Novel extended thiazole [4, 5-d] uridine analogue as well as synthesis and application thereof

Abstract

The invention discloses a novel extended thiazole [4, 5-d] uridine analogue as well as synthesis and application thereof, and belongs to the field of biological medicine and detection. The invention provides a thiazole [4, 5-d] uridine analogue with the following structure, and the thiazole [4, 5-d] uridine analogue has excellent fluorescent property, can be used as a fluorescent probe, such as a polarity detection probe, a pH detection probe, a DNA fluorescent probe and the like, and is widely applied to the fields of biochemistry, medicine and the like. # imgabs0 #

Description

Technical Field

[0001] The invention discloses a novel extended thiazole [4,5-d] uridine analogue, and synthesis and application thereof, and belongs to the field of biomedicine and detection. Background Art

[0002] The research work of nucleic acids mainly relies on technical means such as fluorescence spectroscopy, electrophoresis, nuclear magnetic resonance and X-ray single crystal diffraction. Among them, fluorescence spectroscopy is favored by researchers for its simple operation, high sensitivity and strong selectivity, and has been widely used in the conformational transition process of nucleic acids and their conception dynamics. 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 exert normal cellular functions. According to the requirements of ideal fluorescent nucleosides, the present invention designs and synthesizes this type of isostructural fluorescent nucleoside analogs, and obtains the synthesis conditions of this type of base and its nucleoside precursor by exploring the synthesis method of cytidine analogs.

[0003] Natural nucleosides have almost no emission, so it is necessary to synthesize nucleoside analogs with fluorescent properties and then use their optical signals to study changes in the cellular microenvironment and achieve various detection purposes. Summary of the invention

[0004]

Technical issues

[0005] Provided are a novel thiazole [4,5-d] uridine analog and a preparation method thereof. The thiazole [4,5-d] uridine analog can be used as a probe in various detection and cell imaging fields.

[0006]

Technical solution

[0007] The present invention provides a thiazole [4,5-d] uridine analog, the structure of which is shown below:

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned thiazole [4,5-d] uridine analog, and the reaction scheme thereof is as follows:

[0010]

[0011] The present invention uses 4-amino-5-ethylformyl thiazole as a raw material, and obtains the corresponding ring-extended nucleoside derivative through a ring-forming reaction under the action of diphosgene. After the ring-forming reaction, the present invention generates the target product in two steps, and removes the protective group on the hydroxyl group through ammonia. The reaction conditions are relatively simple and controllable, no by-products are generated, and the yield is high.

[0012] In one embodiment of the present invention, the preparation method comprises the following steps:

[0013] (1) 4-amino-5-carboxylic acid ethyl thiazole reacts with diphosgene and ammonia water to obtain 4-urea-5-carboxylic acid ethyl thiazole;

[0014] (2) Then, 4-urea-5-carboxylic acid ethyl ester thiazole undergoes a cyclization reaction under the action of a base to obtain thiazo[4,5-d]pyrimidine-5,7-dione;

[0015] (3) Thiazolo[4,5-d]pyrimidine-5,7-dione reacts with 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose to obtain tribenzoyloxyuridine;

[0016] (4) Tribenzoyloxyuridine is deprotected under the action of an alkaline reagent to obtain a thiazolyl[4,5-d]uridine analog, denoted as ThU.

[0017] In one embodiment of the present invention, in step (1), the molar ratio of 4-amino-5-ethylcarboxylate thiazole to diphosgene is 1:(1.0-1.5).

[0018] In one embodiment of the present invention, in step (1), the amount of ammonia water relative to 4-amino-5-ethylcarboxylate thiazole and ammonia water is 2-4 mL / mmol, and specifically 2.8 mL / mmol.

[0019] In one embodiment of the present invention, in step (1), the reaction is carried out in an organic solvent, and the organic solvent is dichloromethane.

[0020] In one embodiment of the present invention, in step (1), the reaction temperature is 0-5°C and the reaction time is 1-3h.

