DPP derivative modified deoxyuridine as well as synthesis method and application thereof

Abstract

The invention discloses DPP derivative modified deoxyuridine as well as a synthesis method and application thereof, and belongs to the field of biological medicine and detection. The invention provides DPP derivative modified deoxyuridine shown in the following structure, and the compound has excellent fluorescent property, can be used as a fluorescent probe, and is widely applied in the fields of biochemistry, medicine and the like, such as tools for specific drug delivery, molecular recognition and biosensing, and the like. Action # imgabs0 # in nucleic acid analysis and quantitative detection of fluorescence-labeled oligonucleotides and potential of the fluorescence-labeled oligonucleotides in treatment of human acquired diseases or genetic diseases, capability of specifically regulating gene expression and value of the fluorescence-labeled oligonucleotides in clinical application after chemical modification, as well as application of the fluorescence-labeled oligonucleotides in nucleic acid analysis and quantitative detection of the fluorescence-labeled oligonucleotides

Description

Technical Field

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

[0002] Nucleic acids inevitably interact with other macromolecules, such as proteins, during the course of life. Fluorescence imaging can reveal the interaction patterns between them and is one of the important ways to deeply understand life phenomena and explore the mysteries of life. As one of the most powerful imaging modes, fluorescence imaging provides a sensitive and non-invasive technique for visualizing biomolecules and elucidating biological phenomena in living cells or tissues with high spatial and temporal resolution. In addition, due to the influence of tissue penetration, fluorescence imaging of near-infrared fluorophores is more advantageous than color staining. In the past few decades, extensive research has promoted the construction of many fluorescent probes for biological imaging. Typical sensors include molecular recognition elements that specifically interact with the analyte and signal converters that convert recognition events into fluorescence readouts. As an indispensable tool in molecular biology research, fluorescent nucleic acid probes give full play to the structural characteristics and recognition functions of nucleic acids. These probes convert the hybridization reaction, morphological changes, and specific interactions with metal ions, proteins, and specific cells of nucleic acids into fluorescent signals that can be directly observed, thereby achieving accurate detection and analysis of biomolecules.

[0003] However, the fluorescence intensity of existing fluorescent nucleic acid probes is not high in some different environments, such as pH and polarity, and they are also easily quenched by natural nucleotides. Summary of the invention

[0004]

Technical issues

[0005] In order to solve the above problems, the present invention provides a novel DPP derivative-modified deoxyuridine and a synthesis method thereof. The DPP derivative-modified deoxyuridine can be used as a probe in the field of detection and cell imaging.

[0006]

Technical solution

[0007] The present invention inserts a diketopyrrolopyrrole derivative into the 5-position of deoxyuridine dU to obtain a modified deoxyuridine dU DPPth The modified deoxyuridine extends the conjugated structure of the natural nucleoside.

[0008] The special feature of the fluorescent nucleoside designed in the present invention is that the emission wavelength is in the near-infrared region, which is due to the addition of the thiophene ring. The rigid connection of the alkynyl group between the thiophene ring-modified DPP and deoxyuridine not only extends the conjugated structure of the fluorescent nucleoside, but also keeps the fluorophore away from deoxyuridine. This design makes dUDPPth After insertion into the oligonucleotide, quenching by natural nucleosides can be avoided.

[0009] The present invention provides a DPP derivative modified deoxyuridine (dU DPPth ), the structure is as follows:

[0010]

[0011] The present invention also provides a method for synthesizing the above-mentioned DPP derivative-modified deoxyuridine, and the reaction scheme is as follows:

[0012]

[0013] Compound 2 was prepared by the following method:

[0014]

[0015] The present invention uses a DPP derivative modified deoxyuridine dU prepared from 5-iodo-2'-deoxyuridine as a raw material DPPth The 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, and high yield.

[0016] In one embodiment of the present invention, the preparation method comprises the following steps:

[0017] Compound 2 and compound 3 are used as substrates and react under the action of a catalyst to obtain the target product.

