetic route of cytidine analogs, thereby clarifying the suitable synthesis conditions of this type of base and its nucleoside precursors.

[0004] Natural nucleosides usually exhibit extremely low emissive characteristics. In view of this, preparing nucleoside analogs with fluorescent properties through chemical synthesis and using their unique optical signal properties to deeply explore the dynamic changes of the cellular microenvironment has become a hot topic in the field of biochemical research. Summary of the invention

[0005]

Technical issues

[0006] Provided are a novel fluorescent chromophore DPP derivative-modified uridine analogue and a preparation method thereof. The fluorescent chromophore DPP derivative-modified uridine analogue can be used as a probe in the field of detection and cell imaging.

[0007]

Technical solution

[0008] The present invention provides a uridine analog modified by a fluorescent chromophore DPP derivative, the structure of which is shown below:

[0009]

[0010] The present invention also provides a method for preparing the uridine analog modified by the fluorescent chromophore DPP derivative, comprising the following steps:

[0011]

[0012] The present invention uses a DPP derivative and TIPS as raw materials, and obtains a diketopyrrolopyrrole (DPP) derivative modified uridine analog after reaction. The present invention directly generates the target product, avoiding other reactions such as glycosylation and hydrolysis. It has the characteristics of relatively simple and controllable reaction conditions, no by-product generation, high yield, etc. After obtaining the product, cell activity detection is performed to determine the inhibitory effect.

[0013] In one embodiment of the present invention, the preparation method comprises the following steps:

[0014] (1) Using iodine glycoside and ((4-ethynylphenyl)ethynyl)triisopropylsilane as substrates, in the presence of catalyst 1, compound 1 is obtained;

[0015] (2) Compound 1 reacts with tetrabutylammonium fluoride to obtain compound 2;

[0016] (3) Compound 2 and compound 4 are used as substrates and react in the presence of catalyst 2 to obtain the target product.

[0017] In one embodiment of the present invention, in step (1), the molar ratio of iodine glycoside to ((4-ethynylphenyl)ethynyl)triisopropylsilane is 1:(1-2). Specifically, it can be 1:1.5.

[0018] In one embodiment of the present invention, the catalyst 1 in step (1) is CuI and Pd(PPh3)4.

[0019] In one embodiment of the present invention, in step (1), the molar ratio of iodine glycoside to CuI is 1:(0.1-0.5), and specifically 1:0.2.

[0020] In one embodiment of the present invention, in step (1), the molar ratio of iodine glycoside to Pd(PPh3)4 is 1:(0.05-0.2). Specifically, it can be 1:0.1.

[0021] In one embodiment of the present invention, the reaction in step (1) is carried out in a solvent DMF.

[0022] In one embodiment of the present invention, in step (1), the amount of DMF relative to iodine glycoside is 10-15 mL / mmol, and specifically 12 mL / mmol.

[0023] In one embodiment of the present invention, the reaction in step (1) further comprises adding triethylamine.

[0024] In one embodiment of the present invention, in step (1), the amount of triethylamine relative to iodine is 2-5 mL / mmol.

[0025] In one embodiment of the present invention, in step (2), the molar ratio of compound 1 to tetrabutylammonium fluoride is 1:(1.0-1.5).

[0026] In one embodiment of the present invention, in step (2), the reaction is carried out in a solvent THF.

[0027] In one embodiment of the present invention, in step (3), the molar ratio of compound 2 to compound 4 is 1:(1-2); specifically, it can be 1:1.5.

[0028] In one embodiment of the present invention, in step (3), catalyst 2 is CuI and Pd(PPh3)4.

[0029] In one embodiment of the present invention, in step (3), the molar ratio of compound 2 to CuI is 1:(0.1-0.5), and specifically 1:0.2.

[0030] In one embodiment of the present invention, in step (3), the molar ratio of compound 2 to Pd(PPh3)4 is 1:(0.05-0.2). Specifically, it can be 1:0.1.

[0031] In one embodiment of the present invention, the reaction in step (3) is carried out in a solvent DMF.

[0032] In one embodiment of the present invention, in step (3), the amount of DMF used relative to compound 2 is 10-15 mL / mmol.