[0021] In one embodiment of the present invention, in step (2), the base is selected from 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, in step (2), the reaction is carried out in a solvent, and the solvent is any one of ethanol and water or a combination of both.

[0023] In one embodiment of the present invention, in step (2), the reaction temperature is 60-90° C. and the reaction time is 1-3 h.

[0024] In one embodiment of the present invention, in step (3), the molar ratio of thiazolo[4,5-d]pyrimidine-5,7-dione to 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose is 1:(1.0-1.5).

[0025] In one embodiment of the present invention, in step (3), the reaction further comprises adding promoters N,O-bistrimethylsilylacetamide (N,O-BSA) and trimethylsilyl trifluoromethanesulfonate (TMSOTf).

[0026] In one embodiment of the present invention, in step (3), the molar ratio of thiazolo[4,5-d]pyrimidine-5,7-dione to N,O-BSA is 1:(2-5), and specifically 1:3.

[0027] In one embodiment of the present invention, in step (3), the molar ratio of thiazolo[4,5-d]pyrimidine-5,7-dione to TMSOTf is 1:(1.0-1.5).

[0028] In one embodiment of the present invention, in step (2), the reaction is carried out in an organic solvent, and the organic solvent is acetonitrile.

[0029] In one embodiment of the present invention, in step (3), the 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.

[0030] In one embodiment of the present invention, in step (4), the alkaline reagent is ammonia gas.

[0031] In one embodiment of the present invention, in step (4), the deprotection is carried out in methanol solvent.

[0032] In one embodiment of the present invention, in step (4), the deprotection temperature is 50-80°C, the time is 20-30h, and the reaction is carried out at 60°C for 24h.

[0033] The present invention also provides the use of the above thiazole [4,5-d] uridine analog in the preparation of detection probes.

[0034] The present invention also provides the use of the thiazole [4,5-d] uridine analog in environmental polarity detection, environmental pH detection, DNA fluorescent probe and cell fluorescence imaging.

[0035] Beneficial effects:

[0036] The present invention relates to a thiazole [4,5-d] uridine analogue and a preparation method thereof. The thiazole [4,5-d] uridine analogue has excellent fluorescence properties and can be used as a fluorescent probe, such as a polarity detection probe, a pH detection probe, a DNA fluorescent probe, etc., and is widely used in the fields of biochemistry and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the UV absorption spectrum of thiazolyl[4,5-d]uridine analogs (c = 10 μM).

[0038] Figure 2 Figure 5 is the fluorescence emission spectrum of thiazolyl[4,5-d]uridine analogs (c = 5 μM).

[0039] Figure 3 The UV absorption spectra of thiazolyl[4,5-d]uridine analogs in solutions with different pH values ​​(c = 100 μM).

[0040] Figure 4 Figure 2 is the fluorescence spectra of thiazolyl[4,5-d]uridine analogs in solutions with different pH values ​​(c = 5 μM).

[0041] Figure 5 Figure 2 is the fluorescence intensity-pH curve of thiazolyl[4,5-d]uridine analogs (c = 5 μM).

[0042] Figure 6 The quenching spectrum of thiazolyl[4,5-d]uridine analogs (c = 5 μM). DETAILED DESCRIPTION

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

[0044] The following formula is the synthetic route of the present invention, as shown below:

[0045]

[0046] Under nitrogen atmosphere, add 4-amino-5-carboxylic acid ethyl thiazole to an eggplant-shaped bottle, and then slowly add diphosgene and ammonia water at -10°C to -30°C to obtain 4-urea-5-carboxylic acid ethyl thiazole;

[0047] Then, NaOH was added to continue the reaction. After the reaction was completed, HCl was added to adjust the pH to neutral, and the intermediate product thiazo[4,5-d]pyrimidine-5,7-dione was separated and purified;

[0048] Thiazolo[4,5-d]pyrimidine-5,7-dione is dissolved in acetonitrile, N,O-BSA, 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose and trimethylsilyl trifluoromethanesulfonate are added, the reaction is completed, and then the acetonitrile is removed and purified to obtain tribenzoyloxyuridine;

[0049] Tribenzoyloxyuridine was added into a 50 mL pressure tube, and 20 mL of anhydrous methanol was added. The mixture was stirred at room temperature, and ammonia was introduced for 1 hour. The mixture was heated to 65° C. and reacted at this temperature for 12 hours. Finally, the final product was separated and purified.

