CN114736260A - Preparation method of nucleotide triphosphate - Google Patents

Preparation method of nucleotide triphosphate Download PDF

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CN114736260A
CN114736260A CN202210326177.5A CN202210326177A CN114736260A CN 114736260 A CN114736260 A CN 114736260A CN 202210326177 A CN202210326177 A CN 202210326177A CN 114736260 A CN114736260 A CN 114736260A
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蔡晓茹
郭传鑫
彭祥然
李松
钱其军
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Maxirna Shanghai Pharmaceutical Co Ltd
Maxirna Zhejiang Technology Co Ltd
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Maxirna Shanghai Pharmaceutical Co Ltd
Maxirna Zhejiang Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07H1/06—Separation; Purification

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Abstract

The invention discloses a preparation method of nucleotide triphosphate. The invention provides a preparation method of nucleotide triphosphate triethylamine salt, which comprises the following steps: separating and preparing the active ingredient to be purified of the nucleotide triphosphate shown as the formula C by adopting a chromatography method to obtain the nucleotide triphosphate triethylamine salt shown as the formula D. The preparation method realizes large-scale automatic preparation of various modified and non-modified nucleotide triphosphates, and has good universality and high separation degree.

Description

Preparation method of nucleotide triphosphate
Technical Field
The invention relates to a preparation method of nucleotide triphosphate.
Background
With the development of nucleic acid drugs, mRNA is considered as a new choice that can be used in drug manufacturing. In 1990, a segment of mRNA was injected into mice and successfully encoded a protein. This mRNA is obtained by a technique known as in vitro transcription. Subsequently, a 1992 study found that injection of the antidiuretic hormone-encoding mRNA successfully induced neuronal activity in the hypothalamus of rats. Although mRNA shows excellent biological activity, mRNA is far from being used in clinical disease therapy due to its instability, strong immunogenicity, and difficulty in vivo delivery.
The addition of the modified nucleotide can reduce the self-immunogenicity of mRNA, improve the self-stability of the mRNA, further enhance the expression time and the expression efficiency of the mRNA in target cells, and ensure that the mRMA can be really used in the pharmaceutical field. Wherein, the Moderna and the BioNTech respectively adopt 1-methyl pseudouridine (CN110511939A, CN104114572A, CN103974724A) and pseudouridine (US10232055B2, US9597380B2, US9163213B2), thereby greatly reducing the immunogenicity of mRNA, improving the time and the total amount of target protein expressed in target cells by the mRNA, and laying a foundation for preparing the mRNA into a COVID-19 vaccine and successfully marketing the mRNA.
However, the preparation process of the modified nucleotide triphosphate for mRNA synthesis is complex, especially the purification is very difficult and the product stability is poor, so that the commodity price of the modified nucleotide is as high as hundreds of thousands or even millions of yuan per gram. The high-purity modified nucleotide triphosphate is mainly prepared by an organic synthesis method at the present stage, and is separated and purified by large-scale preparation liquid chromatography, so that the equipment price is high, a large amount of high-purity organic solvent is consumed, the purification efficiency is poor, and toxic organic waste liquid is generated, which is considered as a main reason for the high price of the modified nucleotide. Therefore, the research on the purification method of nucleotide triphosphates is urgent.
Disclosure of Invention
The invention provides a preparation method of nucleotide triphosphate, aiming at solving the problems of expensive purification method of nucleotide triphosphate compound, low substrate applicability and the like in the prior art. The method successfully realizes large-scale automatic preparation of various modified and non-modified nucleotide triphosphates by using the anion exchange column and the triethylamine carbonate buffer solution, does not need to use a large amount of organic solvent and expensive preparation liquid chromatography, greatly reduces the preparation cost of the nucleotide triphosphates, and has the advantages of high preparation purity of more than 99 percent, good universality and high separation degree.
The invention provides a preparation method of nucleotide triphosphate triethylamine salt, which comprises the following steps: separating and preparing a substance to be purified with the active ingredient of the nucleotide triphosphate shown as the formula C by adopting a chromatography method to obtain the nucleotide triphosphate triethylamine salt shown as the formula D;
Figure BDA0003571669580000021
wherein R is a basic group, R' is-OH, -OCH3Or H;
the chromatographic column is an anion exchange column, the ligand of the anion exchange column packing is quaternary ammonium group or diethylamine ethyl (DEAE), and the mobile phase comprises triethylamine-carbonic acid buffer solution (TEAB).
The separation preparation scheme comprises the following scheme I or scheme II,
the first scheme is as follows: the ligand of the anion exchange column packing is a quaternary ammonium group; separating and preparing by taking water as Buffer A and triethylamine-carbonic acid Buffer solution as Buffer B;
scheme II: the ligand of the anion exchange column packing is diethylamine ethyl; the preparation method is characterized by taking 10mM triethylamine-carbonic acid Buffer solution as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B for separation.
The base may be a natural base or a modified base, for example one of the following structures:
Figure BDA0003571669580000022
the matrix of the anion exchange column packing can adopt a matrix known in the art, and is preferably cross-linked dextran, highly cross-linked agarose with dextran surface extender, Polyacrylate (PMMA), sepharose, monodisperse polystyrene-divinylbenzene (PS/DVB), monodisperse polystyrene or polymethacrylate.
In scheme one, the packing material of the anion exchange column can be UniQ, NanoQ or Generic MC-Q.
In scheme two, the anion exchange column may be a DEAE-Sephadex A250, Capto DEAE, UniDEAE, Unicore-DEAE, DEAE Sepharose6FF or Generic MC-DEAE, such as DEAE-Sephadex A250, Capto DEAE, Unigel-DEAE or DEAE Sepharose6 FF.
The diameter of the anion exchange column may be as conventional in the art, for example 5 to 100mm, further for example 7.7 to 10 mm.
The height of the anion exchange column may be a column length as conventional in the art, preferably 50-400mm, such as 100-300mm, and further such as 200-220 mm.
In the first embodiment, the anion exchange column packing has a particle size of 10 to 30 μm, for example 15 μm.
In variant II, the anion exchange column packing has a particle size of 40 to 120. mu.m, for example 50 to 90 μm.
