Fluorescent nucleoside analogue oxazole [5, 4-d] uridine as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent nucleoside analogue oxazole [5, 4-d] uridine as well as a preparation method and application thereof, and belongs to the field of biological medicine and detection. The structure of the fluorescent nucleoside analogue oxazole [5, 4-d] uridine is shown in the specification, and the fluorescent nucleoside analogue oxazole [5, 4-d] uridine can be used as a fluorescent probe and has important application prospects in various fields of biology, medicine and the like, for example, the fluorescent nucleoside analogue oxazole [5, 4-d] uridine can be used as a DNA fluorescent probe, a polarity detection probe, a pH detection probe and the like. The fluorescent nucleoside analogue disclosed by the invention is wide in applicability, convenient to operate and simple in preparation process, and has wide application in the fields of medicines, pesticides, petrochemical engineering and the like. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and detection, and specifically relates to a fluorescent nucleoside analog oxazole [5,4-d] uridine, and a preparation method and application thereof. Background Art

[0002] Fluorescent nucleoside analogs, whose photophysical properties can change regularly with the changes in microenvironmental conditions, are widely used in the study of the structure and function of nucleic acids. Isostructural fluorescent nucleoside analogs, because of their structure similar to natural nucleosides, have little effect on the structure and function of the parent after being inserted into the nucleic acid sequence, and can therefore replace natural nucleosides to perform normal cellular functions. Fluorescent nucleoside analogs play an important role in the research of the entire field of nucleoside modification. Fluorescent nucleoside analogs can be obtained by extending the conjugated structure on the base or directly introducing fluorescent molecules. Because fluorescent nucleoside analogs have different photophysical properties from natural nucleosides, they play an important role in studying genetic processes in living organisms and oligonucleotide delivery processes.

[0003] Aromatic ring-expanded fluorescent nucleoside analogs are obtained by extending additional aromatic rings to natural nucleobases. This approach expands the conjugated electron system of the nucleobase, thereby red-shifting the emission wavelength of the base into the visible range, a move that generally renders the nucleoside highly fluorescent. The extended aromatic system also has additional sites for electron-donating and electron-withdrawing substituents, which facilitates the design of environmentally sensitive probes based on intramolecular charge or proton transfer reactions.

[0004] From the perspective of atom economics, the structure of natural nucleobases is changed to give them fluorescent properties while maintaining their molecular size to ensure their ability to form Watson-Crick hydrogen bonds with natural bases, so as not to affect the intramolecular and intermolecular interactions between the modified bases and natural bases. Fluorescent nucleoside analogs with this structure are called isostructural fluorescent nucleoside analogs.

[0005] However, there are currently few fluorescent nucleoside analogs that can be effectively used as probes for detection, and much needs to be improved and explored. Summary of the invention

[0006]

Technical issues

[0007] Provided are a novel fluorescent nucleoside analog and a preparation method thereof. The fluorescent nucleoside analog can be used as a probe in the field of detection and cell imaging.

[0008]

Technical solution

[0009] The present invention provides a novel fluorescent nucleoside analog, the structure of which is shown below:

[0010]

[0011] The present invention also provides a method for preparing the fluorescent nucleoside analog, the route of which is as follows:

[0012]

[0013] The present invention uses a fluorescent nucleoside derivative oxazole [5,4-d] uridine (5,4-d) prepared from ethyl 5-aminooxazole-4-carboxylate as a raw material. oz U). 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, and high yield.

[0014] In one embodiment of the present invention, the preparation method comprises the following steps:

[0015] S1, reacting 5-aminooxazole-4-carboxylic acid ethyl ester with diphosgene and triethylamine to generate 5-ureidooxazole-4-carboxylic acid ethyl ester; then, 5-ureidooxazole-4-carboxylic acid ethyl ester is cyclized in an alkaline environment to obtain oxazolo[5,4-d]pyrimidine-5,7(4H,6H)dione;

[0016] S2, using oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione and 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose as raw materials, reacting to obtain tribenzoyloxyuridine; then deprotecting under the action of an alkaline reagent to obtain a fluorescent nucleoside analogue oxazolo[5,4-d]uridine, denoted as oz U.

