A purification method for fluorescein-labeled nucleoside triphosphate

Abstract

The present application discloses a method for purifying a fluorescein-labeled nucleoside triphosphate. In the present application, a liquid chromatography method is used to purify a crude product of a fluorescein-labeled nucleoside triphosphate, and a target fraction is collected; wherein, the mobile phase comprises a first component and a second component, the first component is a hexylamine acetate aqueous solution, and the second component is selected from acetonitrile or methanol. The method for purifying a fluorescein-labeled nucleoside triphosphate of the present invention can separate a nucleoside diphosphate, which is similar in structure to the nucleoside triphosphate and difficult to separate, from the target product in the crude product of the fluorescein-labeled nucleoside triphosphate, and obtain a fluorescein-labeled nucleoside triphosphate with a purity greater than 99%. The fluorescein-labeled nucleoside triphosphate purified by the method for purifying a fluorescein-labeled nucleoside triphosphate of the present invention has good fluorescence intensity and high sensitivity, meeting the requirements of fluorescence in situ hybridization experiments.

Description

Technical Field

[0001] Embodiments of the present invention relate to the field of biomedicine, and particularly to a purification method for fluorescein-labeled nucleoside triphosphate. Background Art

[0002] Fluorescence in situ hybridization (FISH) is a non-radioactive in situ hybridization technique developed on the basis of the original radioactive in situ hybridization technique in the early 1980s. Bauman labeled RNA with fluorescence and used it as a probe for DNA detection. Its basic principle is to use a known labeled single-stranded nucleic acid as a probe, and according to the principle of base complementarity, specifically bind to the unknown single-stranded nucleic acid in the material to be detected to form a detectable hybrid double-stranded nucleic acid. Since DNA molecules are linearly arranged along the longitudinal axis of chromosomes, probes can directly hybridize with chromosomes to localize specific genes on chromosomes. Compared with traditional radioactive-labeled in situ hybridization, fluorescence in situ hybridization has the advantages of safety, rapidity, high sensitivity, strong detection signal, high hybridization specificity, and the ability to display multiple colors simultaneously. It can not only display metaphase spreads but also interphase nuclei.

[0003] In terms of methods, fluorescence in situ hybridization (FISH) mainly includes chromosome painting, multicolor in situ primed labeling, comparative genomic hybridization, fluorescence in situ hybridization on DNA fibers, etc. Among them, multicolor in situ primed labeling uses oligonucleotides as primers to in situ PCR amplify the sequence to be detected, and during this process, nucleoside triphosphates directly or indirectly labeled with fluorescein are incorporated, so that the amplified sequences are all labeled. Through several rounds of such amplification, the in situ amplification products of several sequences to be detected are labeled with different fluoresceins, realizing the simultaneous detection of multiple chromosomal micro-changes such as micro-deletions and mutations. This method has been routinely used to detect the localization of specific microsatellite sequences in chromosomes and interphase nuclei. Among them, the nucleoside triphosphates incorporated with direct fluorescein labeling are more common, which can ensure that each specific nucleotide incorporated has fluorescein modification and better signal stability.

[0004] The inventors found that there are at least the following problems in the prior art: When preparing fluorescein-labeled nucleoside triphosphate, reverse-phase high-performance liquid chromatography (HPLC) is used to purify and separate the crude product, but the final product after separation has poor fluorescence emission intensity and insufficient sensitivity, and cannot meet the needs of fluorescence in situ hybridization experiments. Therefore, it is necessary to develop a more effective method for purifying the crude product of fluorescein-labeled nucleoside triphosphate. Summary of the Invention

[0005] The purpose of the embodiments of the present invention is to provide a purification method for fluorescein-labeled nucleoside triphosphate, so that the prepared fluorescein-labeled nucleoside triphosphate has higher purity.

[0006] To solve the above technical problems, an embodiment of the present invention provides a method for purifying a fluorescein-labeled nucleoside triphosphate, comprising the following steps:

[0007] Purify the crude product of the fluorescein-labeled nucleoside triphosphate by liquid chromatography, collect the target fraction, and remove the solvent to obtain the product; wherein the mobile phase comprises a first component and a second component, the first component is an aqueous solution of n-hexylamine acetate, and the second component is acetonitrile or methanol.