[0050] The substrate 4-amino-5-ethylformate thiazole of the present invention can be prepared by the following method:

[0051]

[0052] Synthesis of cyanoimide disulfide potassium salt: Weigh 4.2g (0.1mol) cyanamide and 4mL ethanol, stir until dissolved, and then use a syringe to extract 8.36g (0.11mol) carbon disulfide and add it to the reaction solution. The above mixture is cooled to 0°C in an ice salt bath prepared in advance, and 22.44g (0.4mol) potassium hydroxide is dissolved in 36mL ethanol and slowly dripped in, while stirring rapidly. The reaction is carried out below 10°C. After the addition is completed, the reaction is continued at room temperature for 12h. Then the reaction solution is filtered, rinsed with ice ethanol in small amounts several times, and vacuum dried at 45°C to obtain 12.2g of yellow-green solid with a yield of 63%.

[0053] Synthesis of cyanoimide dithiocarboxylic acid potassium salt (2): Under nitrogen protection, 1.944 g (0.01 mmol) cyanoimide dithiocarboxylic acid potassium salt, 7 mL acetone and 8 mL water were added to a 50 mL three-necked flask. The mixed solution was cooled to below 0°C, and a solution of iodomethane (1.42 g, 0.01 mol) in acetone (3 mL) was added dropwise with rapid stirring. The mixture was reacted in an ice-water bath for 30 min, and the temperature was raised to 25°C for 3.5 h. The solvent was removed by rotary evaporation, and the residue was dissolved in acetone (20 mL). The insoluble organic salt was removed by suction filtration. The filtrate was dried by rotary evaporation and dissolved in ethyl acetate (10 mL) again. The residual potassium iodide and potassium thiocyanate that may have been formed were removed by suction filtration. The filtrate was dried by rotary evaporation and vacuum dried at 45°C to obtain 1.43 g of a milky white solid with a yield of 84%. 1 H NMR (400 MHz, DMSO) δ 2.26.

[0054] Synthesis of 2-methylthio-4-amino-5-carboxylic acid ethyl ester thiazole (3): Add 1.70g (0.01mol) of compound 2 and 2mL of ethanol to a 25mL three-necked flask, stir to dissolve compound 2, and then slowly add 1.67g (0.01mol) of ethyl bromoacetate, and the solution becomes turbid. Heat the reaction mixture to 80°C and react for 1h. Stop heating, add 2.02g (0.02mol) of triethylamine after cooling, and continue to react at 80°C for 3h. Remove the solvent by rotary evaporation, add cooled deionized water to the reaction solution, and after a period of time, a dark brown solid precipitates. Filter it with suction, rinse with deionized water, and vacuum dry it to remove water. Solid 3 (eluent is V 石油醚 :V 乙酸乙酯 =15:3), evaporate the solvent to dryness to obtain 1.86 g of light brown solid, with a yield of 85.3%. 1 HNMR (400MHz, DMSO): δ5.78 (s, 2H), 4.26 (q, J=7.1Hz, 2H), 2.63 (s, 3H), 1.33 (t, J=7.1Hz, 3H).

