A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier

Abstract

The present invention relates to a preparation method of a fluorescent dye-modified deoxynucleoside solid phase carrier, and the steps are as follows: dissolving deoxynucleoside in a first organic solvent, adding 4,4'-dimethoxytriphenylchloromethane, and obtaining compound 2; dissolving compound 2 in a second organic solvent, adding a first condensing agent, a diamine trifluoroacetamide derivative, and obtaining compound 3; dissolving compound 3 in a methanol solution, adding an alkaline substance, and obtaining compound 4 after reaction; dissolving compound 4 in a third organic solvent, adding a fluorescent dye activated ester, and obtaining compound 5; dissolving compound 5 in a fourth organic solvent, adding anhydride and an organic base in sequence, and obtaining compound 6; then dissolving compound 6 in a fifth organic solvent, adding a second condensing agent and a solid phase carrier in sequence, and separating the fluorescent dye-modified deoxynucleoside solid phase carrier after reaction. The method is simple, has a high yield, and achieves the purpose of efficient fluorescence diagnosis.

Description

Technical Field

[0001] The invention belongs to the field of biomolecular diagnosis, relates to a method for synthesizing a fluorescent group in a fluorescent probe, and specifically relates to a method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier. Background Art

[0002] Fluorescent probes use fluorescent substances as indicators. When excited by a certain wavelength, the fluorescent substances produce fluorescence, and the detected substances are quantitatively or qualitatively analyzed by detecting the generated fluorescence. In biomedical testing, fluorescent probes have the advantages of high sensitivity, high selectivity, fast response, good repeatability, simple operation, and low cost compared to other detection methods. Therefore, they are widely used in molecular diagnosis, genetic testing, antibody immunoassay, etc.

[0003] Fluorescent probes are composed of three parts: recognition group, fluorescent group and linker. The recognition group determines the selectivity and specificity of the probe, the fluorescent group determines the sensitivity of the probe, and the linker part plays the role of a connecting hub in the probe. Fluorescent dyes exist as fluorescent groups in fluorescent probes, and their photophysical properties are an important manifestation of the performance of the probe. At present, the fluorescent probes used for labeling or derivatization mainly include rhodamine, fluorescein, o-phthalaldehyde, etc. Among them, fluorescein compounds occupy an extremely important position in the field of biological research. However, the preparation method of deoxynucleoside solid phase carriers modified with fluorescent dyes in China is still immature. Summary of the invention

[0004] The present application mainly solves the problem that there is currently no method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier.

[0005] Based on the above problems, this application proposes the following solutions:

[0006] A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier comprises the following steps:

[0007] S1, fully dissolving the deoxynucleoside in a first organic solvent, adding 4,4'-dimethoxytriphenylmethane, and separating compound 2 after sufficient reaction;

[0008] S2, fully dissolving the compound 2 in step S1 in a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative, and separating the compound 3 after sufficient reaction;

[0009] S3, fully dissolving the compound 3 in step S2 into a methanol solution, adding an alkaline substance, and separating the compound 4 after sufficient reaction;

[0010] S4, fully dissolving the compound 4 in step S3 into a third organic solvent, adding a fluorescent dye activated ester, and separating the compound 5 after sufficient reaction;

[0011] S5, fully dissolving the compound 5 in step S4 into a fourth organic solvent, adding anhydride and an organic base in sequence, and separating the compound 6 after sufficient reaction;

[0012] S6, fully dissolving the compound 6 in step S5 into the fifth organic solvent, adding the second condensing agent and the solid phase carrier in sequence, and separating the fluorescent dye-modified deoxynucleoside solid phase carrier after sufficient reaction.

[0013] As a further improvement of the present application, in step S1, the first organic solvent may be, but is not limited to, any one of pyridine, DMF, dichloromethane, ethyl acetate, and tetrahydrofuran.

[0014] As a further improvement of the present application, in step S2, the second organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, and acetonitrile.

[0015] As a further improvement of the present application, in step S2, the first condensing agent may be, but is not limited to, any one of DCC, EDC, HOBt, HBTu, HATu, and PyBop.

[0016] As a further improvement of the present application, in step S2, the diamine trifluoroacetamide derivative can be but is not limited to any one of ethylenediamine trifluoroacetamide, propylenediamine trifluoroacetamide, butylenediamine trifluoroacetamide, pentanediamine trifluoroacetamide, and hexamethylenediamine trifluoroacetamide.

[0017] As a further improvement of the present application, in step S3, the alkaline substance may be but is not limited to at least one of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonia water, and methylamine.

[0018] As a further improvement of the present application, in step S4, the third organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, ether, and acetonitrile.