[0018] In one embodiment of the present invention, the molar ratio of compound 2 to compound 3 is (1-2):1; specifically, it can be 1:1.5.

[0019] In one embodiment of the present invention, the catalyst is CuI and Pd(PPh 3 ) 4 .

[0020] In one embodiment of the present invention, the molar ratio of compound 3 to CuI is 1:(0.1-0.5), and specifically 1:0.2.

[0021] In one embodiment of the present invention, compound 3 and Pd(PPh 3 ) 4 The molar ratio is 1:(0.05-0.2). Specifically, 1:0.1 can be selected.

[0022] In one embodiment of the present invention, the reaction is carried out in a solvent DMF.

[0023] In one embodiment of the present invention, the amount of DMF relative to compound 3 is 10-20 mL / mmol.

[0024] In one embodiment of the present invention, the reaction further comprises adding triethylamine.

[0025] In one embodiment of the present invention, the amount of triethylamine used relative to compound 3 is 2-5 mL / mmol.

[0026] In one embodiment of the present invention, the preparation route of compound 2 is as follows:

[0027]

[0028] In one embodiment of the present invention, in step A, 5-iodo-2′-deoxyuridine and trimethylsilyl acetylene are reacted with cuprous iodide and bistriphenylphosphine palladium dichloride to obtain compound 1.

[0029] In one embodiment of the present invention, step A further comprises adding an organic solvent and triethylamine.

[0030] In one embodiment of the present invention, in step A, the organic solvent used is acetonitrile.

[0031] In one embodiment of the present invention, in step B, compound 1 is reacted with a base to obtain compound 2.

[0032] In one embodiment of the present invention, in step B, the base used is any one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

[0033] In one embodiment of the present invention, the preparation route of compound 3 is as follows:

[0034]

[0035] The present invention also provides the use of the above-mentioned DPP derivative-modified deoxyuridine in the preparation of detection probes.

[0036] The present invention also provides the use of the above-mentioned DPP derivative-modified deoxyuridine in environmental polarity detection, environmental pH detection, DNA fluorescent probe and cell fluorescence imaging.

[0037] Beneficial effects:

[0038] The present invention is a method for preparing a novel nucleoside derivative. Such a product has excellent fluorescence properties and can be used as a fluorescent probe in a wide range of applications in the fields of biochemistry and medicine, such as as a tool for specific drug delivery, molecular recognition, and biosensing, as well as its potential in treating human acquired diseases or genetic diseases, its ability to specifically regulate gene expression, and its value in clinical applications after chemical modification. In addition, fluorescently labeled oligonucleotides play a role in nucleic acid analysis and quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 dU DPPth UV-Vis spectra in six solvents (c=25 μM).

[0040] Figure 2 dU DPPth Fluorescence spectra in solutions of different polarities (c=5 μM).

[0041] Figure 3 dU DPPth Solvent E T (30)Relationship with Stocks shift.

[0042] Figure 4 dU DPPth UV-Vis spectra in pH solutions (c=100 μM).

[0043] Figure 5 dU DPPth Fluorescence spectra in pH solutions (c=10 μM).

[0044] Figure 6 dU DPPth Normalized fluorescence intensity-pH curve (c=5μM).

[0045] Figure 7 dU DPPth Quenching spectrum of (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 following formula, and the synthesis is carried out in three steps:

[0048] The first step is to weigh cuprous iodide, raw material 5-iodo-2′-deoxyuridine, and bistriphenylphosphine palladium dichloride, add them to an eggplant-shaped bottle, use a double-row tube to replace the nitrogen three times, insert a nitrogen balloon, add anhydrous acetonitrile and anhydrous triethylamine, stir at 25°C, and add trimethylsilyl acetylene to the reactor. After heating the reactor to 50°C, continue the reaction. The reaction formula is:

[0049]