[0055] Synthesis of 4-amino-5-carboxylic acid ethyl thiazole (4): 2.18g (0.01mol) of compound 3 and 40mL of methanol (if insoluble, heat appropriately) were added to a 250mL three-necked flask, 0.975g (0.15mol) of zinc powder was added to the solution at room temperature, and 7mL (0.02mol) of 3N hydrochloric acid solution was added dropwise (due to the generation of hydrogen during the reaction, the hydrochloric acid should be added dropwise slowly in batches. It was found through experiments that it should be added dropwise three times with a half-hour interval), and the reaction was carried out at 25°C for 3h. After the reaction was completed, 100mL of saturated NaHCO3 aqueous solution was added to adjust the reaction solution to pH=7, the insoluble matter was filtered, and then washed with saturated NaCl aqueous solution, extracted with dichloromethane (60mL×2), the lower layer solution was dried with anhydrous NaSO4, the solvent was filtered and dried, and the product 4 (eluent was V 石油醚 :V 乙酸乙酯 =15:1) to give 1.36 g of a yellow solid with a yield of 79%. 1 H NMR (400MHz, CDCl3) δ8.55 (s, 1H), 5.94 (s, 2H), 4.31 (q, J = 7.1Hz, 2H), 1.35 (t, J = 7.1Hz, 3H).

[0056] Example 1

[0057] S1. Synthesis of 4-urea-5-carboxylic acid ethyl thiazole

[0058] 0.86g of 4-amino-5-carboxylic acid ethyl thiazole (5mmol) and 30mL of dichloromethane were added to a 100mL three-necked flask. After the reaction temperature was maintained at 0°C, 1.09g (5.5mmol) of diphosgene was slowly added. After stirring the reaction at this temperature for 1h, 14mL of ammonia water was added dropwise, and the reaction was continued at 0°C for 1.5h. The solid produced was removed by suction filtration, and the obtained mixed solution was concentrated and washed with a saturated aqueous NaCl solution, extracted with ethyl acetate, and the upper layer solution was dried with anhydrous NaSO4. Sodium sulfate was removed by suction filtration, and the obtained liquid was evaporated to dryness and then column chromatography was performed to obtain the product 4-urea-5-carboxylic acid ethyl thiazole (eluent: V 石油醚 :V 乙酸乙酯 =10:3), to give 0.301 g of a yellow solid with a yield of 28%.

[0059] S2. Synthesis of Thiazolo[4,5-d]pyrimidine-5,7-dione

[0060] In a 100mL three-necked flask, add 35mL ethanol and 1.076g (5mmol) 4-urea-5-carboxylic acid ethyl thiazole, then add 6mL 1N NaOH aqueous solution, heat to 80℃ and react for 2h. Then cool to 0℃ with an ice bath, adjust pH to neutral with 5% hydrochloric acid, and remove most of the solvent by rotary evaporation. Wash the remaining mixture with saturated NaCl aqueous solution, extract with ethyl acetate, and dry the upper layer with anhydrous NaSO4. After filtering the insolubles, evaporate to dryness to obtain 1.218g of the product with a yield of 72%. 1 H NMR (400MHz, d-DMSO): δ12.38 (s, 1H), 11.40 (s, 1H), 9.42 (s, 1H).

[0061] S3. Synthesis of tribenzoyloxyuridine

[0062] Under N2 protection, add 0.169g (1mmol) thiazo[4,5-d]pyrimidine-5,7-dione and 15mL acetonitrile to a 50mL eggplant-shaped bottle and stir. At this time, the solid is not completely dissolved. Add 0.610g (3mmol) N,O-BSA dropwise and stir at 25°C until the solid is dissolved. Add 0.756g (1.5mmol) 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-furanose and 0.244g (1.1mmol) trimethylsilyl trifluoromethanesulfonate (TMSOTf), heat to 60°C, react overnight, and TLC (developing solvent is CH2Cl2:CH3OH=10:1) shows that the reactants are completely reacted. Cool to room temperature, remove acetonitrile by rotary evaporation, add dichloromethane to dissolve, neutralize the reaction solution with saturated NaHCO3 solution, wash the lower layer solution three times with saturated NaCl solution, and dry with anhydrous Na2SO4. The obtained mixed solution was filtered and evaporated to dryness. The intermediate compound was obtained by column chromatography as a white sugar-like solid with a yield of 0.344 g and a yield rate of 48%.