[0019] As a further improvement of the present application, in step S4, the fluorescent dye activated ester may be but is not limited to any one of FAM activated ester, HEX activated ester, TET activated ester, cyanine dye activated ester, and rhodamine dye activated ester.

[0020] As a further improvement of the present application, in step S4, the reaction temperature is 60°C-90°C.

[0021] As a further improvement of the present application, in step S5, the fourth organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, and acetonitrile.

[0022] As a further improvement of the present application, in step S5, the organic base may be but is not limited to any one of triethylamine, pyridine, diisopropylamine, and diisopropylethylamine.

[0023] As a further improvement of the present application, in step S5, the acid anhydride may be, but is not limited to, any one of succinic anhydride, glutaric anhydride, and diglycolic anhydride.

[0024] As a further improvement of the present application, in step S6, the fifth organic solvent may be, but is not limited to, any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, and acetonitrile.

[0025] As a further improvement of the present application, in step S6, the second condensing agent may be, but is not limited to, any one of DCC, EDC, HOBt, HBTu, HATu, and PyBop.

[0026] As a further improvement of the present application, in step S6, the solid phase carrier may be, but is not limited to, any one of controlled glass bead powder CPG and aminomethyl polystyrene resin.

[0027] As a further improvement of the present application, the deoxynucleotide may be, but is not limited to, any one of adenine deoxynucleotide, thymine deoxynucleotide, cytosine deoxynucleotide and guanine deoxynucleotide.

[0028] As a further improvement of the present application, the deoxynucleoside may be but is not limited to 5-(2-carboxyvinyl)-2'-deoxyuridine.

[0029] The beneficial effect of the present application is that the present application provides a method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier, the method is simple, the yield is high, and the purpose of efficient fluorescence diagnosis is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The synthetic reaction process of the deoxynucleoside solid phase carrier modified with fluorescent dye. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be clearly and completely described below in combination with the specific embodiments and drawings of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments, and are not intended to limit the scope of the present invention. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present application.

[0032] The present application provides a method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier, comprising the following steps:

[0033] S1. Fully dissolve the deoxynucleoside in a first organic solvent, add 4,4'-dimethoxytriphenylchloromethane, and separate compound 2 after sufficient reaction; preferably, the reaction is carried out at room temperature, and the separation step is to carry out the first decompression and the first extraction in sequence after sufficient reaction, collect the first organic phase and remove the solvent, and then purify the residue by the first step to obtain compound 2; further, the extracting liquid used in the first extraction is a mixed solution of water and dichloromethane, the process for removing the solvent in this step is to remove by reduced pressure evaporation, and the process for the first purification can be but is not limited to chromatography, column chromatography, etc.

[0034] S2, fully dissolving the compound 2 in step S1 in a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative, and separating the compound 3 after sufficient reaction; preferably, the reaction is carried out at room temperature, and the separation step is to sequentially carry out the first step of quenching and the second step of extraction after sufficient reaction, and then remove the solvent after collecting the second organic phase, and then purify the residue by the second step to obtain compound 3; further, the solution used for the first step of quenching is water, and the extracting liquid used in the second step of extraction is ethyl acetate. The process for removing the solvent in this step is distillation under reduced pressure, and the process for the second step of purification can be but is not limited to chromatography, column chromatography, etc.

[0035] S3, fully dissolving the compound 3 in step S2 into a methanol solution, adding an alkaline substance, and separating the compound 4 after sufficient reaction; preferably, the reaction is carried out at room temperature, and the separation step is concentrating and drying after sufficient reaction.

[0036] S4, fully dissolving the compound 4 in step S3 in a third organic solvent, adding a fluorescent dye activated ester, and separating the compound 5 after sufficient reaction; preferably, the reaction is carried out under heating conditions, and the separation step is to remove the solvent after sufficient reaction, add dichloromethane to the residue, wash it with water three times, collect the third organic phase, concentrate it, and finally purify it in the third step to obtain compound 5; further, the process for removing the solvent in this step is to remove it by distillation under reduced pressure, and the process for purification in the third step can be but is not limited to chromatography, column chromatography, etc.

[0037] S5. Fully dissolving the compound 5 in step S4 in a fourth organic solvent, adding anhydride and an organic base in sequence, and separating the compound 6 after sufficient reaction; preferably, the reaction is carried out at room temperature, and the separation steps are sequentially quenched in the second step, extracted in the third step, and the solvent is removed after the fourth organic phase is collected to obtain compound 6; further, the liquid used for the second step quenching is water, and the process for removing the solvent is distillation under reduced pressure.

[0038] S6, fully dissolving the compound 6 in step S5 into the fifth organic solvent, adding the second condensing agent and the solid phase carrier in sequence, and separating the fluorescent dye-modified deoxynucleoside solid phase carrier after sufficient reaction; preferably, the reaction is carried out at room temperature for 10 hours, and the separation step is filtering to collect the solid.