[0008] In some preferred embodiments, the crude product of the fluorescein-labeled nucleoside triphosphate is obtained by reacting fluorescein and nucleoside triphosphate in a borate solution and purified by reverse-phase liquid chromatography, and the mobile phase in the reverse-phase liquid chromatography is triethylamine acetate and acetonitrile.

[0009] In some preferred embodiments, the method for removing the solvent comprises the step of rotary evaporation of the target fraction.

[0010] In some preferred embodiments, the mobile phase consists of a first component and a second component.

[0011] In some preferred embodiments, the first component is an aqueous solution of n-hexylamine acetate, and the second component is acetonitrile.

[0012] In some preferred embodiments, relative to the total volume of the mobile phase, the volume ratio of the first component is not less than 50%; more preferably not less than 60%, more preferably not less than 65%, and more preferably not less than 70%.

[0013] In some preferred embodiments, relative to the total volume of the mobile phase, the volume ratio of the first component is not more than 70%, more preferably not more than 65%, more preferably not more than 60%, and more preferably not more than 50%.

[0014] In some preferred embodiments, relative to the total volume of the mobile phase, the volume ratio of the first component is not less than 50% and not more than 70%.

[0015] In some preferred embodiments, the liquid chromatography uses an isocratic elution program.

[0016] In some preferred embodiments, in the isocratic elution program, the composition of the mobile phase is as follows: the volume of the first component: the volume of the second component = a’:b’, where a’ is 60-80, b’ is 20-40, and a’ + b’ = 100.

[0017] In some preferred embodiments, the liquid chromatography uses a gradient elution program.

[0018] In some preferred embodiments, the gradient elution program at least includes an elution stage, and the elution stage at least includes a first stage with a constant composition of the mobile phase and a second stage with a uniformly changing composition of the mobile phase;

[0019] In the first stage, the mobile phase has a first mobile phase composition, and the first mobile phase composition is: the volume of the first component: the volume of the second component = a 1 :b 1 ;

[0020] In the second stage, the mobile phase uniformly changes from the first mobile phase composition to the second mobile phase composition, and the second mobile phase composition is: the volume of the first component: the volume of the second component = a 2 :b 2 ;

[0021] wherein, a 1 is 75 - 85, b 1 is 15 - 25, a 2 is 55 - 65, b 2 is 35 - 45, and a 1 +b 1 = 100, a 2 +b 2 = 100.

[0022] In some preferred embodiments, the time of the first stage is not less than 1 minute; more preferably not less than 2 minutes.

[0023] In some preferred embodiments, the time of the second stage is not less than 10 minutes, more preferably not less than 12 minutes; more preferably not less than 15 minutes; more preferably not less than 19 minutes; more preferably not less than 20 minutes; more preferably not less than 23 minutes.

[0024] In some preferred embodiments, a third stage is further included after the second stage. In the third stage, the mobile phase uniformly changes from the second mobile phase composition to the third mobile phase composition, and the third mobile phase composition is: the volume of the first component: the volume of the second component = a 3 :b 3 ; wherein, a 3 is 8 - 12, b 3 is 88 - 92, and a 3 +b 3 = 100;

[0025] A fourth stage is further included after the third stage. In the fourth stage, the mobile phase is the third mobile phase composition.

[0026] In some preferred embodiments, the time of the third stage is 0.05 - 0.15 minutes.

[0027] In some preferred embodiments, the time of the fourth stage is 3 to 10 minutes, preferably 4 to 6 minutes.

[0028] In some preferred embodiments, a equilibration stage is included after the elution stage, and the equilibration stage includes a fifth stage in which the composition of the mobile phase changes uniformly and a sixth stage in which the composition of the mobile phase is constant;

[0029] In the fifth stage, the mobile phase changes uniformly from the composition of the third mobile phase to the composition of the first mobile phase;

[0030] In the sixth stage, the mobile phase is the composition of the first mobile phase.