[0017] In one embodiment of the present invention, in S1, the molar ratio of ethyl 5-aminooxazole-4-carboxylate to diphosgene is 1:(1.5-3.0), and specifically 1:2.

[0018] In one embodiment of the present invention, in S1, the molar ratio of ethyl 5-aminooxazole-4-carboxylate to triethylamine is 1:(5-8), and specifically 1:6.

[0019] In one embodiment of the present invention, in S1, the reaction of 5-aminooxazole-4-carboxylic acid ethyl ester with diphosgene to generate 5-ureidoxazole-4-carboxylic acid ethyl ester is carried out in an organic solvent, and the organic solvent is dichloromethane.

[0020] In one embodiment of the present invention, in S1, during the process of generating 5-ureidoxazole-4-carboxylic acid ethyl ester from 5-aminooxazole-4-carboxylic acid ethyl ester with diphosgene, the reaction temperature is -20°C to 5°C, and the reaction time is 1-3h.

[0021] In one embodiment of the present invention, the base used in the alkaline environment in S1 is any one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, potassium tert-butoxide, and sodium tert-butoxide.

[0022] In one embodiment of the present invention, the cyclization in S1 is carried out in an organic solvent, and the organic solvent is anhydrous ethanol.

[0023] In one embodiment of the present invention, the reaction temperature of the cyclization in S1 is 60-90°C, and can be 80°C.

[0024] In one embodiment of the present invention, after the cyclization described in S1 is completed, a yellow insoluble precipitate appears in the solution, which is stirred in an ice bath, and concentrated hydrochloric acid is slowly added dropwise during the process to adjust the pH to 3-4; the insoluble matter is then filtered off and dried to obtain an off-white solid oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione.

[0025] In one embodiment of the present invention, in S2, the molar ratio of oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose is 1:(0.8-1.5), and specifically 1:1.1.

[0026] In one embodiment of the present invention, in S2, the process of obtaining tribenzoyloxyuridine by reaction also includes adding promoters N,O-bistrimethylsilylacetamide (N,O-BSA) and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

[0027] In one embodiment of the present invention, the molar ratio of oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to N,O-BSA is 1:(1.5-2.5). Specifically, it can be 1:2.

[0028] In one embodiment of the present invention, the molar ratio of oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to TMSOTf is 1:(1.0-1.5), and specifically 1:1.2.

[0029] In one embodiment of the present invention, in S2, during the reaction to obtain tribenzoyloxyuridine, the reaction temperature is 60-80°C and the reaction time is 8-15 hours, specifically, the reaction can be carried out at 70°C for 12 hours.

[0030] In one embodiment of the present invention, in S2, the process of obtaining tribenzoyloxyuridine by reaction is carried out in an organic solvent, and the organic solvent is acetonitrile.

[0031] In one embodiment of the present invention, in S2, the alkaline reagent used in the deprotection process is ammonia gas.

[0032] In one embodiment of the present invention, in S2, the deprotection is carried out in an organic solvent, and the organic solvent is methanol.

[0033] In one embodiment of the present invention, in S2, the deprotection reaction temperature is 50-80°C and the reaction time is 5-15 hours. Specifically, the reaction temperature can be 60°C for 8 hours.

[0034] The present invention also provides the use of the fluorescent nucleoside analog in the preparation of detection probes.

[0035] The present invention also provides the use of the fluorescent nucleoside analogs in environmental polarity detection, environmental pH detection, DNA fluorescent probes and cell fluorescence imaging.

[0036] Beneficial effects:

[0037] The present invention relates to a novel fluorescent nucleoside derivative and a preparation method thereof. Such compound 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

[0038] Figure 1 For the present invention oz UV-visible spectra of U in six solvents (c=1 mM).

[0039] Figure 2 For the present invention oz Fluorescence spectra of U in six solvents (c = 10 μM).

[0040] Figure 3 For the present invention oz UV absorption spectrum of U in pH solution (c=100 μM).

[0041] Figure 4 For the present invention oz Fluorescence spectra of U in solutions with different pH values ​​(c=10 μM).