[0033] In one embodiment of the present invention, the reaction in step (3) further comprises adding triethylamine.

[0034] In one embodiment of the present invention, in step (3), the amount of triethylamine used relative to compound 2 is 2-5 mL / mmol.

[0035] In one embodiment of the present invention, ((4-ethynylphenyl)ethynyl)triisopropylsilane is prepared by the following reaction route:

[0036]

[0037] The present invention also provides the use of the uridine analog modified by the fluorescent chromophore DPP derivative in the preparation of a detection probe.

[0038] The present invention also provides the use of the uridine analog modified by the fluorescent chromophore DPP derivative in environmental polarity detection, environmental pH detection, DNA fluorescent probe and cell fluorescence imaging.

[0039] Beneficial effects:

[0040] The present invention discloses a novel fluorescent chromophore DPP derivative-modified uridine analog and a synthesis method thereof. The uridine analog has excellent fluorescent properties and can be used as a fluorescent probe and is widely used in the fields of biochemistry and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 UV absorption spectra of uridine analogs modified with DPP derivatives in six solvents (c=25 μM).

[0042] Figure 2 Fluorescence spectra of uridine analogs modified with DPP derivatives in six polar solutions (c=5 μM).

[0043] Figure 3 Solvent E T (30) There is a linear relationship between the value and the Stocks shift.

[0044] Figure 4 Figure 5 is the fluorescence spectrum of uridine analogs modified with DPP derivatives in pH solution (c=5 μM).

[0045] Figure 5 The quenching spectra of uridine analogs modified with DPP derivatives (c=5 μM). DETAILED DESCRIPTION

[0046] The following are specific embodiments of the present invention.

[0047] The following is the synthesis route of the present invention, as shown in the accompanying drawings, the synthesis is carried out in three steps:

[0048] (1) Synthesis of uridine analog 1

[0049] Weigh iodine glycoside, CuI, Pd(PPh3)4, pass N2, add DMF and Et3N, stir, then add ((4-ethynylphenyl)ethynyl)triisopropylsilane, react at room temperature, filter, and distill to obtain uridine analog 1.

[0050] (2) Synthesis of uridine analog 2

[0051] The entire compound 2 was dissolved in 10 mL of THF in an eggplant-shaped bottle, and then tetrabutylammonium fluoride (TBAF) THF solution was added thereto. The mixture was stirred for reaction and distilled to obtain uridine analog 2

[0052] (3) Synthesis of DPP derivative-modified uridine analog 3

[0053] Compound 4, CuI, compound 8, and Pd(PPh3)4 were mixed, vacuumed, passed through N2, DMF (5 ml) and Et3N (1 ml) were added, stirred at room temperature, insoluble matter was removed by filtration, and then distilled to obtain DPP derivative-modified uridine analog 3.

[0054] Preparation of the intermediate ((4-ethynylphenyl)ethynyl)triisopropylsilane

[0055] Synthesis of triisopropyl((4-((trimethylsilyl)ethynyl)phenyl)ethynyl)silane: 4-bromoiodobenzene (3 g, 10.6 mmol, 1 equiv), Pd(PPh3)2Cl2 (0.3 g, 0.04 mol, 0.0041 equiv), CuI (43 mg, 0.04 mol, 0.004 equiv) were weighed in a 100 mL two-necked round-bottom flask, and then 50 mL of diisopropylamine was added, and then triisopropylsilylacetylene (TIPSA) (2.7 mL, 12 mmol, 1.2 equiv) was slowly added and stirred at room temperature for 24 h. Then trimethylsilylacetylene (TMSA) (14 mmol, 1.3 equiv) was added dropwise and stirred at 60°C overnight. The mixture was dried under vacuum and the crude product was purified by column chromatography (hexane as eluent) to give 1.9 g of triisopropyl((4-((trimethylsilyl)ethynyl)phenyl)ethynyl)silane.