[0031] In some preferred embodiments, the time of the fifth stage is 0.05 to 0.15 minutes.

[0032] In some preferred embodiments, the time of the sixth stage is 3 to 10 minutes, preferably 3 to 5 minutes.

[0033] In some preferred embodiments, when the flow rate is not more than 1.3 ml / min and not less than 0.7 ml / min, the gradient elution program is as follows:

[0034] Time (min) First Component (%) Second Component (%) 0 80 20 2 80 20 25 60 40 25.1 10 90 30 10 90 30.1 80 20 35 80 20

[0035] In some preferred embodiments, when the flow rate is 1.5 ml / min, the gradient elution program is as follows:

[0036]

[0037]

[0038] In some preferred embodiments, when the flow rate is 0.5 ml / min, the gradient elution program is as follows:

[0039] Time (min) First Component (%) Second Component (%) 0 80 20 3 80 20 40 60 40 40.1 10 90 50 10 90 50.1 80 20 60 80 20

[0040] In some preferred embodiments, the concentration of the n-hexylamine acetate aqueous solution is 80 to 120 mM, such as 100 mM.

[0041] In some preferred embodiments, the flow rate of the mobile phase is 0.5 to 1.5 ml / min, more preferably 0.7 to 1.3 ml / min.

[0042] In some preferred embodiments, the chromatographic column is a C 18 chromatographic column, for example: Waters Xbridge BEH 4.6 x 250 mm, 5 um.

[0043] In some preferred embodiments, the column temperature of the chromatographic column is 24 to 26 °C.

[0044] The embodiments of the present invention have at least the following advantages over the prior art:

[0045] (1) The purification method of fluorescein-labeled nucleoside triphosphate provided by the embodiments of the present invention has high efficiency in purifying fluorescein-labeled nucleoside triphosphate, and can separate the target product of nucleoside diphosphate, which is difficult to separate from the crude product of fluorescein-labeled nucleoside triphosphate and has a structure similar to that of nucleoside triphosphate, to obtain fluorescein-labeled nucleoside triphosphate with a purity greater than 99%.

[0046] (2) The fluorescein-labeled nucleoside triphosphate purified by the purification method of fluorescein-labeled nucleoside triphosphate provided by the embodiments of the present invention has good fluorescence intensity and high sensitivity, meeting the requirements of fluorescence in situ hybridization experiments.

[0047] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0048] One or more embodiments are illustrated by way of example in the pictures in the corresponding drawings, and these exemplary illustrations do not constitute a limitation on the embodiments.

[0049] Figure 1 is the chromatogram of the Fluorescein-12-dUTP product prepared by the method of Example 1 of the present invention;

[0050] Figure 2 is the chromatogram of the refined Fluorescein-12-dUTP product prepared by the method of Example 2 of the present invention;

[0051] Figure 3 is the mass spectrum of the impurities obtained in the method of Example 2 of the present invention;

[0052] Figure 4 is the mass spectrum of Fluorescein-12-dUTP obtained in the method of Example 2 of the present invention;

[0053] Figure 5 is the chromatogram of the refined Fluorescein-12-dUTP product prepared by the method of Example 3 of the present invention;

[0054] Figure 6 is the chromatogram of the Tetramethyl-Rhodamine-5-dUTP product prepared by the method of Example 4 of the present invention;

[0055] Figure 7 It is the chromatogram of the refined product of Tetramethyl - Rhodamine - 5 - dUTP prepared by the method of Example 5 of the present invention;

[0056] Figure 8 It is the mass spectrum of the impurities obtained in the method of Example 5 of the present invention;

[0057] Figure 9 It is the mass spectrum of the Tetramethyl - Rhodamine - 5 - dUTP product obtained in the method of Example 5 of the present invention. Detailed implementation mode

