Preparation method of coumarin derivative labeled nucleoside triphosphate and application thereof
By optimizing reaction conditions and reagent selection, coumarin derivatives labeled with nucleoside triphosphates were synthesized efficiently, solving the problem of low labeling efficiency in existing technologies and improving the labeling efficiency of FISH probes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGZHOU LBP MEDICINE SCI & TECH
- Filing Date
- 2023-12-25
- Publication Date
- 2026-07-24
AI Technical Summary
The lack of efficient methods for synthesizing fluorescein-labeled nucleoside triphosphates with inserted carbon chains in the current technology leads to low labeling efficiency.
Coumarin derivative-labeled nucleoside triphosphates were prepared by reacting compound AA-dUTP with Boc-6-Ahx-Osu and a base, followed by treatment with acid and base solutions, and then reacting with DEAC-NHS. The reaction conditions were optimized by controlling parameters such as solution concentration and temperature.
It improves the synthesis yield of fluorescently labeled nucleoside triphosphates and enhances the labeling efficiency, making it suitable for the preparation of FISH probes.
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Figure CN117801041B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing coumarin derivative-labeled nucleoside triphosphates and its application. Background Technology
[0002] Fluorescence in situ hybridization (FISH), a molecular cytogenetics technique for visualizing specific DNA sequences, boasts advantages such as high sensitivity, strong signal, low background, and speed. It is particularly effective in gene qualitative and quantitative analysis, integration, and expression studies, and is currently widely used in many fields including genetic disease diagnosis, viral infection analysis, prenatal diagnosis, tumor genetics, and genomics research, playing a vital role in clinical testing, teaching, and research.
[0003] FISH probe labeling typically employs both direct and indirect labeling methods. Compared to the cumbersome two-step indirect labeling method, the direct labeling method, which incorporates fluorescein-dUTP (deoxyuridine triphosphate) during nick translation labeling, offers advantages such as simpler detection steps and lower background, making it widely applicable in molecular biology and cytogenetics research. Fluorescein-dUTP is commonly synthesized through the coupling reaction of allylamine-deoxyuridine triphosphate (AA-dUTP) with N-succinimide ester of fluorescein. The fluorophore in the resulting product is attached to a base; the steric hindrance of the fluorophore can affect DNA strand formation, reducing labeling efficiency.
[0004] Blue light probes are best suited for direct imaging of high-abundance targets such as cellular targets, cell tracking applications, substrates for detecting enzyme activity, and certain multicolor fluorescence applications—including immunofluorescence, nucleic acid and protein microarrays, in situ hybridization, and neuron tracking. Coumarin and its derivatives are UV-excited blue fluorescent dyes with short emission wavelengths in the 390–480 nm range. Compared to fluorescein, rhodamine, and anthocyanins, coumarin produces moderate fluorescence due to its relatively low extinction coefficient and exhibits good photostability. A blue fluorescent conjugate prepared from 7-(diethylamino)-coumarin-3-carboxylic acid-N-succinimide ester (DEAC-NHS) exhibits very high fluorescence intensity, is easily excited under UV light, and has a longer excitation wavelength due to the 7-diethylamino fluorophore.
[0005] When DEAC-dUTP, generated by the direct coupling of DEAC-NHS and AA-dUTP, is applied to a probe, the small distance between the DEAC group and the bases on the dUTP creates steric hindrance for base pairing, reducing labeling efficiency. Inserting a carbon chain between the base and the fluorescent group increases the space between them, reducing steric hindrance during base pairing and increasing the activity of the enzymatic reaction, thus achieving higher labeling efficiency. However, current technology does not provide a clear and efficient method for synthesizing related compounds.
[0006] CN201210441732.5 discloses a method for preparing fluorescein-labeled nucleotides, specifically a method for preparing fluorescein-rhodamine B-labeled deoxyuridine triphosphates. The process includes the preparation of the starting material solution, coupling reaction, and purification. This method can be used to synthesize DEAC-dUTP, but it cannot achieve the preparation of products with a carbon chain inserted between DEAC and dUTP.
[0007] Therefore, how to provide a method for efficiently synthesizing fluorescently labeled nucleoside triphosphates with elongated carbon chains has become an urgent problem to be solved. Summary of the Invention
[0008] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing coumarin derivative-labeled nucleoside triphosphates and its applications. The preparation method provided by the present invention can efficiently synthesize the target product and has the advantage of high synthesis yield.