[0056] Synthesis of ((4-ethynylphenyl)ethynyl)triisopropylsilane: Weigh ((4-ethynylphenyl)ethynyl)triisopropylsilane (1.76 g, 5 mmol, 1 equiv) in an eggplant-shaped bottle equipped with a magnetic bar, evacuate, and pass N2. Each operation is repeated three times. Under the N2 state, open the bottle stopper and add DCM (60 mL) and methanol (60 mL) to dissolve compound 5. Add K2CO3 (1.5 g, 11 mmol, 1 equiv) and stir at room temperature for 2 h. Filter out the solid residue and remove the volatile solvent from the filtrate. Dissolve and dilute with CH2Cl2, then wash it with a saturated NaCl aqueous solution, dry the lower layer solution with anhydrous MgSO4, filter, and evaporate to dryness to obtain a colorless oil, which is further purified by column chromatography using hexane as the eluent. ((4-ethynylphenyl)ethynyl)triisopropylsilane (0.812 g, 58%) is obtained. 1 H NMR (400MHz, CDCl3) δ7.34 (s, 4H), 3.08 (s, 1H), 1.05 (s, 21H).

[0057] Example 1

[0058]

[0059] S1. Synthesis of uridine analogs (1)

[0060] Weigh iodine (0.354 g, 1 mmol, 1 equiv), CuI (0.040 g, 0.2 mmol, 0.2 equiv), Pd(PPh3)4 (0.116 g, 0.1 mmol, 0.1 equiv), evacuate, pass N2, repeat each operation three times, add DMF (12 ml) and Et3N (3 ml), stir at room temperature for 0.5 h to completely dissolve the solid. Then add ((4-ethynylphenyl)ethynyl)triisopropylsilane (0.423 g, 1.5 mmol, 1.5 equiv), react at room temperature for 3 h, track the reaction by TLC, the developing solvent ratio is dichloromethane: methanol = 15:1. After filtering, use an oil pump and cold hydrazine to vacuum decompress the solvent in a 65°C oil bath to remove DMF. Column chromatography (eluent ratio is V 二氯甲烷 :V 甲醇 =80:1~25:1) to obtain 0.376 g of a light yellow solid uridine analog (1) with a yield of 74%.

[0061] S2. Synthesis of uridine analogs (2)

[0062] Compound 1 (0.470 g, 0.92 mmol, 1 equiv) was weighed and dissolved in 10 mL of THF in an eggplant-shaped bottle, and then 1 M tetrabutylammonium fluoride (TBAF) THF solution (1.2 mL, 1.3 equiv) was added. The mixture was stirred for 12 h and the reaction was followed by TLC. The developing solvent ratio was V 二氯甲烷 :V 甲醇 =10:1. The solvent was removed in vacuo and the resulting crude material was purified by column chromatography (eluent ratio: V 二氯甲烷 :V 甲醇 =50:1~10:1) to obtain 0.250 g of light yellow solid uridine analog (2) with a yield of 78%.

[0063] S3, Synthesis of DPP Derivative Modified Uridine Analogs (3)

[0064] Weigh compound 4 (0.213 g, 0.444 mmol, 1.5 equiv), CuI (0.012 g, 0.06 mmol, 0.2 equiv), compound 2 (0.104 g, 0.296 mmol, 1 equiv), and Pd(PPh3)4 (0.035 g, 0.03 mmol, 0.1 equiv). Vacuum and pass N2. Repeat each operation three times. Add DMF (5 ml) and Et3N (1 ml) under N2. Stir at room temperature for 4 h. Follow the reaction by TLC (developer ratio V 二氯甲烷 :V 甲醇 =10:1). Remove the insoluble matter by filtration. In a 65°C oil bath, use an oil pump connected to cold hydrazine to vacuum decompress the solvent to remove DMF. Then, column chromatography (eluent: V 二氯甲烷 :V 甲醇 =50:1~20:1). A total of 0.1 g of red solid uridine analog (3) was obtained with a yield of 45%.