[0063] S4. Synthesis of Thiazolo[4,5-d]Uridine Analogs

[0064] Take 0.3g (0.49mmol) of tribenzoyloxyuridine in a 100mL sealed pressure tube, add 60mL of methanol, stir at room temperature, pass ammonia for 40min, and keep the vent tube below the liquid surface to facilitate the dissolution of ammonia. After 40min, seal the bottle and react at 60℃ for 12h. TLC tracking shows that if the reaction is not complete, wait for the reaction tube to cool to room temperature and pass ammonia again, and continue to react at 60℃ for 12h. After the reaction is complete, cool down, evaporate the solvent, and obtain the final product by column chromatography. The final product is an off-white powder with a yield of 0.053g and a yield of 36.1%.

[0065] 1 H NMR (400MHz, d-DMSO) δ11.83(s,1H),9.51(s,1H),6.29(d,J=4.7Hz,1H),5.21(d,J=5.4Hz,1H),5.01(d,J=6.0Hz,1H),4.72(dd,J=10.6 ,5.2Hz,1H),4.67(t,J=5.8Hz,1H),4.17(q,J=5.7Hz,1H),3.79(dd,J=9.8,5.7Hz,1H),3.64(dt,J=11.5,4.4Hz,1H),3.50–3.42(m,1H).

[0066] 13C NMR (101MHz, d-DMSO) δ162.33(s), 157.94(s), 156.69(s), 151.01(s), 107.64(s), 90.88(s), 85.25(s), 71.16(s), 70.53(s), 62.61(s).

[0067] Example 2 Application of Thiazolo[4,5-d]Uridine (ThU) as a Probe in the Field of Detection

[0068] When cells carry out life activities, almost all chemical processes are carried out in cell solutions, and the same is true for the life chemical activities of nucleic acids. Different life activities in cells have different requirements for the environment. Therefore, the following test simulates the effect of a nearly harsh environment on the photophysical properties of nucleoside analogs. The photophysical signals obtained through the test directly reflect the changes in the surrounding environment. Due to the diversity and complexity of the internal environment of cells, before inserting the analog into the oligonucleotide chain, the compound synthesized in this chapter, thiazole [4,5-d] uridine analog, explores its photophysical properties through different polarities, pH values ​​and natural nucleosides, and preliminarily determines whether it has the potential to become an excellent fluorescent nucleoside probe.

[0069] 1. Thiazolo[4,5-d]uridine analogs as probes for detecting and identifying polar environments

[0070] Test solution for UV absorption spectrum: weigh 3 mg of 0.01 mmol fluorescent thiazole [4,5-d] uridine analog with an analytical balance, add an appropriate amount of solvent to dissolve the solute, then pour into a 100 mL 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 100 μmol / L. According to this method, prepare solutions of six different solvents of thiazole [4,5-d] uridine analogs. After preparation, shake well and use an ultrasonic cleaner to further mix the test solution.

[0071] Fluorescence emission spectrum test solution: Pipette 500 μL of the previously prepared 100 μmol / L test solution of the compound thiazole [4,5-d] uridine analog into a 10 mL volumetric flask, add solvent to 10 mL, and shake the test solution in the volumetric flask to obtain a solution with a concentration of 5 μmol / L. According to this method, prepare solutions of six different polar solvents of thiazole [4,5-d] uridine analogs. After preparation, shake well and use an ultrasonic cleaner to further mix the test solution.