[0009] To achieve this objective, the present invention adopts the following technical solution: On one hand, the present invention provides a method for preparing coumarin derivative-labeled nucleoside triphosphates, the preparation method comprising the following steps: (1) Compound AA-dUTP was mixed with compound Boc-6-Ahx-Osu and a base and reacted in the dark to obtain compound 1; (2) Compound 1 was mixed with an acid solution, and then mixed with an alkaline solution to obtain compound 2; (3) Compound 2 and compound DEAC-NHS were mixed and reacted in the dark to obtain compound DEAC-12-dUTP, which is the coumarin derivative labeled nucleoside triphosphate.
[0010] The reaction process is as follows: .
[0011] The above method can efficiently synthesize the target product and has the advantage of high synthesis yield.
[0012] Preferably, the base in step (1) includes triethylamine.
[0013] Preferably, in step (1), AA-dUTP and Boc-6-Ahx-Osu are mixed in solution form. The concentration of the AA-dUTP solution is 10-30 mM, and the concentration of the Boc-6-Ahx-Osu solution is 35-45 mM. The concentration of the AA-dUTP solution can be 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, or 30 mM, etc., and the concentration of the Boc-6-Ahx-Osu solution can be 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, or 45 mM, etc., but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0014] Preferably, the solvent of the AA-dUTP solution is methanol.
[0015] The above method effectively reduces the hydrolysis reactivity of the raw materials by mixing the reactants in solution and controlling the solution concentration and solvent selection, thereby improving the coupling reaction activity and yield, and can effectively increase the reaction yield.
[0016] Preferably, the molar ratio of AA-dUTP to alkali and Boc-6-Ahx-Osu in step (1) is 1:(15-25):(0.5-0.9), wherein the number of parts of alkali can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, and the number of parts of Boc-6-Ahx-Osu can be 0.5, 0.6, 0.7, 0.8 or 0.9, but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0017] Preferably, the reaction temperature in step (1) is 10-30℃ and the time is 4-8 h. The temperature can be 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃ or 30℃, etc., and the time can be 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h or 8 h, etc., but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0018] Preferably, the acid solution in step (2) is a hydrochloric acid-ethyl acetate mixture.
[0019] Preferably, the alkaline solution in step (2) is a triethylamine-methanol mixture.
[0020] The above method, by using specific acid and alkaline solutions for de-Boc and desalting, can easily remove the primary amino group, allowing the next reaction to continue.
[0021] Preferably, the molar ratio of DEAC-NHS to AA-dUTP in step (3) is (1.2-1.5):1, 1.2:1, 1.3:1, 1.4:1 or 1.5:1, etc., but not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0022] Preferably, the reaction temperature in step (3) is 10-30℃ and the time is 4-8 h. The temperature can be 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, 22℃, 24℃, 26℃, 28℃ or 30℃, etc., and the time can be 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h or 8 h, etc., but is not limited to the values listed above. Other unlisted values within the above range are also applicable.
[0023] On the other hand, the present invention also provides the application of the preparation method described above in the preparation of FISH probes.
[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing coumarin derivative-labeled nucleoside triphosphates, which can efficiently synthesize the target product and has the advantage of high synthesis yield; and by adopting specific operation methods and reagents, as well as controlling relevant parameters, the reaction yield is further improved. Attached Figure Description
[0025] Figure 1 These are graphs showing the yield results of the examples and comparative examples; Figure 2 This is a graph showing the signal values of DEAC-12-dUTP and DEAC-dUTP as FISH markers. Detailed Implementation
[0026] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
[0027] Example 1 This embodiment provides a method for preparing coumarin derivative-labeled nucleoside triphosphates, the specific steps of which are as follows: (1) Dissolve 10 mg of AA-dUTP solid in anhydrous methanol to obtain a solution A with a concentration of 20 mM; (2) Weigh a certain amount of Boc-6-Ahx-Osu and dissolve it in DMSO to obtain a solution B with a concentration of 40 mM; (3) The materials were added according to the molar ratio of AA-dUTP:triethylamine:Boc-6-Ahx-Osu = 1:20:0.77. The feeding process was as follows: a certain amount of triethylamine was added to solution A, mixed well, and solution B was slowly added under stirring at 20°C in the dark. The reaction solution was sealed and incubated for 5 hours. (4) The solvent in the reaction solution is removed by rotary evaporation at 65°C; (5) Add 2 mL of 2% hydrochloric acid and ethyl acetate mixture, and acidify at 20°C for 2 hours. Remove the solvent by rotary evaporation. (6) Add 2 mL of 5% triethylamine-methanol mixture and stir vigorously to dissolve the solid; (7) Weigh out DEAC-NHS, which is 0.3 times higher in molar amount than the added AA-dUTP, and dissolve it in DMSO to obtain solution C; (8) After dissolving in step (6), slowly add solution C to the solution at 20°C in the dark with stirring. Continue stirring for 5 hours after the addition is complete. (9) After removing most of the organic solvent by rotary evaporation at 65°C, the reaction solution was diluted to 3 mL with purified water, purified by HPLC, and compared with the standard sample to confirm the successful synthesis of the product.