[0065] 1 H NMR (400MHz, DMSO) δ11.77(s,1H),8.51(s,1H),7.97(d,J=8.5Hz,2H),7.93-7.86(m,2H),7.82(d,J=8.5 Hz,2H),7.69(s,1H),7.66(dd,J=6.3,3.6Hz,4H),7.61(s,1H),7.59(s,1H),6.20(t,J=6.5Hz,1H),5.40 (d,J=32.7Hz,2H),4.35(d,J=3.5Hz,1H),3.89(q,J=3.2Hz,1H),3.78(dd,J=13.8,6.7Hz,4H),3.70(d,J =13.1Hz,2H),2.29-2.20(m,2H),1.54-1.40(m,4H),1.20(dp,J=14.2,7.2Hz,4H),0.81(q,J=7.3Hz,6H).

[0066] 13C NMR(101MHz,DMSO)δ161.95(s),161.92(s),150.03(s),148.89(s),147.10(s),145.74(s),139.84(s),133.11 (s),132.78(s),131.46(s),131.13(s),130.23(s),129.87(s),128.59(s),128.33(s),125.08(s),125.00(s) ,123.55(s),123.47(s),122.28(s),122.20(s),109.72(s),109.25(s),98.35(s),91.84(s),91.24(s),85.59 (s),79.89(s),79.56(s),79.22(s),71.11(s),61.26(s),41.04(s),40.72(s),29.89(s),19.75(s),14.43(s).

[0067] Example 2: Application of DPP derivative-modified uridine analogs as probes in detection fields

[0068] 1. DPP derivative-modified uridine analogs as probes to detect and identify polar environments

[0069] Test solution for UV absorption spectrum: Weigh the DPP derivative-modified uridine analog with an analytical balance, add an appropriate amount of solvent to dissolve the solute, then pour into a 100mL volumetric flask, rinse the small beaker with a small amount of solvent several times, then add the rinse solution to the volumetric flask, and shake to obtain a compound solution with a concentration of 25μmol / L. According to this method, prepare six different solvent solutions of thiazole [4,5-d] uridine analogs. After preparation, shake well and use an ultrasonic cleaner to further mix the test solution.

[0070] Fluorescence emission spectrum test solution: Pipette 500 μL of the previously prepared 25 μmol / s test solution of the compound thiazole [4,5-d] uridine analogue into a 10 mL volumetric flask, add solvent to 10 mL, and shake the test solution in the volumetric flask. Prepare six solutions of DPP derivative-modified uridine analogues in solvents of different polarity according to this method. After preparation, shake well and use an ultrasonic cleaner to further mix the test solution.

[0071] (1) Ultraviolet absorption spectra of different polarities

[0072] The UV absorption spectra of uridine analogs modified by DPP derivatives in six different polarities are as follows Figure 1As shown, in the near-ultraviolet region ranging from 200nm to 400nm, there is an absorption peak around 310nm, which is produced by π-π* electron transition, and is produced by the large conjugation formed by the benzene ring, alkynyl group and uridine base. There is another absorption peak around 480nm, which is produced by n-π* transition, and is the peak of the DPP derivative introduced into the compound. With the increase of polarity, the absorption peak has a slight blue shift phenomenon.

[0073] (2) Fluorescence emission spectra under different polarity conditions

[0074] The maximum absorption wavelength of the DPP derivative-modified uridine analog in six solvents was used as the excitation wavelength, and the fluorescence spectrum was repeatedly scanned until the excitation wavelength and emission wavelength were stable. The corresponding fluorescence emission spectrum was as follows: Figure 2 shown.

[0075] like Figure 2 As shown in the fluorescence emission spectrum, the compound has strong fluorescence spectrum emission in six solvents of different polarities, and the fluorescence intensity is the strongest in dichloromethane solvent. Specific fluorescence spectrum data are shown in Table 1.

[0076] Table 1 Fluorescence spectra of uridine analogs modified by DPP derivatives in six polar solutions

[0077]

[0078] (3) Stokes shift and solvent E T (30) Relationship

[0079] Stokes shift is an important reference data in the study of spectral phenomena. Stokes shift is caused by the vibration relaxation or dissipation of solvent molecules and solvent reorganization. One of the factors affecting it is the polarity of the solution. In different solvents, the Stokes shift of the same compound will be very different due to the difference in their solvent polarity. From the wavelength data of the maximum peak obtained from the absorption and emission spectra, the Stokes shift and the solution polarity constant E can be judged. T (30) to determine whether there is a linear relationship between the two, thereby determining the sensitivity of the compound to polarity. The formula for calculating the Stokes shift is:

[0080]

[0081] The Stokes shift values ​​of thiazolyl[4,5-d]uridine analogs were calculated using the above formula and are shown in Table 2.