[0072] (1) Ultraviolet absorption spectra of different polarities

[0073] like Figure 1As shown in the figure, the fluorescent nucleoside analog thiazole [4,5-d] uridine analog has a strong absorption peak near 290nm in the near-ultraviolet region (200-400nm), which is generated by the n→π* transition of C=C and C=N conjugation. Due to the influence of solvent effect, the maximum absorption wavelength and absorption intensity of the fluorescent nucleoside analog thiazole [4,5-d] uridine analog in different solvents have changed. Except for dichloromethane solvent, the solvent has little effect on the maximum absorption wavelength of thiazole [4,5-d] uridine analog, which is around 293nm. The maximum absorption wavelength of thiazole [4,5-d] uridine analog in dioxane solvent is 295nm, and the maximum absorption wavelength in dichloromethane solvent is blue-shifted to 267nm. In strong polar solvents, although the ultraviolet absorption peak shift of the fluorescent nucleoside analog thiazole [4,5-d] uridine analog is not obvious, the intensity of the absorption peak of the fluorescent nucleoside analog thiazole [4,5-d] uridine analog in these six solvents changes significantly. In solvents with higher polarity, the absorbance decreases with increasing polarity, acetonitrile > methanol > water, and in weak polar solvents, the absorbance increases with increasing polarity, ethyl acetate > dioxane > dichloromethane.

[0074] (2) Fluorescence emission spectra of different polarities

[0075] like Figure 2 As shown, at the optimal excitation wavelength, the compound thiazolyl[4,5-d]uridine analog has good emission in six different solvents. Except for water with the highest polarity and dichloromethane with the lowest polarity, the fluorescence intensity of the thiazolyl[4,5-d]uridine analog gradually increases with the increase of solvent polarity. This indicates that the polarity of the solvent changes the energy of the electron n-π transition, which in turn affects the fluorescence intensity of the thiazolyl[4,5-d]uridine analog. Therefore, it is proved that the compound thiazolyl[4,5-d]uridine analog is sensitive to solvent polarity, indicating that it may become an environmentally sensitive fluorescent probe.

[0076] 2. Thiazolo[4,5-d]uridine analogs as probes for detection of pH response

[0077] Currently, only a few pH-sensitive fluorescent nucleoside analogs have been reported. Generally, the pH of the solution only affects the fluorescence intensity of the emission spectrum of fluorescent nucleoside analogs, and generally does not affect the maximum emission wavelength.

[0078] 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.

[0079] UV absorption spectrum test solution: Use an analytical balance to accurately weigh 0.01mmol of thiazole [4,5-d] uridine analog, add deionized water, shake well in a small beaker to dissolve the solute, add to a 10ml volumetric flask, and then wash the beaker three times with an appropriate amount of deionized water, collect the liquid and add it to the volumetric flask, fix the volume, shake well, and further mix the test solution with an ultrasonic cleaner to obtain a thiazole [4,5-d] uridine analog aqueous solution with a concentration of 1mmol / L. 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 a pH of 1 to 12, fix the volume to 10ml, shake well, and obtain a thiazole [4,5-d] uridine analog test solution with a concentration of 100μmol / L and 12 pH values.

[0080] Fluorescence emission spectrum test solution: Use a pipette to transfer 500 μL of the previously prepared 100 μmol / L aqueous solution of thiazole [4,5-d] uridine analog into a volumetric flask, dilute to the 10 ml mark of the volumetric flask with a pH buffer solution of pH = 1 to 12, shake the test solution in the volumetric flask, and obtain a 5 μmol / L test solution of thiazole [4,5-d] uridine analog fluorescence emission spectrum after ultrasonic treatment.

[0081] (1) UV absorption spectra of thiazolyl[4,5-d]uridine analogs in solutions with different pH values

[0082] like Figure 3 , is the UV spectrum of the compound thiazole [4,5-d] uridine analog in aqueous solution at different pH values, and its specific data are shown in Table 1 below. In an acidic environment, as the pH gradually decreases, the maximum absorption wavelength of the compound does not change, and the absorbance decreases in an acidic environment, but the absorbance intensity increases at pH = 1; in an alkaline environment, as the pH gradually increases, the maximum absorption wavelength only fluctuates around 293nm, but the absorbance increases with the increase of pH.