[0042] Figure 5 For the present invention oz Quenching spectrum of U (c = 5 μM). DETAILED DESCRIPTION

[0043] The following are specific embodiments of the present invention.

[0044] The following is the synthesis route of the present invention, as shown in the following formula, and the synthesis is carried out in two steps:

[0045]

[0046] The first step is to prepare oxazolo[5,4-d]uridine oz U.

[0047] 5-aminooxazole-4-carboxylic acid ethyl ester is added in a three-necked flask, dichloromethane is added as solvent under a nitrogen atmosphere, and stirring and dissolving is performed at room temperature, and triethylamine is added during the process. After dissolving, the reactor is placed in a constant temperature ice salt bath, and diphosgene is added dropwise using a constant pressure dropping funnel, and reacts for a period of time, and the reaction time is 6-8 hours. Finally, ammonia is slowly added dropwise to quench. Filter, wash, extract, through column chromatography, purification and separation obtain intermediate product 5-urea-based oxazole-4-carboxylic acid ethyl ester.

[0048] Place 5-ureidoxazole-4-carboxylic acid ethyl ester and sodium hydroxide in a three-necked flask, add anhydrous ethanol as a solvent under a nitrogen atmosphere, and react at 80°C. After the reaction is completed, a yellow insoluble precipitate appears in the solution. Place the three-necked flask in an ice bath and stir, and add concentrated hydrochloric acid dropwise to adjust the pH to 3-4. Filter the insoluble matter, dry it in a vacuum oven, and obtain an off-white solid oxazolo[5,4-d]pyrimidine-5,7(4H,6H)dione.

[0049] Step 2: Preparation of oxazolo[5,4-d]uridine analogs oz U.

[0050] Add oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to a three-necked flask. Add acetonitrile as solvent under nitrogen protection, add N,O-BSA dropwise, heat to 60°C and stir until the reaction solution is clear. Then add 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-furanose under nitrogen atmosphere and stir to dissolve, add trimethylsilyl trifluoromethanesulfonate dropwise, heat the reaction to 70°C, react for 12 hours, and after the reaction stops, remove the solvent by rotary evaporation; separate and purify by column chromatography to obtain the intermediate product tribenzoyloxyuridine. Put tribenzoyloxyuridine in a pressure-resistant tube and add 20mL of methanol to dissolve it. Then put the pressure-resistant tube in an ice bath and stir while passing ammonia. At this time, ensure that the vent tube is always below the liquid level to prevent methanol from being sucked back. After half an hour, seal the bottle stopper tightly, slowly warm it to room temperature, and then heat it to 60°C to react overnight. After the reaction, the product oxazole [5,4-d] uridine analogue was obtained by column chromatography separation and purification as a light yellow powder. oz U.

[0051] The intermediate (ethyl 5-aminooxazole-4-carboxylate) can be prepared by the following method:

[0052] Preparation of ethyl 2-oxime cyanoacetate: To an aqueous solution of ethyl cyanoacetate (3.00 g, 30.0 mmol, 1 equiv) and sodium nitrite (2.48 g, 36.0 mmol, 1.2 equiv) was added dropwise acetic acid (2.5 mL) at 0-5°C, stirred for 1 hour, and the resulting mixture was allowed to react at room temperature for 1 hour. The ratio of V 石油醚 :V乙酸乙酯 =1:1 developing solvent, after confirming the completion of the reaction by TLC, the obtained mixture was extracted with petroleum ether and the organic layer was concentrated to dryness. The residue obtained by rotary evaporation was washed with cyclohexane and filtered to obtain 3.60 g of light yellow solid, with a yield of 93.8%.

[0053] Preparation of ethyl 2-amino-2-cyanoacetate: Dissolve ethyl 2-oximecyanoacetate (10.00 g, 78.0 mmol, 1 equiv) in a mixed solution of water (240.0 mL) and ethanol (110.0 mL) and stir to dissolve. Add sodium dithionite (61.11 g, 351.0 mmol, 4.5 equiv) in batches at room temperature and then heat to 50°C with stirring. Use a ratio of V 石油醚 :V 乙酸乙酯 = 1:1 developing solvent for TLC. After the reaction was completed, the mother liquor was extracted with dichloromethane, the organic phase was concentrated after separation, and the insoluble matter was removed by filtration to finally obtain 7.35 g of ethyl 2-amino-2-cyanoacetate in the form of a red oil with a yield of 82.6%. 1 H NMR (400MHz, CDCl3) δ4.41 (s, 1H), 4.24 (q, J = 7.2Hz, 1H), 2.05 (s, 2H), 1.27 (t, J = 7.2Hz, 2H).