[0058] In the prior art, the fluorescence emission effect of the prepared fluorescein - labeled nucleoside triphosphate is poor and the sensitivity is low. The inventor speculated that it might be due to the insufficient purity of the fluorescein - labeled nucleoside triphosphate product. However, when the inventor used the traditional liquid chromatography method (the mobile phase is a methanol - water system), no impurities were found. After further research, the inventor found that by improving the mobile phase system and using the two - component mobile phase system with the first component being n - hexylamine acetate aqueous solution and the second component being acetonitrile or methanol, the target fluorescein - labeled nucleoside triphosphate product can be separated from the impurities. The inventor determined the specific structures of each peak after separation by mass spectrometry (the impurity peak is fluorescein - labeled nucleoside diphosphate and the target peak is fluorescein - labeled nucleoside triphosphate), while the traditional liquid chromatography method (the mobile phase is a methanol - water system) cannot separate the impurity peak and the target peak.

[0059] Based on the above - mentioned research findings, the inventor developed a purification method for fluorescein - labeled nucleoside triphosphate to obtain a product with better fluorescence emission effect and high sensitivity to meet the needs of in - situ fluorescence hybridization experiments.

[0060] Terms

[0061] As used herein, the term "nucleoside triphosphate" refers to a nucleotide containing three phosphate groups, including natural nucleoside triphosphates and deoxynucleoside triphosphates.

[0062] As used herein, the term "fluorescein" refers to: well - known to those skilled in the art, a chemical compound that re - emits light after being photo - excited. Non - limiting examples are CY5, EDANS, xanthine derivatives (e.g., fluorescein, rhodamine, Oregon green, HIV, Texas red), penem derivatives (e.g., indocyanine, oxazepam, merocyanine), amber derivatives (e.g., Seta, Se Tau, Square pigment), etc.

[0063] As used herein, the term "fluorescein-labeled nucleoside triphosphate" refers to a product obtained by directly or indirectly labeling a nucleoside triphosphate with fluorescein, and non-limiting examples include nucleoside triphosphates labeled with Fluorescein SFX.

[0064] As used herein, the term "liquid chromatography" refers to a method for separating components of a mixture by taking advantage of the different affinities of the components in the mixture for the two phases of the stationary phase and the mobile phase. For example, in the present invention, a high-performance liquid chromatograph is used to separate the impurities and the main product in the crude product of fluorescein-labeled nucleoside triphosphate by virtue of the different affinities of the components for the stationary phase and the mobile phase.

[0065] As used herein, the term "gradient elution program" refers to a means of separating and analyzing a sample by continuously changing the concentration ratio of the mobile phase to a certain extent within the same analysis cycle.

[0066] As used herein, the term "isocratic elution program" refers to a program for separating and analyzing a sample with a constant mobile phase composition within the same analysis cycle.

[0067] As used herein, the term "uniform change in the composition of the mobile phase" means that the composition of the mobile phase changes uniformly with time. For example, if the initial mobile phase composition is component A (90%) - component B (10%), and within one minute, it uniformly changes to component A (80%) - component B (20%), that is, within one minute, component A decreases by 1.67% per second, and at the same time, component B increases by 1.67% per second.

[0068] As used herein, the term "C 18 chromatographic column" refers to a chromatographic column in which the reverse-phase column packing is based on silica gel and has a non-polar octadecyl functional group structure bonded to its surface.

[0069] In high-performance liquid chromatography (HPLC), a pressurized liquid solvent containing a sample mixture is passed through a column filled with a solid adsorbent material, causing the components of the sample to interact with the adsorbent material. Since different components interact with the adsorbent material differently, this results in the separation of the components when they flow out of the column. A method for purifying a fluorescein-labeled nucleoside triphosphate provided by the present invention includes the following steps:

[0070] Purifying the crude product of fluorescein-labeled nucleoside triphosphate by liquid chromatography, collecting the target fraction, and removing the solvent to obtain the product; wherein the mobile phase includes a first component and a second component, the first component is an aqueous solution of n-hexylamine acetate, and the second component is acetonitrile or methanol.