[0050] In the second step, the raw material deoxyuridine analog (1) and anhydrous potassium carbonate are weighed and added into an eggplant-shaped bottle, the air in the reactor is replaced with nitrogen using a double-row tube, a nitrogen balloon is inserted, anhydrous methanol is added, and then the reaction is stirred at 25°C. The reaction formula is:

[0051]

[0052] The third step is to weigh deoxyuridine analog (2), cuprous iodide, and tetratriphenylphosphine palladium and add them to the reaction eggplant-shaped flask. The air in the reactor is replaced with nitrogen using a double-row tube, and a nitrogen balloon is inserted to balance the air pressure. DMF and Et 3 N, and the reactor was placed under room temperature and stirred. The DPP derivative (3) was dissolved in DMF and added to the reactor to continue the reaction to obtain the target product. The reaction formula is:

[0053]

[0054] Preparation of intermediate DPP derivative (3):

[0055]

[0056] Preparation of 3-(5-bromothiophen-2-yl)-6-phenyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione:

[0057] Weigh sodium tert-amyl alcohol (17g, 156mmol, 3equiv) and add it to a 500mL three-necked flask. Set up a reflux device and replace the nitrogen three times. Then add 200mL of tert-amyl alcohol under nitrogen atmosphere and stir to dissolve. After the solution is clarified, add 5-oxo-2-phenyl-4,5-dihydro-1H-pyrrole-3-carboxylic acid ethyl ester (12g, 51.89mmol, 1equiv) and 5-bromo-thiophene-2-carbonitrile (13.05g, 71.61mmol, 1.38equiv) under nitrogen atmosphere, and then seal the bottle mouth with a reverse stopper. Stir and reflux at 110°C for 20h, and the reaction solution gradually turns dark red. Finally, 5.8g of dark red solid compound 3-(5-bromothiophene-2-yl)-6-phenyl-2,5-dihydropyrrole [3,4-c] pyrrole-1,4-dione is obtained with a yield of 30.6%.

[0058] Preparation of DPP derivative 3: 3-(5-bromothiophene-2-yl)-6-phenyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (1.86 g, 5 mmol, 1 equiv) and anhydrous potassium carbonate (3.5 g, 25 mmol, 5 equiv) were placed in a 200 mL three-necked flask. Nitrogen replacement was performed three times, and 80 mL of DMF was added to the bottle cap under nitrogen atmosphere. The temperature was then raised to 100°C and the reactants were stirred for 1 hour. After adding 1-bromobutane (4.1 g, 30 mmol, 6 equiv), the temperature was set to 120°C. After the reaction was completed, a red powder solid was obtained by column chromatography, with a total of 850 mg and a yield of 36%. 1 H NMR (400 MHz, CDCl 3 )δ1.59(s,1H),1.51(s,1H),1.34(s,1H),1.28(s,0H),1.19(s,2H),0.86(s,2H),0.76(s,2H) 3.78(d,J=46.1Hz,2H),7.71(s,1H),7.14(s,0H),7.42(d,J=6.3Hz,3H)8.60(d,J=4.2Hz,1H).

[0059] Embodiment 1:

[0060] S1. Synthesis of deoxyuridylic acid analogs (1)

[0061] Weigh cuprous iodide (44.6 mg, 0.235 mmol, 0.05 equiv), raw material 5-iodo-2′-deoxyuridine (1.64 g, 4.7 mmol, 1 equiv), bistriphenylphosphine palladium dichloride (0.66 g, 0.94 mmol, 0.2 equiv), add to an eggplant-shaped bottle, use a double-row tube to replace the nitrogen three times, insert a nitrogen balloon, add 15 mL of anhydrous acetonitrile and anhydrous triethylamine, stir at 25°C for 20 minutes, weigh trimethylsilyl acetylene (1.84 g, 18.8 mmol, 4 equiv) with a disposable syringe and add to the reactor. After the reactor is heated to 50°C, the reaction is continued for 3 hours. After TLC shows that the reaction is complete, the insoluble salts and catalyst are removed by suction filtration. Separation and purification give 1.17 g of deoxyuridine analog (1) in the form of light yellow solid powder with a yield of 77%.