[0083] (2) Fluorescence spectra under different pH conditions

[0084] The fluorescence emission spectra of the compound thiazole [4,5-d] uridine analogue were scanned with an excitation wavelength of 291 nm. Figure 4 , the compound thiazole [4,5-d] uridine analogue obtained different fluorescence emission spectra in 12 different pH solutions. As the pH value changes, the maximum emission wavelength of the compound does not show regular changes with the pH, but the fluorescence intensity changes significantly. The fluorescence intensity of the compound thiazole [4,5-d] uridine analogue increases with the increase of pH value in the range of pH = 1 to 6, and there is no regular change in the range of pH = 8 to 12. The fluorescence intensity is weakest at pH = 1, indicating that a strong acid environment will destroy the fluorescence properties of the compound. The fluorescence intensity is strongest at pH = 9, indicating that the compound has strong tolerance in an alkaline environment.

[0085] Depend on Figure 4 It can be seen that within the range of pH = 1 to 9, the emission peak intensity of the fluorescent nucleoside analogue weakens as the pH of the solution decreases. It can be concluded that the fluorescent nucleoside analog thiazole [4,5-d] uridine analogue has the characteristic of identifying the change of solution acidity and alkalinity within a certain pH range and can be used to detect pH changes within a certain range. See Table 2, pH = 1 to 12 is the horizontal axis, the normalized value of fluorescence intensity is the vertical axis, and the linear fitting is obtained. Figure 5 With the increase of pH, the fluorescence intensity of the compound thiazole [4,5-d] uridine analogue increases first and then decreases; in phase analysis, the overall fluorescence intensity of the compound thiazole [4,5-d] uridine analogue in pH = 1 to 9 solution gradually increases, and the fluorescence intensity decreases regularly at pH = 10 to 12. Judging from the trend of the spectrum change, the compound thiazole [4,5-d] uridine analogue can be used as a fluorescent probe to study RNA protonation.

[0086] Table 2 Emission peaks of compounds in different pH solutions

[0087]

[0088] Among them, 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, and the corresponding structure is (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 ofuridine and cytidine.J Heterocycl Chem.1992,2,29,343-354.).

[0089] By comparison, it was found that the emission wavelength (430±5nm) of the thiazole [4,5-d] uridine analog under the same pH conditions is much greater than that of 1-β-D-arabino-yl pyrrolo [3,2-D] pyrimidine-2,4 (3H, 5H) -dione (270±3nm) reported in the literature, and is in the violet region (380-450nm) of the optical spectrum, exhibiting purple fluorescence, and can be used as a fluorescence detection probe; while the reference substance does not have fluorescence visible to the naked eye, 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.

[0090] 3. Fluorescence quenching effect of thiazolyl[4,5-d]uridine analogs as probes

[0091] In 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. Excellent fluorescent nucleoside analogs lose fluorescence under the action of adjacent bases and cannot become good fluorescent probes.

[0092] The present invention adds the monophosphate of natural nucleoside to the phosphate buffer solution of thiazole [4,5-d] uridine analog by titration, and obtains the emission spectrum of thiazole [4,5-d] uridine analog by scanning with a fluorescence spectrophotometer. By studying the fluorescence spectrum of thiazole [4,5-d] uridine analog, we can further understand the photophysical properties of thiazole [4,5-d] uridine analog, understand the influence of different natural nucleotides on thiazole [4,5-d] uridine analog fluorescent probe, and judge whether thiazole [4,5-d] uridine analog can play a normal function in the oligonucleotide sequence.

[0093] 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: F0 / F=1+K SV *[NMPs];

[0094] 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.

[0095] In order to further understand the photophysical properties of thiazolyl[4,5-d]uridine analogs and preliminarily understand the effects of four natural bases on thiazolyl[4,5-d]uridine analogs, a buffer solution was prepared, and the emission spectrum was scanned and observed using a fluorescence instrument to draw conclusions.

[0096] Preparation of quenching solution: The fluorescence quenching test solution needs to be mixed with phosphate buffer solution and natural nucleotide solution.

[0097] 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.