[0071] In some preferred embodiments, the crude product of fluorescein-labeled nucleoside triphosphate is obtained by reacting fluorescein and nucleoside triphosphate in a borate solution and purifying by reverse-phase liquid chromatography, wherein the mobile phase in the reverse-phase liquid chromatography is triethylamine acetate and acetonitrile.

[0072] In some preferred embodiments, the method for removing the solvent includes the step of rotary evaporation of the target fraction.

[0073] In some preferred embodiments, the mobile phase consists of a first component and a second component.

[0074] In some preferred embodiments, the first component is an aqueous solution of n-hexylamine acetate, and the second component is acetonitrile.

[0075] In some preferred embodiments, relative to the total volume of the mobile phase, the volume ratio of the first component is not less than 50%; more preferably not less than 60%, more preferably not less than 65%, and more preferably not less than 70%.

[0076] In some preferred embodiments, relative to the total volume of the mobile phase, the volume ratio of the first component is not more than 70%, more preferably not more than 65%, more preferably not more than 60%, and more preferably not more than 50%.

[0077] In some preferred embodiments, relative to the total volume of the mobile phase, the volume ratio of the first component is not less than 50% and not more than 70%.

[0078] In some preferred embodiments, the liquid chromatography uses an isocratic elution program.

[0079] In some preferred embodiments, in the isocratic elution program, the composition of the mobile phase is as follows: the volume of the first component: the volume of the second component = a':b', where a' is 60 - 80, b' is 20 - 40, and a' + b' = 100.

[0080] In some preferred embodiments, the liquid chromatography uses a gradient elution program.

[0081] In some preferred embodiments, the gradient elution program at least includes an elution stage, and the elution stage at least includes a first stage with a constant composition of the mobile phase and a second stage with a uniformly changing composition of the mobile phase;

[0082] In the first stage, the mobile phase has a first mobile phase composition, and the first mobile phase composition is: the volume of the first component: the volume of the second component = a 1 :b 1 ;

[0083] In the second stage, the mobile phase changes uniformly from the first mobile phase composition to the second mobile phase composition, and the second mobile phase composition is: the volume of the first component: the volume of the second component = a 2 :b 2 ;

[0084] wherein, a 1 is 75 - 85, b 1 is 15 - 25, a 2 is 55 - 65, b 2 is 35 - 45, and a 1 +b 1 = 100, a 2 +b 2 = 100.

[0085] In some preferred embodiments, the time of the first stage is not less than 1 minute; more preferably not less than 2 minutes.

[0086] In some preferred embodiments, the time of the second stage is not less than 10 minutes, more preferably not less than 12 minutes; more preferably not less than 15 minutes; more preferably not less than 19 minutes; more preferably not less than 20 minutes; more preferably not less than 23 minutes.

[0087] In some preferred embodiments, after the second stage, there is further a third stage. In the third stage, the mobile phase changes uniformly from the second mobile phase composition to the third mobile phase composition, and the third mobile phase composition is: the volume of the first component: the volume of the second component = a 3 :b 3 ; wherein, a 3 is 8 - 12, b 3 is 88 - 92, and a 3 +b 3 = 100;

[0088] After the third stage, there is further a fourth stage. In the fourth stage, the mobile phase is the third mobile phase composition.

[0089] In some preferred embodiments, the time of the third stage is 0.05 - 0.15 minutes.

[0090] In some preferred embodiments, the time of the fourth stage is 3 - 10 minutes, preferably 4 - 6 minutes.

[0091] In some preferred embodiments, after the elution stage, there is further a balance stage. The balance stage includes a fifth stage in which the composition of the mobile phase changes uniformly and a sixth stage in which the composition of the mobile phase is constant;

[0092] In the fifth stage, the mobile phase changes uniformly from the composition of the third mobile phase to the composition of the first mobile phase;

[0093] In the sixth stage, the mobile phase is the composition of the first mobile phase.

[0094] In some preferred embodiments, the time of the fifth stage is 0.05 - 0.15 minutes.

[0095] In some preferred embodiments, the time of the sixth stage is 3 - 10 minutes, preferably 3 - 5 minutes.