[0062] S2. Synthesis of deoxyuridine analogs (2)

[0063] The raw material deoxyuridine analog 1 (1.8 g, 5.55 mmol, 1 equiv) and anhydrous potassium carbonate (1.68 g, 12.2 mmol, 2 equiv) were weighed and added to an eggplant-shaped bottle, a nitrogen balloon was inserted, and 30 mL of anhydrous methanol was added, followed by stirring and reacting at 25°C. After TLC showed that the reaction was complete, the insoluble anhydrous potassium carbonate was removed by suction filtration. 0.58 g of a light yellow solid was obtained by separation and purification, with a yield of 50%.

[0064] S3, DPP derivative-modified deoxyuridine (dU DPPth )

[0065] Deoxyuridine analog 2 (0.213 g, 0.444 mmol, 1.5 equiv), cuprous iodide (0.012 g, 0.06 mmol, 0.2 equiv), and tetrakistriphenylphosphine palladium (0.035 g, 0.03 mmol, 0.1 equiv) were weighed and added to a reaction flask. 5 mL of DMF and Et 3 N 1mL, the reactor was placed at room temperature and stirred for 20 minutes. DPP derivative 3 (0.145g, 0.3mmol, 1equiv) was added, DMF was used as solvent, and the reaction was carried out at room temperature. After TLC confirmed that the reaction was complete, the insoluble matter was removed by suction. The red solid DPP derivative modified deoxyuridine (dU DPPth ) total 0.24g, yield 32%.

[0066] 1 H NMR (400 MHz, DMSO-d 6 )δ11.78(s,1H),8.77(d,J=4.1Hz,1H),8.46(s,1H),7.82(dd,J=6.6,3.1Hz,2H),7.57(dd,J=7.5,3.8Hz,4H),6.13(t,J=6.5Hz,1H) ,2.19(s,1H),1.58(t,J=7.7Hz,2H),1.38(dt,J=36.6,7.4Hz,3H),1.22–1.08(m,3H),0.89(t,J=7.3Hz,3H),0.75(t,J=7.3Hz,3H).

[0067] Example 2dU DPPth Application as probe in detection field

[0068] Although the environment inside a healthy human body is relatively stable, there are also extreme environments in some parts of the body, such as the acidic environment inside cancerous cells. How to identify the microenvironment inside somatic cells through effective and intuitive means has become a topic of concern for researchers, and nucleosides, as an indispensable part of cell life and metabolism, have become a hot topic of research. This example uses synthetic dU DPPth As a probe, its fluorescence changes under different polarity and pH environments were tested.

[0069] 1.dU DPPth Used as a probe to detect and identify polar environments

[0070] UV-visible absorption spectroscopy solution: weigh a certain amount of dU DPPth Place in a 10mL beaker and add appropriate amounts of solvents of various polarities to dissolve the solute. After complete dissolution, pour the solution into the corresponding 10mL volumetric flask. Rinse the small beaker repeatedly with various corresponding polar solvents and then pour into the corresponding volumetric flask. After the solution is diluted to 10mL, use an ultrasonic cleaner to further mix the test solution to obtain six different polarities of 25μM dU DPPth of the UV test solution.

[0071] Fluorescence emission spectrum test solution: Use a pipette to add 0.1 mL of the above 25 μM UV test solution to a 10 mL volumetric flask. When the volumetric flask is fixed to 10 mL, a solvent of corresponding polarity should be used. After shaking, use an ultrasonic cleaner to further mix to obtain six polarities of fluorescent test solutions.

[0072] (1)dU DPPth UV absorption spectra in solvents of different polarities

[0073] Ud DPPth The UV absorption spectra of six different polarities are as follows Figure 1 As shown, dU DPPth There are two absorption peaks between 300nm and 600nm. The absorption peak around 360nm is caused by π-π*, while the maximum absorption peak is caused by n-π* transition. As for the maximum absorption wavelength, it red-shifts when the polarity increases.