[0054] Preparation of ethyl 2-cyano-2-formamidoacetate: Formic acid (2.3 g, 50 mmol, 2 equiv) and acetic anhydride (5.1 g, 50 mmol, 2 equiv) were mixed, heated and stirred at 55°C for 2 h to prepare formic acid acetic anhydride. Ethyl 2-amino-2-cyanoacetate (3.2 g, 25 mmol, 1 equiv) was added to a three-necked flask, replaced with nitrogen, and then tetrahydrofuran was added as a solvent. The formic acid acetic anhydride prepared previously was slowly added dropwise at 0°C, and then the ice bath was removed and the reaction was carried out at room temperature for 8 hours. The ratio of V 二氯甲烷 :V 甲醇 =10:1 developing solvent for TLC. After the reaction is completed, tetrahydrofuran is removed by rotary evaporation. The mother liquor is dissolved in ethyl acetate and washed with water to remove excess formic acid and acetic anhydride. After separation using a separatory funnel, the aqueous phase is discarded, the organic phase is collected and dried over anhydrous magnesium sulfate, and allowed to stand at room temperature. After 3 hours, the anhydrous magnesium sulfate is removed by suction filtration. The solvent is removed by rotary evaporation, and the temperature is lowered to crystallize to obtain 3.2 g of a red needle-shaped solid with a yield of 82%. 1 H NMR (400MHz, CDCl3) δ1.26 (t, J = 7.2Hz, 3H), 4.26 (q, J = 7.1Hz, 2H), 5.45 (d, J = 7.7Hz, 1H), 7.83 (d, J = 7.7Hz, 1H), 8.22 (s, 1H).

[0055] Preparation of ethyl 5-aminooxazole-4-carboxylate: Weigh the above ethyl 2-cyano-2-formamidoacetate (3.06 g, 20 mmol, 1 equiv), add it to a three-necked flask, and replace it with nitrogen three times. Add dichloromethane as a solvent under nitrogen atmosphere, stir and dissolve at room temperature, put the reactor into an ice bath pot and continue stirring, then slowly add trifluoroacetic acid (6.84 g, 60 mmol, 3 equiv) dropwise. The reaction is carried out for 3 hours. After the addition is completed, remove the ice bath and keep at room temperature. The ratio of use is V 石油醚 :V 乙酸乙酯 =1:1 developing solvent for TLC. After the reaction was completed, dichloromethane was removed by rotary evaporation, the obtained material was placed in an ice bath, and ammonia water was slowly added under stirring to adjust the pH of the system to 7. Insoluble solids appeared, and after suction filtration, a yellow powder solid was dried in a vacuum drying oven, with a yield of 68.6%. 1 H NMR (400MHz, Acetonitrile-d3) δ7.31 (s, 1H), 6.01 (s, 2H), 4.23 (p, J = 7.5Hz, 3H), 1.27 (t, J = 7.1Hz, 3H).