[0039] The purpose of quenching the reaction in the above steps: In an organic chemical reaction, a certain reaction substrate is in excess. When the chemical reaction proceeds to a certain extent, the target product has been obtained. The continued existence of the excess reaction substrate will further react to generate by-products or affect the post-processing operation, so it needs to be quenched. Quenching is to use another compound that is more likely to react with the excess substance to react with it, by reducing the reaction rate or destroying the reaction system, removing it from the system and terminating the reaction, so as to reduce the generation of by-products and ensure safe and convenient post-experimental processing.

[0040] In the present application, since 5-(2-carboxyvinyl)-2'-deoxyuridine is a kind of deoxynucleoside, 5-(2-carboxyvinyl)-2'-deoxyuridine is taken as an example for description, and the specific embodiments are as follows:

[0041] A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier, the overall process of the synthesis reaction is as follows Figure 1 As shown, the following steps are included:

[0042] S1. Place 40 g of compound 1 in a dry flask, wherein compound 1 is: 5-(2-carboxyvinyl)-2'-deoxyuridine, add 300 ml of pyridine to the flask to dissolve, then add 45 g of 4,4'-dimethoxytriphenylmethane, react at room temperature until complete, evaporate pyridine under reduced pressure, then extract with water and 300 ml of dichloromethane, collect the organic phase, evaporate the solvent under reduced pressure, and purify the residue by column chromatography to obtain compound 2, which is 52 g of pure white solid. Compound 1 is obtained by reaction a to obtain compound 2, and reaction a is

[0043]

[0044] The HNMR analysis of compound 2 is as follows: 1H NMR(DMSO) δ 2.16-2.25(m,1H),2.30-2.36(m,1H),3.16(dd,1H),3.24(dd,1H),3.72(dd,6H),3.88-3.92(m,1H),4.21-4.26(m ,1H),5.29(d,1H),6.17(dd,1H),6.78-6.87(m,5H),7.17-7.38(m,10H),8.03(s,1H),11.67(s,1H),12.1(w,1H).

[0045] S2, 52 g of compound 2 in step S1 was placed in a dry flask, 300 ml of DMF was added to dissolve, 27 g of DCC, 18 g of HOBt, 24 ml of triethylamine and 22 g of 1,6-hexanediamine trifluoroacetamide were added in sequence, the reaction was completed at room temperature, 800 ml of water was added to quench, 800 ml of ethyl acetate was used to extract and separate, the organic phase was collected, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography to obtain 50 g of white solid as compound 3. Compound 2 was obtained by reaction b to obtain compound 3, and reaction b was

[0046]

[0047] The HNMR analysis of compound 3 is as follows: 1 H NMR(DMSO)δ1.23-.128(m,4H),1.38-1.48(m,4H),2.14-2.20(m,1H),2.30 -2.36(m,1H),2.99-3.03(m,1H),3.10-3.19(m,5H),3.72(dd,6H),3.88-3 .92(m,1H),4.21-4.26(m,1H),5.29(d,1H),6.17(t,1H),6.85-6.91(m,4H ),7.05-7-4(m,11H),7.94(s,1H),8.01(t,1H),9.39(t,1H),11.60(s,1H).

[0048] S3, 50 g of compound 3 in step S2 is placed in a dry flask, 100 ml of methanol is added to dissolve, and then 100 ml of concentrated ammonia is added, and the reaction is completed at room temperature, and then concentrated to dryness to obtain compound 4, which is directly used in the next step. Compound 3 is reacted to obtain compound 4 through reaction c, and reaction c is

[0049]

[0050] S4, 44 g of compound 4 in step S3 was placed in a flask, 400 ml of DMF was added to dissolve it, and then 40 g of FAM-NHS was added, and the reaction was heated to 80°C overnight. After the reaction was completed, the solvent DMF was evaporated under reduced pressure, and the residue was dissolved in 500 ml of dichloromethane, washed with water 3 times, the organic phase was collected and concentrated, and the residue was purified by column chromatography to obtain 40 g of compound 5. Compound 4 was obtained by reaction d to obtain compound 5, and reaction d was

[0051]

[0052] The HNMR analysis of compound 5 is as follows: 1 H NMR(DMSO) δ 1.23-.128(m,4H),1.32(s,18H),1.38-1.48(m,4H),2.14-2.20(m,1H),2.30-2.36(m ,1H),2.99-3.03(m,1H),3.10-3.19(m,5H),3.72(dd,6H),3.88-3.92(m,1H),4.21-4 .26(m,1H),5.29(d,1H),6.17(t,1H),6.8(dd,4H),6.85-6.91(m,4H),7.05-7.4(m,1 4H),7.94(s,1H),8.01(t,1H),8.40(d,1H),8.73(s,1H),9.39(t,1H),11.60(s,1H).