[0074] (2) Fluorescence emission spectra under different polarity conditions

[0075] The excitation wavelength was set to dU DPPth At the maximum absorption wavelength in different polar solvents, the fluorescence spectrum was scanned repeatedly before the emission wavelength and excitation wavelength stabilized. The obtained fluorescence spectrum data was exported and plotted using origin to obtain Figure 2 .

[0076] Fluorescence spectroscopy Figure 2As shown in , when the compound is dissolved in an organic solvent, it shows a very high fluorescence intensity. The specific data are shown in Table 1.

[0077] Table 1dU DPPth Emission wavelength and intensity in six polar solutions

[0078]

[0079] (3) Stokes shift and solvent E T (30) Relationship

[0080] The photophysical properties of the compound are affected by the polarity of the solvent, the Stokes shift value of the compound and the solvent E T The relationship between the values ​​of (30) can well describe the degree of this influence. Select the corresponding data and use origin to draw the graph to get Figure 3 , specific values ​​are shown in Table 2.

[0081] Table 2dU DPPth Stokes shift in six solvents

[0082]

[0083] Select E T (30) is the horizontal axis and the Stokes shift value is the vertical axis to obtain a scatter plot. The data of methanol, acetonitrile, dichloromethane, ethyl acetate, and dioxane were linearly fitted and the results showed that the fluorescent deoxyuridine dU DPPth It is sensitive to changes in polarity, and its Stokes shift value and solvent E T (30) is linear and R 2 =0.925.

[0084] 2.dU DPPth As a probe to detect the corresponding pH environment

[0085] As an important indicator for judging the presence of somatic cells, pH is often considered when designing probes. pH does not have much effect on the emission wavelength of fluorescent molecules, but mainly on the emission intensity.

[0086] Preparation method of different pH solutions: In order to accurately and quickly prepare different pH test solutions, the buffer solution should be prepared first. Accurately weigh 0.01mmol of phosphoric acid and sodium hydroxide, then pour them into a beaker, dissolve them with deionized water and pour them into a 100mL volumetric flask. Then wash the small beakers together and pour them into the corresponding volumetric flasks. Add deionized water to 100mL to complete the volume, and use an ultrasonic cleaner to further dissolve. Prepare 0.1mM Na in an alkaline system according to the above method. 2 HPO4 NaH in aqueous solution and acidic system 2 PO 4 When preparing a buffer solution with a pH value of 1-6, use an acidic system and use phosphoric acid solution and sodium hydroxide solution to 2 PO 4 When preparing alkaline solution, the Na 2 HPO 4 The aqueous solution is fine-tuned by adding phosphoric acid and sodium hydroxide solution. After fine-tuning, a pH meter is used to read the pH value, and the preparation is completed after the reading stabilizes.

[0087] Preparation of UV absorption spectrum test solution: Add an accurate amount of 0.01mmol of dU to a small beaker. DPPth , use deionized water to shake and dissolve it in an ultrasonic cleaner. Then pour the solution into a 10mL volumetric flask, rinse the small beaker several times and pour the cleaning solution into the volumetric flask. After the volume is fixed, use a wave cleaner to shake and mix to obtain 10mL of 1mM dU DPPth Buffer solution. Prepare 12 5 mL centrifuge tubes numbered 1 to 12, and use a pipette to draw 0.5 mL of the above solution into each tube. When the volume is adjusted to 5 mL, use a phosphate buffer solution with the pH value corresponding to the number.

[0088] Preparation of fluorescence emission spectroscopy test solution: Use a pipette to draw the above 100 μM dU DPPth 0.5 mL of each UV test solution was added to a 5 mL empty centrifuge tube with the corresponding pH number. The volume was adjusted to 5 mL using buffer solutions of various corresponding pH values, and finally 10 μM dU was obtained. DPPth of fluorescent test solution.