[0028] Example 2 This embodiment provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for step (1) as follows, the other steps are the same as in Example 1.
[0029] (1) Dissolve 10 mg of AA-dUTP solid in anhydrous methanol to obtain a solution A with a concentration of 10 mM.
[0030] Example 3 This embodiment provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for step (1) as follows, the other steps are the same as in Example 1.
[0031] (1) Dissolve 10 mg of AA-dUTP solid in anhydrous methanol to obtain a solution A with a concentration of 30 mM.
[0032] Comparative Example 1 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for step (1) as follows, the other steps are the same as in Example 1.
[0033] (1) Dissolve 10 mg of AA-dUTP solid in anhydrous methanol to obtain a solution A with a concentration of 1 mM.
[0034] Comparative Example 2 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. In the specific steps, except for step (3) where the molar ratio of AA-dUTP:triethylamine:Boc-6-Ahx-Osu is 1:10:0.77, the rest are the same as in Example 1.
[0035] Comparative Example 3 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. The specific steps are consistent with Example 1, except that the molar ratio of AA-dUTP:triethylamine:Boc-6-Ahx-Osu in step (3) is 1:20:1.
[0036] Comparative Example 4 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for steps (1) and (6) as follows, the rest are the same as in Example 1.
[0037] (1) Dissolve 10 mg of AA-dUTP solid in a 0.5 mM sodium borate buffer solution with a pH of 8.5 to obtain a solution A with a concentration of 20 mM; (6) Add 2 mL of 0.5 mM sodium borate buffer solution with pH 9.0 and stir vigorously to dissolve the solid.
[0038] Comparative Example 5 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for step (1) as follows, the other steps are the same as in Example 1.
[0039] (1) Dissolve 10 mg of AA-dUTP solid in a 0.5 mM sodium borate buffer solution with pH 8.5 to obtain a solution A with a concentration of 20 mM.
[0040] Comparative Example 6 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for step (6) as follows, the other steps are the same as in Example 1.
[0041] (6) Add 2 mL of 0.5 mM sodium borate buffer solution with pH 9.0 and stir vigorously to dissolve the solid.
[0042] Comparative Example 7 This comparative example provides a method for preparing coumarin derivative-labeled nucleoside triphosphates. Except for step (5) as follows, the other steps are the same as in Example 1.
[0043] (5) Add 2 mL of 10% trifluoroacetic acid in dichloromethane solution, and acidify at 20°C for 2 hours. Remove the solvent by rotary evaporation.
[0044] Effect test: Take 10 μL of the reaction solution diluted with purified water in step (9) of Examples 1-3 and Comparative Examples 1-7, and then add 290 μL of purified water for further dilution. HPLC was used to detect the concentration of the product. The HPLC detection conditions are as follows: The HPLC test conditions are as follows: (1) Chromatographic column: InfinityLab Poroshell 120 SB-C18 column, 120 Å, 4 μm, 4.6*250 mm; (2) Mobile phase A: 0.05 M, pH=6.0, sodium phosphate buffer solution; Mobile phase B: methanol; (3) Column temperature: 30℃; (4) Flow rate: 1.0 mL / min; (5) Gradient elution: (6) Detector and detection wavelength: Variable Wavelength Detector, 426 nm.
[0045] The results are as follows: Another DEAC-12-dUTP reference standard was injected into the HPLC system at different concentrations to obtain working curves. The results are as follows: The working curve is y = 1402x - 0.977, R0 2 =0.997.
[0046] The overall reaction yield was calculated, and the results are as follows: Figure 1 As shown in the figure, the yields of Examples 1-3 are similar, and the concentration of solution A can be within the range of 10-30 mM. However, when the concentration is reduced to 1 mM (Comparative Example 1), the yield decreases significantly. In Comparative Examples 2 and 3, the yield decreases significantly after changing the molar ratio of the reactants, and reducing the amount of triethylamine has a significant impact on the yield. In Comparative Examples 4-6, the yield decreases significantly after changing the solvent system from an organic solvent system to a buffer salt solvent system. In Comparative Example 7, the DEAC-12-dUTP cannot be synthesized by changing the acid hydrolysis reaction conditions.