[0056] Example 1: Synthesis of oxazolo[5,4-d]pyrimidine-5,7(4H,6H)dione

[0057] 5-aminooxazole-4-carboxylic acid ethyl ester (0.78 g, 5 mmol, 1 equiv) was added to a three-necked flask and replaced with nitrogen three times. 20 mL of dichloromethane was added as a solvent under a nitrogen atmosphere, and the mixture was dissolved by stirring at room temperature. During the process, triethylamine (3.03 g, 30 mmol, 6 equiv) was added. After dissolution, the reactor was placed in a constant temperature ice salt bath, and 20 mL of a dichloromethane solution of diphosgene (2.96 g, 10 mmol, 2 equiv) was slowly added dropwise using a constant pressure dropping funnel. The ratio of use was V 石油醚 :V 乙酸乙酯 =1:2 developing solvent for TLC. After the reaction product disappears, slowly add ammonia water to quench, and continue stirring for 1 hour in an ice bath. Collect the reaction solution and remove the solvent by rotary evaporation. Column chromatography (eluent: V 石油醚 :V 乙酸乙酯 =1:1) to obtain white powdery solid 5-ureidoxazole-4-carboxylic acid ethyl ester. Then, 5-ureidoxazole-4-carboxylic acid ethyl ester (200 mg, 1 mmol, 1 equiv) and sodium hydroxide (40 mg, 2 mmol, 2 equiv) were weighed in a three-necked flask, and the atmosphere was replaced with nitrogen three times. Anhydrous ethanol was added as a solvent under nitrogen atmosphere, and the reactor was sealed with a reverse stopper, and the temperature was set to 80°C for reaction. The ratio of V 石油醚 :V 乙酸乙酯=1:2 developing solvent. After the reaction is completed, a yellow insoluble precipitate appears in the solution. Place the three-necked flask in an ice bath and stir, slowly add concentrated hydrochloric acid to adjust the pH to 3-4. At this time, there are still insolubles. Filter the insolubles and dry them in a vacuum oven to obtain 150 mg of off-white solid oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione. The total yield is 23%.

[0058] NMR data: 1 H NMR (400MHz, DMSO-d6) δ9.71(s,1H),10.99(s,1H),8.24(s,1H).

[0059] Example 2: Oxazolo[5,4-d]uridine analogs oz Synthesis of U

[0060] Add oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione (150 mg, 1 mmol, 1 equiv) to a 50 mL eggplant-shaped bottle, replace the air with nitrogen using a double-row tube, add acetonitrile (20 mL) under a nitrogen atmosphere, and then seal the bottle mouth with a reverse stopper and stir. Add N,O-BSA (400 mg, 2 mmol, 2 equiv) dropwise, and after the addition is completed, heat to 60 °C and stir until the reaction solution is clear. Then add 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-furanose (555.4 mg, 1.1 mmol, 1.1 equiv) under a nitrogen atmosphere and stir to dissolve, then use a syringe to weigh trimethylsilyl trifluoromethanesulfonate (TMSOTf) (226.7 mg, 1.2 mmol, 1.2 equiv). Heat the reaction to 70 °C for 12 hours, using a ratio of V 二氯甲烷 :V 甲醇 =10:1 developing solvent for TLC. After showing that the reactants have disappeared, cool to room temperature and remove the solvent by rotary evaporation. Column chromatography (eluent is V dichloromethane: V methanol = 80:1) gives tribenzoyloxyuridine, a white sugar-like solid compound. Then put tribenzoyloxyuridine (179 mg, 0.3 mmol, 1 equiv) into a pressure tube and add 20 mL of methanol to dissolve it. Then put the pressure tube into an ice bath and stir while passing ammonia. At this time, ensure that the vent tube is always below the liquid level, and pay attention to the backflow of methanol. After half an hour, quickly seal the bottle stopper, slowly warm it to room temperature, and then heat it to 60°C for reaction overnight. After the reactor is cooled to room temperature, use a ratio of V 二氯甲烷 :V 甲醇 =10:1 developing solvent for TLC. The solvent was removed by rotary evaporation and column chromatography (eluent: V 二氯甲烷 :V 甲醇 =100:1~V 二氯甲烷 :V 甲醇=10:1), and a pale yellow powdery substance oxazole [5,4-d] uridine analogue was obtained. oz U. The total yield was 58%.

[0061] NMR data: 1 H NMR (400MHz, DMSO-d6) δ8.35(s,1H),6.62(s,1H),5.46(s,1H),5.32(s,1H),4.53(s,1H),4.45(s,2H),3.48(d,J=6.2Hz,2H),3.42(d,J=5.8Hz,2H).

[0062] Example 3 Application of oxazolo[5,4-d]uracil as a probe in the detection field

[0063] 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 oxazole [5,4-d] uridine ( OZ U) was used as a probe to test its fluorescence changes under different polarities, pH values ​​and environments.