[0089] (1) dU at different pH values DPPth UV-Visible spectrum

[0090] The relationship between the absorption coefficient of DPP-th modified deoxyuridine and the maximum absorption wavelength and different pH conditions is shown in Figure 4 The absorption coefficient at the maximum absorption wavelength reaches its maximum value between pH 6-7, and with the increase of acidity and alkalinity, the maximum absorption wavelength will show a blue shift trend. When pH = 12, the absorption wavelength is the smallest. The absorption coefficient does not reflect a strict rule, but the absorption coefficient is significantly reduced under over-acid or over-alkaline conditions, reflecting the compound's certain sensitivity to acid and base.

[0091] (2) Fluorescence spectra under different pH conditions

[0092] The fluorescence emission spectra of the compounds at pH values ​​of 1-12 were scanned sequentially to obtain Figure 5According to Table 3, the excitation wavelength was set to 570 nm. The emission peak shift of the compound changes between 598 and 603 nm as the pH value increases. When pH = 8, the maximum emission wavelength is 603 nm. When the pH value increases or decreases from 8, the maximum emission wavelength has a slight blue shift, and the shift value is no more than 3 nm. It can be considered that the pH value has little effect on the emission wavelength. Under different pH conditions, dU DPPth The fluorescence intensity of the compound varies between 190 and 655 a.u. Similarly, under neutral conditions such as pH 6-8, the fluorescence intensity hardly changes significantly, but when the environment tends to be acidic or alkaline, the fluorescence intensity decreases, which indicates that acidic or alkaline conditions have a significant effect on the fluorescence intensity of the compound, which means that the compound can be used as a probe for pH environments.

[0093] Table 3 Emission wavelength and corresponding fluorescence intensity under pH solution

[0094]

[0095] After processing the fluorescence intensity values ​​using the normalization method, we get Figure 6 The pH value is taken as the horizontal axis and the normalized fluorescence intensity is taken as the vertical axis. It can be seen that in the pH range of 1-12, when the environment is closer to neutral, the dU DPPth The higher the fluorescence intensity, the lower the fluorescence intensity will be. When the environment is too acidic or too alkaline, this indicates that dU DPPth Sensitivity to changes in pH conditions.

[0096] The control The emission wavelength and corresponding fluorescence intensity results in different pH solutions are shown in Table 4:

[0097] Table 4

[0098]

[0099] 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.

[0100] 3. dU DPPth Fluorescence quenching effect as a probe

[0101] The quenching process is a process that competes with the luminescence process. The addition of quenchers will shorten the luminescence process of fluorescent molecules, that is, reduce the fluorescence lifetime. Due to the complex environment in the human body, fluorescent molecules must have relatively strong anti-quenching properties in order to be used in probe-level applications.

[0102] For a qualified nucleoside fluorescent probe, it must not only have the ability to recognize various environments, but also must not be quenched by the influence of natural nucleosides and lose its excellent photophysical properties. When a fluorescent nucleoside analog is inserted into an oligonucleotide, the adjacent natural base may undergo a single electron transfer with it, thereby quenching its fluorescence. DPPth It is very important to determine whether quenching occurs due to the influence of natural nucleosides.

[0103] According to the Stern-Volmer equation It was judged that the process of quenching the extended fluorescent uridine analogs using four natural nucleosides conforms to the principle of dynamic quenching.

[0104] Use [NMPs], F 0 / F Make a Stern-Volmer plot. The slope of the straight line in the plot 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 it cannot achieve the performance that a fluorescent probe should have.

[0105] Preparation of quenching solution: Use fixed concentration solutions of natural nucleosides and dU DPPth The fluorescence quenching test solution was prepared by mixing with phosphate buffer.