[0082] Table 2 Stock shift of uridine analogs modified by DPP derivatives in six solvents

[0083]

[0084] E T (30) is the horizontal axis, and Stocks Shift is the vertical axis. Figure 3 Linear fitting of methanol, acetonitrile, ethyl acetate, dioxane and dichloromethane shows that the stock shift of uridine analogs modified by DPP derivatives is similar to that of E T (30) There is a linear relationship between 2 The value is 0.71459, which is sensitive to the polarity of the organic solvent, indicating that the uridine analog modified by the DPP derivative of the present invention is a better polarity probe.

[0085] 2. DPP derivative-modified uridine analogs as probes responding to pH environment

[0086] Preparation of buffer solutions with different pH values: 0.1mmol / L of Na2HPO4, NaH2PO4, H3PO4 and NaOH aqueous solutions are required to prepare buffer solutions, where Na2HPO4 and NaH2PO4 are alkaline and acidic, respectively. Therefore, NaH2PO4 solution should be used to prepare buffer solutions with pH values ​​of 1 to 6. Finally, use a few drops of NaOH or H3PO4 solution to fine-tune the pH value, subject to the stability of the pH meter value. Use Na2HPO4 solution to prepare a buffer solution with a pH value of 7 to 12, and finally add NaOH or H3PO4 solution to fine-tune the pH value. After the indication value of the pH meter is finally stabilized, a pH buffer solution with pH = 1 to 12 is obtained.

[0087] Preparation of aqueous solution of DPP derivative-modified uridine analog: accurately weigh 0.01mmol DPP derivative-modified uridine analog with analytical balance, add deionized water, shake well in a small beaker to dissolve the solute, add to a 10ml volumetric flask, wash the beaker three times with appropriate amount of deionized water, collect the liquid and add to the volumetric flask, fix the volume, shake well, and further mix the solution to be tested with ultrasonic cleaning agent to obtain a 1mmol / L DPP derivative-modified uridine analog aqueous solution. Use a pipette to accurately draw 1ml of the prepared solution and put it into a 10ml centrifuge tube, then add a buffer solution with pH=1-12, fix the volume to 10ml, shake well, and obtain a 100μmol / L DPP derivative-modified uridine analog test solution with 12 pH values.

[0088] Fluorescence emission spectrum test solution: Use a pipette to transfer 500 μL of the previously prepared 100 μmol / L DPP derivative-modified uridine analog aqueous solution into a volumetric flask, dilute to the 10 ml mark of the volumetric flask with a pH buffer solution of pH=1-12, shake the test solution in the volumetric flask, and obtain a 5 μmol / L DPP derivative-modified uridine analog fluorescence emission spectrum test solution after ultrasonication.

[0089] Fluorescence spectra under different pH conditions

[0090] With 500 nm as the excitation wavelength, the fluorescence emission spectra of the compounds in different pH solutions were scanned in turn. Figure 4 . With the change of pH value, the displacement of the compound's fluorescence emission peak changes between 585nm and 623nm. The maximum emission wavelength is 585nm when pH=12 and 653nm when pH=6, indicating that the strong alkaline environment will cause the compound's emission peak to blue shift. The fluorescence intensity varies between 303 and 653a.u., with the weakest fluorescence intensity at pH=2 and the strongest fluorescence intensity at pH=6, indicating that the strong acid environment will weaken the compound's fluorescence effect. In an alkaline environment, the compound's emission intensity will decrease as the alkalinity of the solution increases. The specific values ​​are shown in Table 3.

[0091] Table 3 Emission wavelength and corresponding fluorescence intensity under pH solution

[0092]

[0093] It can be seen that in the ranges of pH 3 to pH 5, pH 6 to pH 9, and pH 10 to pH 12, the fluorescence intensity gradually weakened as the alkalinity increased, indicating that the uridine analog modified with the compound DPP derivative can be used as a fluorescent probe within a specific pH range.