[0096] In some preferred embodiments, when the flow rate is not greater than 1.3 ml / min and not less than 0.7 ml / min, the gradient elution program is as follows:

[0097] Time (min) First Component (%) Second Component (%) 0 80 20 2 80 20 25 60 40 25.1 10 90 30 10 90 30.1 80 20 35 80 20

[0098] In some preferred embodiments, when the flow rate is 1.5 ml / min, the gradient elution program is as follows:

[0099] Time (min) First Component (%) Second Component (%) 0 80 20 1 80 20 16 60 40 16.1 10 90 19 10 90 19.1 80 20 22 80 20

[0100] In some preferred embodiments, when the flow rate is 0.5 ml / min, the gradient elution program is as follows:

[0101] Time (min) First Component (%) Second Component (%) 0 80 20 3 80 20 40 60 40 40.1 10 90 50 10 90 50.1 80 20 60 80 20

[0102] In some preferred embodiments, the concentration of the n-hexylamine acetate aqueous solution is 80 - 120 mM, such as 100 mM.

[0103] In some preferred embodiments, the flow rate of the mobile phase is 0.5 - 1.5 ml / min, more preferably 0.7 - 1.3 ml / min.

[0104] In some preferred embodiments, the chromatographic column is a C 18 chromatographic column, for example: Waters XbridgeBEH4.6x250mm, 5um.

[0105] In some preferred embodiments, the column temperature of the chromatographic column is 24 - 26 °C. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0106] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0107] Example 1. Preparation of Fluorescein-12-dUTP

[0108] Dissolve 5 mg of Fluorescein SFX (ThermoFisher) in 100 μl of DMSO solution and shake for 5 min to dissolve; dissolve 1 mg of AA-dUTP (biotium) in 300 μl of 0.1 M borate at pH 9.0 and shake for 5 min to dissolve. Take 35 μl of Fluorescein SFX and add it to the above-mentioned borate solution of AA-dUTP, and shake at room temperature for 2 hours to end the reaction. Coarse purification of the reaction solution is carried out as follows:

[0109] Filter the above reaction solution through a 0.22-μm organic filter membrane, and then purify it by reverse-phase C 18 (Chromatographic column: Waters Xbridge BEH 4.6x250 mm, 5 μm, mobile phase A: 100 mM triethylamine acetate (TEAA), B: acetonitrile). Inject three times, collect the target fractions and concentrate them to obtain the purified product of Fluorescein-12-dUTP.

[0110] The purification conditions of the above-mentioned Fluorescein-12-dUTP are shown in Table 1:

[0111] Table 1

[0112]

[0113]

[0114] The obtained chromatogram is shown in Figure 1 .

[0115] Under the condition of using triethylamine acetate (TEAA) as the mobile phase, the purification chromatogram is a single peak and no impurity peaks appear.

[0116] However, subsequent experiments found that the product prepared by the above method has poor fluorescence emission intensity and insufficient sensitivity, and cannot meet the needs of fluorescence in situ hybridization experiments. After a large number of tests and data analysis, the inventor suspects that it is caused by the presence of unknown impurities in the purified product of Fluorescein-12-dUTP.

[0117] Example 2. Refinement of Fluorescein-12-dUTP

[0118] Dissolve the purified product of Example 1 Fluorescein-12-dUTP in 500 μl of deionized water and refine it according to the conditions in Table 2 below:

[0119] Table 2

[0120]

[0121] The obtained chromatogram is shown in Figure 2 . Among them, the retention time of 16.294 min is the nucleoside diphosphate impurity labeled with Fluorescein SFX, and the retention time of 18.927 min is the nucleoside triphosphate product labeled with Fluorescein SFX. It can be seen that the resolution between the product and the impurity is good.

[0122] Detect the nucleoside diphosphate impurity labeled with Fluorescein SFX with a retention time of 16.294 min (negative ion mode) by LC-MS (the mass spectrum is shown in Figure 3 ), and the nucleoside triphosphate product labeled with Fluorescein SFX with a retention time of 18.927 min (negative ion mode) (the obtained mass spectrum is shown in Figure 4 ).