[0064] 1. Oxazole [5,4-d] uracil ( oz U) As a probe to specifically detect and recognize polar environments

[0065] UV-visible absorption spectroscopy solution: weigh 0.01 mmol oxazolidinone [5,4-d] uracil oz U is placed in a 10mL beaker, and appropriate amounts of solvents of various polarities are added to dissolve the solute. After complete dissolution, the solution is poured into the corresponding 10mL volumetric flask. The small beaker is repeatedly rinsed with various corresponding polar solvents, and then poured into the corresponding volumetric flask. After the solution is diluted to 10mL, the test solution is further mixed with an ultrasonic cleaner to obtain six 1mM UV test solutions of different polarities.

[0066] Fluorescence emission spectrum test solution: Use a pipette to add 0.1 mL of the above 1 mM 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 10 μM fluorescent test solutions of six polarities.

[0067] (1) oz UV absorption spectra of U in different polar solvents

[0068] like Figure 1 As shown, C=N and C=C conjugation leads tooz U produces an n→π* transition at around 300nm, resulting in a strong absorption peak. The solvation effect causes the maximum absorption wavelength and absorbance to change with the polarity of the solvent. In general, as the polarity increases, oz The maximum absorption wavelength of U is slightly red-shifted, ranging from 300nm to 310nm, of which the maximum absorption wavelength in water is 310nm, while the maximum absorption wavelength in dichloromethane is 300nm.

[0069] (2) oz Fluorescence emission spectra of U in different solvents

[0070] Use a fluorescence spectrophotometer, set 600nm as the excitation wavelength to scan the absorption spectrum to obtain the maximum absorption wavelength, and then scan the fluorescence spectrum with the obtained absorption wavelength to obtain the emission wavelength. This cycle is repeated many times until the two values ​​tend to be stable and no longer change. At this time, the maximum wavelength of the absorption spectrum is used as the excitation wavelength for scanning. The final result is as follows Figure 2 shown.

[0071] oz U has good emission properties in solvents of different polarities. The change in emission intensity of fluorescent base analogs does not form a regular relationship with the change in polarity, but in organic solvents, its emission intensity is relatively high. Combined with the test data in Table 1, it is found that the oxazole [5,4-d] uracil base exhibits a higher emission wavelength in solvents with greater polarity, reaching about 430nm. In organic solvents with lower polarity, its emission wavelength is smaller, concentrated at 410nm.

[0072] Table 1 Stokes shift and solvent E T (30)

[0073]

[0074] As shown in Table 2, oz The fluorescence yield of U in dichloromethane and acetonitrile is very high, while the yield in methanol and ethyl acetate is lower, which indicates that in low polarity solvents, oz U has better fluorescence performance. Comparison of the fluorescence yield differences in solvents of different polarity shows that oz U can be used as a fluorescent reporter to distinguish certain low-polarity solvents (such as dichloromethane and acetonitrile).

[0075] Table 2 Quantum yields of compounds in six solvents

[0076]

[0077]

[0078] 2. Oxazole [5,4-d] uracil ( oz U) as a probe to detect the pH environment

[0079] 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.

[0080] Method for preparing solutions of different pH values: First, prepare the buffer solution. 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 beaker together and pour them into the corresponding volumetric flasks. Add deionized water to 100mL to complete the volume adjustment, and use an ultrasonic cleaner to further dissolve. According to the above method, prepare a 0.1mM Na2HPO4 aqueous solution under an alkaline system and a NaH2PO4 aqueous solution under an acidic system. 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 fine-tune the NaH2PO4 aqueous solution. When preparing an alkaline solution, add phosphoric acid and sodium hydroxide solution to the Na2HPO4 aqueous solution for fine-tuning. After fine-tuning, use a pH meter to read the reading, and complete the configuration after the reading stabilizes.

[0081] Preparation of UV absorption spectrum test solution: Add an accurate amount of 0.01mmol of oz U, use deionized water to shake and dissolve it in an ultrasonic cleaner. Then pour the solution into a 10mL volumetric flask, wash 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 oxazole [5,4-d] uridine buffer solution. Prepare 12 5mL centrifuge tubes numbered 1 to 12, use a pipette to draw 0.5mL of the above solution and add it into them. When it is fixed to 5mL, use a phosphate buffer solution with a pH value corresponding to the label.