[0094] The control The emission wavelength and corresponding fluorescence intensity results in different pH solutions are shown in Table 4:

[0095] Table 4

[0096]

[0097] The fluorescence intensity of the reference substance is weak, which will significantly affect its probe application. Moreover, as the pH value increases, the fluorescence intensity of the reference substance increases first and then decreases, and in solutions with a pH of 11 and 12, its absorbance becomes negative. The possible reason is that under alkaline conditions, the amide bond in the reference substance is hydrolyzed, and its structure is destroyed, causing the fluorescence intensity to decrease or even become negative.

[0098] 3. Fluorescence quenching effect of uridine analogs modified with DPP derivatives as probes

[0099] The photophysical properties of fluorescent nucleoside analogs are not only affected by the surrounding environment, but also by the interaction with adjacent natural bases. For fluorescent probes, they need to be highly sensitive to the environment and maintain good photophysical properties when inserted into the target oligonucleoside chain. The interaction between adjacent natural nucleosides may quench them, affecting their role as oligonucleotide probes. Therefore, it is very important to understand whether the fluorescence intensity of uridine analogs modified by DPP derivatives will be quenched by the four natural bases.

[0100] During the dynamic quenching process, the excited molecules of the fluorescent compound return to the ground state by colliding with the quenching molecules, and their excitation energy is lost through energy transfer or charge transfer mechanisms. Therefore, changes in the concentration of the quencher will change the efficiency of dynamic quenching. The factors that affect the photophysical properties of fluorescent compounds are many and complex. Due to the influence of other substances, the photophysical properties of fluorescent compounds are prone to change and have great uncertainty. After forming an oligonucleotide in a certain sequence, whether its fluorescence intensity will be weakened and whether it can play a normal role also depends on whether the adjacent bases will affect the fluorescent nucleoside analogs. If a fluorescent nucleoside analog with excellent performance loses fluorescence under the action of adjacent bases, it cannot become a good fluorescent probe.

[0101] The monophosphate of natural nucleosides is added to the phosphate buffer solution of the DPP derivative-modified uridine analog by titration, and the emission spectrum of the uridine analog is obtained by scanning with a fluorescence spectrophotometer. By studying the fluorescence spectrum of uridine analogs, we can further understand the photophysical properties of uridine analogs, understand the effects of different natural nucleotides on uridine analog fluorescent probes, and determine whether uridine analogs can function normally in oligonucleotide sequences.

[0102] The fluorescence quenching process produced by the interaction between the extended fluorescent cytidine analogs and the four natural bases belongs to dynamic quenching and conforms to the Stern-Volmer equation:

[0103] F0, F, [NMPs], and K SV Respectively represent the emission intensity of the compound without quencher, the emission intensity of the compound when quencher is added, the quencher concentration and the quenching constant. Use [NMPs] and F0 / F to make a Stern-Volmer plot. The slope of the straight line in the figure is K SV . K SV The larger the value, the better the quenching effect of the natural base on the fluorescent nucleoside analog. After being inserted into the target oligonucleotide chain, it is likely to be affected by the natural base, causing its fluorescence characteristics to deteriorate and fail to achieve the performance that a fluorescent probe should have.

[0104] Preparation of quenching solution: The fluorescence quenching test solution needs to be mixed with phosphate buffer solution and natural nucleotide solution.

[0105] Phosphate buffer solution: Use 0.1 mol / L NaOH solution, H3PO4 solution, Na2HPO4 solution, NaH2PO4 and NaCl aqueous solution to prepare phosphate buffer solution. When preparing, add the above solutions to the NaCl aqueous solution to fine-tune the pH value until the pH meter reading stabilizes at 7.4.