[0053] S5, 40 g of compound 5 in step S4 was placed in a dry flask, 400 ml of dichloromethane was added to dissolve, 4.9 g of succinic anhydride and 9 ml of triethylamine were added in sequence, the reaction was completed at room temperature, 200 ml of water was added to quench, extraction was performed, the organic phase was collected, and the solvent was evaporated under reduced pressure to obtain compound 6, which was directly used in the next step reaction. Compound 5 was subjected to reaction e to obtain compound 6, and reaction e was

[0054]

[0055] S6, 10 g of compound 6 in step S5 was placed in a dry flask, dichloromethane was added to dissolve, and then 8 g of DCC, 5 g of HOBt, 10 ml of triethylamine, and controlled glass bead powder CPG were added in sequence, stirred at room temperature for 10 hours, filtered, and the solid was collected to obtain compound 7, i.e., FAM-dT-CPG. Compound 6 was obtained by reaction f, and reaction f was:

[0056]

[0057] In summary, the present application provides a method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier, which has a simple method, high yield, and achieves the purpose of efficient fluorescence diagnosis. And FAM is a common fluorescent dye, with an excitation wavelength of 498nm and an emission wavelength of 525nm, which is commonly used in fluorescent probes. FAM can also be modified on deoxynucleoside dT to form a FAM-dT phosphoramidite monomer, which can be marked to any site of the probe in the synthesis of the DNA probe, or it can be loaded on a solid phase carrier to form a FAM-dT-solid phase carrier, such as: FAM-dT-CPG (PS), which is used for 3' end modification of the DNA probe. The present invention especially introduces in detail the synthesis method of the solid phase carrier of FAM-dT-CPG (PS).

[0058] Although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each implementation mode may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0059] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent implementation methods or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent dye-modified deoxynucleoside solid phase carrier, characterized in that: The steps include: S1, fully dissolving the deoxynucleoside in a first organic solvent, adding 4,4'-dimethoxytriphenylmethane, and separating compound 2 after sufficient reaction; the deoxynucleoside is 5-(2-carboxyvinyl)-2'-deoxyuridine, and the first organic solvent is any one of pyridine, DMF, dichloromethane, ethyl acetate, and tetrahydrofuran; S2, fully dissolving the compound 2 in step S1 in a second organic solvent, adding a first condensing agent and a diamine trifluoroacetamide derivative, and separating the compound 3 after sufficient reaction, wherein the second organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, ether, and acetonitrile, and the first condensing agent is any one of DCC, EDC, HOBt, HBTu, HATu, and PyBop; S3, fully dissolving the compound 3 in step S2 into a methanol solution, adding an alkaline substance, and separating the compound 4 after sufficient reaction; S4, fully dissolving the compound 4 in step S3 into a third organic solvent, adding a fluorescent dye activated ester, and separating the compound 5 after sufficient reaction at a temperature of 60°C-90°C; the fluorescent dye activated ester is a FAM activated ester, and the third organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, ether, and acetonitrile; S5, fully dissolving the compound 5 in step S4 in a fourth organic solvent, adding anhydride and an organic base in sequence, and separating the compound 6 after sufficient reaction; the anhydride is any one of succinic anhydride, glutaric anhydride, and diethanol anhydride; the organic base is any one of triethylamine, pyridine, diisopropylammonium, and diisopropylethylamine; the fourth organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, diethyl ether, and acetonitrile; S6. Fully dissolve the compound 6 in step S5 in a fifth organic solvent, add a second condensing agent and a solid phase carrier in sequence, and separate the fluorescent dye-modified deoxynucleoside solid phase carrier after sufficient reaction; the second condensing agent is any one of DCC, EDC, HOBt, HBTu, HATu, and PyBop; the solid phase carrier is any one of controllable glass bead powder CPG and aminomethyl polystyrene resin, and the fifth organic solvent is any one of DMF, dichloromethane, chloroform, ethyl acetate, ether, and acetonitrile.

2. The method for preparing the fluorescent dye-modified deoxynucleoside solid phase carrier according to claim 1, characterized in that: In step S2, the diamine trifluoroacetamide derivative is any one of ethylenediamine trifluoroacetamide, propylenediamine trifluoroacetamide, butylenediamine trifluoroacetamide, pentanediamine trifluoroacetamide, and hexanediamine trifluoroacetamide.

3. The method for preparing the fluorescent dye-modified deoxynucleoside solid phase carrier according to claim 1, characterized in that: In step S3, the alkaline substance is at least one of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, ammonia water, and methylamine.

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