[0082] Preparation of fluorescence emission spectrum test solution: Use a pipette to draw 0.5 mL of each of the above 100 μM oxazole [5,4-d] uridine UV test solutions of different pH values, and then add them to the 5 mL empty centrifuge tubes with the corresponding pH numbers. Use buffer solutions of various corresponding pH values ​​to make it equal to 5 mL, and finally obtain 10 μM oxazole [5,4-d] uridine fluorescence test solution.

[0083] (1) Different pH values oz UV-Vis spectrum of U

[0084] The UV absorption spectra of the compounds in different pH solutions are shown in Figure 3As shown. It can be seen that there is an absorption peak around 270nm, but the absorption intensity is not high. The effect of pH change on the maximum absorption wavelength can be ignored, and its influence is mainly reflected in the oz The absorbance of U changes in magnitude, but since the absorption peak around 270nm is difficult to identify, it is difficult to see the pattern of change.

[0085] (2) oz Fluorescence spectra of U under different pH conditions

[0086] Since the maximum absorption wavelength is difficult to confirm from the UV absorption spectrum, when measuring the fluorescence spectrum, a larger wavelength is first selected to reverse scan the absorption spectrum to obtain its maximum absorption wavelength, and then the wavelength is used as the excitation wavelength to scan the emission spectrum. Figure 4 .

[0087] Table 3 Emission wavelength and intensity of compounds in different pH solutions

[0088]

[0089] The reference substances mentioned in the present invention all refer to 1-β-D-arabino-pyrrolo[3,2-D]pyrimidine-2,4(3H,5H)-dione, which has the structure (See existing reportsSynthesis of5,7-disubstituted-4-β-D-ribofuranosylpyrazolo[4,3-d]-pyrimidines and2,4-disubstituted-1-β-D-ribofuranosylpyrrolo[3,2-d]-pyrimidines as congeners of uridine andcytidine.J Heterocycl Chem.1992,2,29,343-354.).

[0090] By comparison, it was found that the emission wavelength (401±5nm) of oxazolidinone [5,4-d] uracil (ozU) under the same pH conditions is much larger than that of 1-β-D-arabino-pyrrolo [3,2-D] pyrimidine-2,4 (3H, 5H) -dione (270±3nm) reported in the literature, and is in the violet region of the optical spectrum (380-450nm), showing purple fluorescence, and can be used as a fluorescence detection probe; while the reference substance does not have visible fluorescence, and the fluorescence intensity of the reference substance is much weaker than that of the target product of the present invention, and cannot be used as an effective fluorescence probe. Combined with Table 3, as the pH changes, ozThe maximum emission wavelength of U does not change much between 1 and 12. The compound has a relatively good fluorescence intensity under neutral conditions, and as the pH of the solution changes to acidic and alkaline, the fluorescence intensity has a relatively obvious downward trend. This shows oz U can be used to detect pH.

[0091] 3. Oxazole [5,4-d] uracil ( oz U) Fluorescence quenching effect as a probe

[0092] 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.

[0093] 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 fluorescent nucleoside analogs are inserted into oligonucleotides, the adjacent natural bases may undergo single electron transfer with them, thereby quenching their fluorescence. oz It is very important to determine whether U will be affected by natural nucleosides and undergo quenching.

[0094] Preparation of phosphate buffer solution: Use aqueous solutions of NaOH, NaCl, H3PO4, NaH2PO4, and Na2HPO4, all with a concentration of 0.1 mM. After measuring the pH of the NaCl aqueous solution, if the pH is not far from 7.4, add an aqueous solution of NaH2PO or an aqueous solution of Na2HPO4 to make fine adjustments based on the acidity and alkalinity. If the difference is too large, add NaOH or H3PO4 solution first to make fine adjustments. Use the above phosphate buffer solution to oz U is prepared into a quenching solution with a concentration of 100 μM.