[0106] Natural nucleotide solution: Accurately weigh 0.2mmol of natural nucleosides (NMP): adenosine (0.0694g), cytidine (0.0646g), guanosine (0.0814g) and thymidine (0.0732g) in a small beaker. Add a small amount of prepared phosphate buffer solution to fully disperse the natural nucleosides, and then add it to a 10ml volumetric flask. Clean the beaker with phosphate buffer solution in small amounts several times and return it to the volumetric flask. Finally, set the volume to 10ml and shake evenly to obtain 20mmol / L adenylic acid (AMP), cytidine (CMP), guanosine (GMP) and thymidine (TMP) nucleotide solutions.

[0107] Fluorescence quenching test solution: number 24 centrifuge tubes: A1-A6, C1-C6, G1-G6 and T1-T6, first add 0.5mL of 100μmol / L fluorescent nucleoside analog solution to each centrifuge tube, then add 0mL-0.5mL of four nucleotide solutions in the order of the numbering of each group of centrifuge tubes, finally add the prepared phosphate buffer solution to make the volume to 10mL, shake well to obtain 4 groups of 24 test solutions of four nucleotides, among which the concentration of the fluorescent nucleoside analog is 5μmol / L.

[0108] The fluorescence quenching spectrum obtained is as follows Figure 5 shown.

[0109] like Figure 5 Adenosine (AMP), cytidylic acid (CMP), guanylic acid (GMP) and thymidylic acid (TMP) have little effect on the fluorescence intensity of uridine analogs modified by DPP derivatives, indicating that it will not be affected by natural nucleoside bases and weaken the fluorescence intensity. It can maintain strong emission when inserted into the target nucleotide chain and can be used as a biological fluorescence probe.

[0110] The fluorescence quenching effect of the reference substance was tested in the same way. It was found that after adding the quencher, the luminescence process of the reference substance was greatly affected by the natural nucleotides. SV It is much larger than the uridine analog modified by the DPP derivative of the present invention. Therefore, it can be seen that the uridine analog modified by the DPP derivative of the present invention is a better biological fluorescence probe.

Claims

1. A uridine analog modified with a fluorescent chromophore DPP derivative, characterized in that: The structure is as follows:

2. A method for preparing the uridine analog modified by the fluorescent chromophore DPP derivative according to claim 1, characterized in that: The steps include:

3. The preparation method according to claim 2, characterized in that: The following steps are involved: (1) Using iodine glycoside and ((4-ethynylphenyl)ethynyl)triisopropylsilane as substrates, in the presence of catalyst 1, compound 1 is obtained; (2) Compound 1 reacts with tetrabutylammonium fluoride to obtain compound 2; (3) Compound 2 and compound 4 are used as substrates and react in the presence of catalyst 2 to obtain the target product.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of iodine glycoside to ((4-ethynylphenyl)ethynyl)triisopropylsilane is 1:(1-2).

5. The preparation method according to claim 3, characterized in that: The catalyst 1 in step (1) is CuI and Pd(PPh3)4; wherein the molar ratio of iodine glycoside to CuI is 1:(0.1-0.5); and the molar ratio of iodine glycoside to Pd(PPh3)4 is 1:(0.05-0.2).

6. The preparation method according to claim 3, characterized in that: The reaction in step (1) further comprises adding triethylamine; the amount of triethylamine used relative to iodine is 2-5 mL / mmol.

7. The preparation method according to claim 3, characterized in that: In step (2), the molar ratio of compound 1 to tetrabutylammonium fluoride is 1:(1.0-1.5).

8. The preparation method according to any one of claims 3 to 7, characterized in that: In step (3), the molar ratio of compound 2 to compound 4 is 1:(1-2); catalyst 2 is CuI and Pd(PPh3)4, wherein the molar ratio of compound 2 to CuI is 1:(0.1-0.5), and the molar ratio of compound 2 to Pd(PPh3)4 is 1:(0.05-0.2); the reaction further comprises adding triethylamine, and the amount of triethylamine relative to compound 2 is 2-5 mL / mmol.

9. Use of the uridine analog modified with the fluorescent chromophore DPP derivative according to claim 1 in the preparation of detection probes.

10. Use of the uridine analog modified with the fluorescent chromophore DPP derivative according to claim 1 in environmental polarity detection, environmental pH detection, DNA fluorescent probe and cell fluorescence imaging.

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