[0047] Then, DEAC-12-dUTP obtained in Example 1 and DEAC-dUTP (preparation method refers to steps (1)-(3) of Example 1, with Boc-6-Ahx-Osu replaced by DEAC-NHS in steps (2) and (3)) were tested as FISH markers. The specific steps are as follows: Fluorescence in situ hybridization (FISH) was performed using the nick-transfer labeling method. Specific experimental steps were as follows: 1. Prepare CSP17 blue probe; (1) Incubate 2 μg of centromere No. 17 in 200 μL of 10× nick translation buffer (0.5 mol / L Tris•Cl (pH 7.2); 0.1 mol / L MgSO4; 10 mmol / L DTT; 100 μg / mL BSA); (2) Add 10 μL of 0.2 mM dNTPs (except dUTP) aqueous solution, 10 μL of 0.2 mM DEAC-12-dUTP aqueous solution (add 10 μL, 15 μL, and 20 μL respectively for DEAC-dUTP testing), and 5 units of DNA polymerase I and deoxyribonuclease I respectively; (3) The mixture was incubated at 25°C for 120 minutes; (4) Detect the labeled product by gel electrophoresis with 8.0 g / L agarose / TBE buffer. The DNA fragment length should be about 300-500 bp. If the fragment is too large, add an appropriate amount of deoxyribonuclease I to continue the enzymatic digestion until the DNA fragment length is appropriate. Then add 2 μL of 0.5 M EDTA and 1.25 μL of 5% SDS to terminate the reaction. (5) The probe precipitate was purified by ethanol-sodium acetate precipitation. 2. FISH experiment: Dissolve the probe precipitate in 20 μL of purified water, add 35 μL of hybridization solution (70% formamide / 2×SSC), denature in a 75℃ water bath for 5 min, take 2 μL of probe hybridization solution and add it to the prepared peripheral blood sample smear for hybridization (incubate overnight at 42℃), then wash and counterstain with DAPI; 3. Observation results: The signal values after probe hybridization with different amounts of DEAC-12-dUTP / DEAC-dUTP were observed and recorded under a fluorescence microscope.
[0048] The results are as follows Figure 2As shown, it can be observed that, with the same amount of fluorescein raw material, when the signal intensity value of DEAC-12-dUTP reaches above 2, the signal intensity value of DEAC-dUTP is only 1. The amount of DEAC-dUTP needs to be twice that of DEAC-12-dUTP for the signal intensity value to reach above 2. Therefore, the labeling efficiency of DEAC-12-dUTP is higher than that of DEAC-dUTP.
[0049] The main sources of raw materials and equipment for each embodiment, comparative example, and test design are as follows: The applicant declares that this invention illustrates the preparation method and application of coumarin derivative-labeled nucleoside triphosphates through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.
[0050] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0051] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
Claims
1. A method for preparing coumarin derivative-labeled nucleoside triphosphates, characterized in that, The preparation method includes the following steps: (1) Compound AA-dUTP was mixed with compound Boc-6-Ahx-Osu and a base and reacted in the dark to obtain compound 1; (2) Compound 1 was mixed with an acid solution, and then mixed with an alkaline solution to obtain compound 2; (3) Compound 2 and compound DEAC-NHS were mixed and reacted in the dark to obtain compound DEAC-12-dUTP, which is the coumarin derivative labeled nucleoside triphosphate; The reaction process is as follows: 。 2. The preparation method according to claim 1, characterized in that, The base used in step (1) is selected from triethylamine.
3. The preparation method according to claim 1 or 2, characterized in that, In step (1), AA-dUTP and Boc-6-Ahx-Osu are mixed in solution form, with the concentration of AA-dUTP solution being 10-30 mM and the concentration of Boc-6-Ahx-Osu solution being 35-45 mM.
4. The preparation method according to claim 3, characterized in that, The solvent for the AA-dUTP solution is methanol.
5. The preparation method according to claim 1, characterized in that, The molar ratio of AA-dUTP to alkali and Boc-6-Ahx-Osu in step (1) is 1:(15-25):(0.5-0.9).
6. The preparation method according to claim 1, characterized in that, The reaction in step (1) is carried out at a temperature of 10-30℃ for 4-8 hours.
7. The preparation method according to claim 1, characterized in that, The acid solution in step (2) is a mixture of hydrochloric acid and ethyl acetate.
8. The preparation method according to claim 1, characterized in that, The alkaline solution in step (2) is a triethylamine-methanol mixture.
9. The preparation method according to claim 1, characterized in that, The molar ratio of DEAC-NHS to AA-dUTP in step (3) is (1.2-1.5):
1.
10. The preparation method according to claim 1, characterized in that, The reaction in step (3) is carried out at a temperature of 10-30℃ for 4-8 hours.
11. The application of the preparation method according to any one of claims 1-10 in the preparation of FISH probes.