[0098] Natural nucleotide solution: Accurately weigh 0.2mmol of natural nucleosides: 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 a small amount of phosphate buffer solution several times and recover it to the volumetric flask. Finally, set the volume to 10ml and shake evenly to obtain a 20mmol / L adenylic acid (AMP), cytidine (CMP), guanosine (GMP) and thymidine (TMP) nucleotide solution. Among them, natural nucleotides (NMR) include adenosine (AMP), cytidine (CMP), guanosine (GMP) and thymidine (TMP) nucleotides.

[0099] Fluorescence quenching test solution: number 24 centrifuge tubes: A1~A6, C1~C6, G1~G6 and T1~T6, first add 0.5mL of 100μmol / L thiazole [4,5-d] uridine 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, and finally add the prepared phosphate buffer solution to make the volume to 10mL. After shaking, 4 groups of 24 test solutions of four nucleotides are obtained, among which the concentration of thiazole [4,5-d] uridine analog is 5μmol / L.

[0100] Fluorescence quenching spectra of thiazo[4,5-d]uridine analogs Figure 6 As shown. There is a good linear relationship between thiazolyl[4,5-d]uridine analogs and the four natural nucleosides. The four natural nucleosides have little effect on the fluorescence intensity of thiazolyl[4,5-d]uridine analogs, and the value of F0 / F has no regularity with the change of concentration. Cytidylic acid (CMP) has a quenching effect on the fluorescence intensity of the compound, and the quenching constant Ksv is 0.0002, which is a very weak quenching effect; and adenosine (AMP), guanylic acid (GMP) and thymidylic acid (TMP) also have an enhancing effect on the fluorescence intensity of thiazolyl[4,5-d]uridine analogs; so the natural nucleosides have little effect on thiazolyl[4,5-d]uridine analogs as a whole. It shows that thiazolyl[4,5-d]uridine analogs can play a normal fluorescent probe function in the in vivo environment in the oligonucleoside sequence.

[0101] 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 thiazolyl[4,5-d]uridine analogue, characterized in that: The structure is as follows:

2. A method for preparing the thiazole [4,5-d] uridine analogue according to claim 1, wherein the reaction scheme is as follows:

3. The preparation method according to claim 2, characterized in that: The steps include: (1) 4-amino-5-carboxylic acid ethyl thiazole reacts with diphosgene and ammonia water to obtain 4-urea-5-carboxylic acid ethyl thiazole; (2) Then, 4-urea-5-carboxylic acid ethyl ester thiazole undergoes a cyclization reaction under the action of a base to obtain thiazo[4,5-d]pyrimidine-5,7-dione; (3) Thiazolo[4,5-d]pyrimidine-5,7-dione reacts with 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose to obtain tribenzoyloxyuridine; (4) Tribenzoyloxyuridine is deprotected under the action of an alkaline reagent to obtain a thiazolyl[4,5-d]uridine analog, denoted as ThU.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of 4-amino-5-ethylformate thiazole to diphosgene is 1:(1.0-1.5); the amount of ammonia water relative to 4-amino-5-ethylformate thiazole and ammonia water is 2-4 mL / mmol.

5. The preparation method according to claim 3, characterized in that: In step (2), the base is selected from 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 step (3), the molar ratio of thiazolo[4,5-d]pyrimidine-5,7-dione to 1-acetoxy-2,3,5-tribenzoyloxy-1-β-D-ribofuranose is 1:(1.0-1.5).

7. The preparation method according to claim 3, characterized in that: In step (3), in step (3), the reaction further includes adding promoters N, O-BSA and TMSOTf; wherein the molar ratio of thiazolo[4,5-d]pyrimidine-5,7-dione to N, O-BSA is 1:(2-5); and the molar ratio of thiazolo[4,5-d]pyrimidine-5,7-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 step (4), the alkaline reagent is ammonia gas.

9. Use of the thiazolyl[4,5-d]uridine analogue of claim 1 in the preparation of a detection probe.

10. Use of the thiazolyl[4,5-d]uridine analogue of claim 1 in environmental polarity detection, environmental pH detection, DNA fluorescent probe and cell fluorescence imaging.

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