[0106] The preparation of phosphate buffer solution requires the use of NaOH, NaCl, H 3 PO 4 、NaH 2 PO 4 、Na 2 HPO 4 The concentration of the aqueous solution is 0.1mM. After measuring the pH of the NaCl aqueous solution, add NaH 2 Aqueous solution of PO or Na 2 HPO 4 If the difference is too large, add NaOH, H 3 PO 4 Use the above phosphate buffer to convert dU DPPth Prepare a quenching solution with a concentration of 100 μM.

[0107] Preparation of natural nucleotide (NMP) solution: Weigh 0.2 mmol each of adenosine (AMP), cytidine (CMP), guanosine (GMP) and thymidine (TMP), dissolve them in a small beaker using the above-mentioned phosphate buffer, then add them to a 10 mL volumetric flask, wash with phosphate buffer solution in small amounts several times, make up to volume, and shake evenly to obtain 20 mM natural nucleotide (NMP) phosphate buffer.

[0108] Fluorescence quenching test solution: Take 6 5mL empty centrifuge tubes and number them T0 to T5. First, add 0.5mL of the 100μM quenching solution prepared above to each empty centrifuge tube. Then, according to the sequence, add 0-2.5mL of the four natural nucleotide buffer solutions to the centrifuge tubes numbered 0-5. Use the previously prepared buffer solution to make the volume constant to obtain a concentration of 10μM dU DPPth Quench the solution to be tested. Prepare A0-A5, C0-C5 and G0-G5 in the same way.

[0109] The concentration of natural nucleosides was used as the horizontal axis, and the ratio of fluorescence intensity before and after quenching with natural nucleosides was used as the vertical axis. A scatter plot was drawn and linear fitting was performed to obtain Figure 7 .

[0110] From the analysis of the figure, adenosine (AMP) and cytosine (CMP) for dU DPPth The fluorescence intensity of dU has almost no quenching effect, while guanosine monophosphate (GMP) and thymidylic acid (TMP) have a strong quenching effect on dU DPPth The quenching effect is strong, and cytidine and dU should be avoided as much as possible when designing oligonucleotide chains. DPPth Directly connected.

[0111] The fluorescence quenching effect of the control sample was tested using the same method. The results showed that after adding the quencher, the luminescence process of the control sample was greatly affected by the four nucleotides and was not suitable as a fluorescent nucleic acid probe.

Claims

1. A deoxyuridine modified with a DPP derivative, characterized in that: The structure is as follows:

2. A method for synthesizing deoxyuridine modified with a DPP derivative according to claim 1, characterized in that: The reaction route is as follows: Compound 2 and compound 3 are used as substrates, and the target product is obtained by reaction under the action of a catalyst.

3. The preparation method according to claim 2, characterized in that: The molar ratio of compound 2 to compound 3 is (1-2):

1.

4. The preparation method according to claim 2, characterized in that: The catalyst is CuI and Pd(PPh3)4; wherein the molar ratio of compound 3 to CuI is 1:(0.1-0.5); and the molar ratio of compound 3 to Pd(PPh3)4 is 1:(0.05-0.2).

5. The preparation method according to claim 2, characterized in that: The reaction is carried out in a solvent DMF, and the amount of DMF used relative to compound 3 is 10-20 mL / mmol.

6. The preparation method according to claim 2, characterized in that: The reaction further comprises adding triethylamine, wherein the amount of triethylamine used relative to compound 3 is 2-5 mL / mmol.

7. The preparation method according to any one of claims 2 to 6, characterized in that: The preparation route of compound 2 is as follows: In step A, 5-iodo-2′-deoxyuridine and trimethylsilyl acetylene are reacted with cuprous iodide and bistriphenylphosphine palladium dichloride to obtain compound 1; In step B, compound 1 is reacted with a base to obtain compound 2.

8. The preparation method according to claim 7, characterized in that: In step A, an organic solvent and triethylamine are added, and the organic solvent used is acetonitrile; in step B, the base used is any one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

9. Use of the DPP derivative-modified deoxyuridine according to claim 1 in the preparation of detection probes.

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

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