[0123] According to the above mass spectrum data, the theoretical molecular weight of the nucleoside triphosphate labeled with Fluorescein SFX is 994.69, Figure 4 among which the molecular ion peak in the negative ion mode is 993.59, and the structure is correct.

[0124] The theoretical molecular weight of the nucleoside diphosphate impurity labeled with Fluorescein SFX is 914.69, Figure 3 among which the molecular ion peak in the negative ion mode is 913.05, and the structure is correct.

[0125] Collect the fractions with a retention time of 17.5 - 19.5 min, and rotary evaporate to remove the mobile phase to obtain the purified nucleoside triphosphate labeled with Fluorescein SFX.

[0126] Example 3. Refinement of Fluorescein-12-dUTP

[0127] Dissolve the purified product of Fluorescein-12-dUTP obtained in Example 1 in 500 μl of deionized water and purify it according to the conditions in Table 3 below:

[0128] Table 3

[0129]

[0130] The obtained chromatogram is shown inFigure 5 Among them, the retention time of 9.501 min is the nucleoside diphosphate impurity labeled with Fluorescein SFX, and the retention time of 13.510 min is the nucleoside triphosphate product labeled with Fluorescein SFX. The resolution between the product and the impurity is good.

[0131] Collect the fractions with a retention time of 12.5 - 16.0 min, and remove the mobile phase by rotary evaporation to obtain the purified nucleoside triphosphate labeled with Fluorescein SFX.

[0132] Example 4. Preparation of Tetramethyl - Rhodamine - 5 - dUTP

[0133] Dissolve 5 mg of Rhodamine NHS in 100 μl of DMSO solution and shake for 5 min to dissolve; dissolve 1 mg of AA - dUTP (biotium) in 300 μl of 0.1 M borate with a pH of 9.0 and shake for 5 min to dissolve. Take 35 μl of Rhodamine NHS and add it to the above AA - dUTP borate solution, and shake at room temperature for 2 hours to end the reaction. Coarse purification of the reaction solution is carried out as follows:

[0134] Filter the above reaction solution through a 0.22 - μm organic filter membrane. After filtration, purification is carried out using reverse - phase C 18 (chromatographic column: Waters Xbridge BEH 4.6x250 mm, 5 μm, mobile phase A: 100 mM triethylammonium acetate (TEAA), B: acetonitrile). Inject the sample three times, collect the target fractions and concentrate them to obtain the purified product of Tetramethyl - Rhodamine - 5 - dUTP.

[0135] The purification conditions of the above - mentioned Tetramethyl - Rhodamine - 5 - dUTP are shown in Table 4:

[0136] Table 4

[0137]

[0138] The obtained chromatogram is shown in Figure 6 .

[0139] The purification chromatogram under the condition of using triethylammonium acetate (TEAA) as the mobile phase is a single peak.

[0140] Example 5. Refinement of Tetramethyl - Rhodamine - 5 - dUTP

[0141] Dissolve the purified product of Tetramethyl-Rhodamine-5-dUTP obtained in Example 4 in 500 μl of deionized water and refine it according to the conditions in Table 5 below:

[0142] Table 5

[0143]

[0144]

[0145] The obtained chromatogram is shown in Figure 7 . Among them, the retention time of 19.025 min is the impurity of Rhodamine NHS-labeled nucleoside diphosphate, and the retention time of 21.884 min is the product of Rhodamine NHS-labeled nucleoside triphosphate. It can be seen that the separation degree between the product and the impurity is good.

[0146] Detect the Rhodamine NHS-labeled nucleoside diphosphate impurity with a retention time of 19.025 min (negative ion mode) by LC-MS (the mass spectrum is shown in Figure 8 ), and the Rhodamine NHS-labeled nucleoside triphosphate product with a retention time of 21.884 min (negative ion mode) (the obtained mass spectrum is shown in Figure 9 ).