[0095] Natural nucleotides (NMP) include adenosine (AMP), cytidine (CMP), guanosine (GMP) and thymidine (TMP). Preparation of natural nucleotide (NMP) solution: Weigh 0.2mmol of each natural nucleotide (NMP), dissolve them in a small beaker using the above phosphate buffer, then add them to a 100mL volumetric flask, wash, dilute and shake evenly according to the above method to obtain a 2mM natural nucleotide phosphate buffer.

[0096] Fluorescence quenching test solution: Take 6 5mL empty centrifuge tubes and add 0.5mL of 100μM quenching solution to each empty centrifuge tube. Then add 0-2.5mL of four natural nucleotide buffer solutions to the centrifuge tubes respectively. Use the previously prepared buffer solution to make the volume constant to obtain a quenching test solution with a concentration of 10μM oxazole [5,4-d] uracil (ozU).

[0097] According to the Stern-Volmer equation The process of quenching the extended fluorescent uridine analogs using four natural nucleosides conforms to the principle of dynamic quenching. The Stern-Volmer plot is made using [NMPs] and F0 / F. 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.

[0098] The present invention oz The quenching curves of U and four natural nucleosides are shown in Figure 5 As shown, in the presence of four natural nucleosides, oz The quenching effect of the fluorescence intensity of U is relatively small. oz U can be used as a probe to be inserted into oligonucleotides to achieve fluorescence detection, such as cell imaging.

[0099] The fluorescence quenching effect of the reference substance 1-β-D-arabino-pyrrolo[3,2-D]pyrimidine-2,4(3H,5H)-dione was tested using the same method. By comparison, it was found that after the addition of the quencher, the luminescence process of 1-β-D-arabino-pyrrolo[3,2-D]pyrimidine-2,4(3H,5H)-dione was greatly affected, the fluorescence lifetime was significantly shortened, and it could not be selected as a good probe application.

Claims

1. A novel fluorescent nucleoside analogue, characterized in that: The structure is as follows:

2. A method for preparing the fluorescent nucleoside analogue according to claim 1, characterized in that: The route looks like this:

3. The preparation method according to claim 2, characterized in that: The steps include: S1, reacting 5-aminooxazole-4-carboxylic acid ethyl ester with diphosgene and triethylamine to generate 5-ureidooxazole-4-carboxylic acid ethyl ester; then, 5-ureidooxazole-4-carboxylic acid ethyl ester is cyclized in an alkaline environment to obtain oxazolo[5,4-d]pyrimidine-5,7(4H,6H)dione; S2. Oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione and 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose are used as raw materials to react to obtain tribenzoyloxyuridine; then, deprotection is carried out under the action of an alkaline reagent to obtain a fluorescent nucleoside analog oxazolo[5,4-d]uridine.

4. The preparation method according to claim 3, characterized in that: In S1, the molar ratio of ethyl 5-aminooxazole-4-carboxylate to diphosgene is 1:(1.5-3.0); the molar ratio of ethyl 5-aminooxazole-4-carboxylate to triethylamine is 1:(5-8).

5. The preparation method according to claim 3, characterized in that: The alkali used in the alkaline environment in S1 is any one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, potassium tert-butoxide, and sodium tert-butoxide.

6. The preparation method according to claim 3, characterized in that: In S2, the molar ratio of oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose is 1:(0.8-1.5).

7. The preparation method according to claim 3, characterized in that: In S2, in S2, the process of obtaining tribenzoyloxyuridine by reaction also includes adding promoters N,O-BSA and TMSOTf; wherein the molar ratio of oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to N,O-BSA is 1:(1.5-2.5), and the molar ratio of oxazolo[5,4-d]pyrimidine-5,7(H,6H)dione to TMSOTf is 1:(1.0-1.5).

8. The preparation method according to any one of claims 3 to 7, characterized in that: In S2, the alkaline reagent used in the deprotection process is ammonia; the deprotection is carried out in an organic solvent, and the organic solvent is methanol.

9. Use of the fluorescent nucleoside analogue according to claim 1 in the preparation of detection probes.

10. Use of the fluorescent nucleoside analogue according to claim 1 in environmental polarity detection, environmental pH detection, DNA fluorescent probe and cell fluorescence imaging.

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