[0147] According to the above mass spectrum data, the theoretical molecular weight of Rhodamine NHS-labeled nucleoside triphosphate is 936.67, Figure 9 in which the molecular ion peak in the negative ion mode is 933.99, and the structure is correct.

[0148] The theoretical molecular weight of Rhodamine NHS-labeled nucleoside diphosphate is 856.67, Figure 8 in which the molecular ion peak in the negative ion mode is 854, and the structure is correct.

[0149] Collect the fractions with a retention time of 21.00 - 22.00 min, rotary evaporate to remove the mobile phase, and obtain the purified Rhodamine NHS-labeled nucleoside triphosphate.

[0150] Those of ordinary skill in the art can understand that the above embodiments are specific examples for implementing the present invention, and in practical applications, various changes can be made in form and details without departing from the spirit and scope of the present invention.

Claims

1. A purification method for fluorescein-labeled nucleoside triphosphate, characterized in that, it comprises the following steps: Purify the crude product of fluorescein-labeled nucleoside triphosphate by liquid chromatography and collect the target fraction; wherein, the mobile phase consists of A and B, wherein mobile phase A is an aqueous solution of n-hexylamine acetate with a concentration of 80 - 120 mM, and mobile phase B is acetonitrile; The chromatographic column is C 18 The chromatographic column; isocratic elution or gradient elution is adopted; detection is carried out at 495 nm; The isocratic elution program is: The gradient elution program is: Wherein, the crude product of fluorescein-labeled nucleoside triphosphate is obtained by reacting fluorescein and nucleoside triphosphate in a borate solution and purifying by reverse-phase liquid chromatography. In the reverse-phase liquid chromatography, the mobile phase is triethylamine acetate and acetonitrile; the fluorescein is Fluorescein SFX, the nucleoside triphosphate is AA-dUTP, and the crude product of fluorescein-labeled nucleoside triphosphate contains fluorescein-labeled nucleoside diphosphate.

2. The method according to claim 1, wherein, the column temperature of the chromatographic column is 25 °C.

3. The method according to claim 1, wherein, The chromatographic column is Waters Xbridge BEH C 18 4.6x250mm, 5um.

4. The method according to claim 1, wherein, mobile phase A is an aqueous solution of 100 mM n-hexylamine acetate.

5. The method according to claim 1, wherein, the liquid chromatography is Agilent 1260.

6. The method according to claim 1, wherein, when the elution program is isocratic elution, the flow rate of the mobile phase is 1.5 ml / min; when the elution program is gradient elution, the flow rate of the mobile phase is 1 ml / min.

7. A purification method for fluorescein-labeled nucleoside triphosphate, characterized in that, it comprises the following steps: Purify the crude product of fluorescein-labeled nucleoside triphosphate by liquid chromatography and collect the target fraction; wherein, the mobile phase consists of A and B, wherein mobile phase A is an aqueous solution of n-hexylamine acetate with a concentration of 80 - 120 mM, and mobile phase B is acetonitrile; The chromatographic column is C 18 Chromatographic column; gradient elution is adopted; the gradient elution program is as follows: Detect at 552 nm; Wherein, the crude product of fluorescein-labeled nucleoside triphosphate is obtained by reacting fluorescein and nucleoside triphosphate in a borate solution and purifying by reverse-phase liquid chromatography. In the reverse-phase liquid chromatography, the mobile phase is triethylamine acetate and acetonitrile; the fluorescein is Rhodamine NHS, the nucleoside triphosphate is AA-dUTP, and the crude product of fluorescein-labeled nucleoside triphosphate contains fluorescein-labeled nucleoside diphosphate.

8. The method according to claim 7, wherein, the column temperature of the chromatographic column is 25 °C; and / or, the chromatographic column is Waters Xbridge BEH C 18 4.6x250mm, 5um; and / or, the liquid chromatography is Agilent 1260; and / or, the flow rate of the mobile phase is 1 ml / min.

9. The method according to claim 7, wherein, mobile phase A is an aqueous solution of 100 mM n-hexylamine acetate.

Images