The anion exchange column may have a column temperature of 4 to 20 deg.C, for example 4 deg.C.
The initial concentration of the mobile phase is less than the concentration at which the triethylamine triphosphate salt of nucleotide triphosphate is eluted.
In the first scheme, the Buffer A is ultrapure water.
In the first scheme, the Buffer B is 1-2M triethylamine-carbonic acid Buffer solution.
In the first embodiment, the separation preparation comprises the following steps: (1) balancing by using A Buffer with 3-5 times of column volume; (2)1-3 column volumes A Buffer wash, e.g., 2-2.5 column volumes; (3) eluting by 5-20 times of column volume, wherein the final proportion of B Buffer is 18% -80%; e.g., 5.5-8 column volumes, the final proportion of B Buffer is 18% -30%; and for example 6.5 column volumes, the final proportion of B Buffer is 20-30%; (4) eluting residual impurities by 1-2 times of the column volume, wherein the proportion of B Buffer is 50-100%.
In scheme two, the separation preparation comprises the following steps: (1) balancing by A Buffer with 3-5 times of column volume; (2) washing impurities by using A Buffer in 0.5-2 times of column volume; (3) eluting by 8-20 times of column volume, wherein the final proportion of the B Buffer is 12-100%; (4) eluting residual impurities by 1-2 times of the column volume, wherein the proportion of B Buffer is 60-100%.
The elution may comprise a linear gradient elution and/or a step gradient elution, for example, by gradually increasing the B Buffer ratio in a linear gradient manner and collecting the sample solution corresponding to the chromatographic peak.
In scheme one, when the elution is combined with linear gradient elution and step gradient elution, it may comprise the following steps: and (3) preliminarily eluting impurities in a step gradient elution mode, wherein the proportion of B Buffer is 20%, and gradually increasing the proportion of B Buffer in a linear gradient mode, wherein the proportion of B Buffer is 30%.
In scheme two, when the elution is combined with linear gradient elution and step gradient elution, it comprises the following steps: the impurities are preliminarily eluted in a step gradient elution mode, wherein the proportion of the B Buffer is 12 percent, and then the proportion of the B Buffer is gradually increased in a linear gradient mode, wherein the proportion of the B Buffer is 30 percent.
The separation preparation can adopt any one of the following methods:
the method 101 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking NanoQ as a filler, ultrapure water as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 80%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 102 comprises the following steps: the method comprises the following steps of (1) performing separation preparation by using UniQ as a filler, using ultrapure water as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 80%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 103 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 80%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 104 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) performing linear gradient elution by 8 times of column volume, wherein the final proportion of B Buffer is 22.5%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 105 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3)5.5 times of column volume, and the final proportion of B Buffer is 22.5%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 106 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by Buffer in 2.5 times of column volume A; (3) performing step gradient elution by 0.5 times of column volume, wherein the proportion of B Buffer is 20 percent, performing linear gradient elution by 6 times of column volume, and the final proportion of B Buffer is 30 percent; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 107 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume; (3) performing linear gradient elution by 8 times of column volume, wherein the final proportion of B Buffer is 22.5%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 108 comprises the following steps: the method comprises the following steps of (1) performing separation preparation by taking Generic MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume; (3) performing linear gradient elution by 8 times of column volume, wherein the final proportion of B Buffer is 18%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 201: using DEAE-Sephadex A250 as a filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A, and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1)5 times of column volume of A Buffer balance; (2) washing impurities by using Buffer in 2 times of column volume A; (3) gradient elution is carried out by 1 time of column volume, and the proportion of B Buffer is 12%; then linear gradient elution is carried out by 8 times of column volume, and the final proportion of B Buffer is 30 percent; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 60 percent or 100 percent;
the method 202 comprises the following steps: the separation preparation method comprises the following steps of (1) balancing A Buffer with 5 times of column volume by taking Capto DEAE as a filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution is carried out by 20 times of column volume, the initial proportion of B Buffer is 10 percent, and the final proportion is 100 percent; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 203 comprises the following steps: using UniGel-DEAE as filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, and the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 100%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 204 comprises the following steps: using DEAE Sepharose6FF as a filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A, and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1)5 times of column volume of A Buffer balance; (2) washing impurities by using Buffer in 2 times of column volume A; (3) performing linear gradient elution by 10 times of column volume, wherein the initial proportion of B Buffer is 10 percent, and the final proportion is 100 percent; (4) the remaining impurities were eluted at 1 column volume, the B Buffer ratio was 100%.
The said purified material may be pre-treated before elution according to the routine in the art to meet the standard of injection. The pretreatment may include the steps of: and dissolving the substance to be purified in a TEAB solution to obtain a sample to be loaded. The concentration of the substance to be purified in the TEAB solution may be 0-100mg/mL, for example 10-20 mg/mL.
The said purified product is synthesized by Yoshikawa method or Ludwig-Eckstein method.
The synthesis of the substance to be purified may comprise the following steps:
(i) in methyl triphosphate, carrying out phosphorylation reaction on the compound shown in the formula A and phosphorus oxychloride as shown in the specification to obtain a compound shown in the formula B;
(ii) tri-n-butylamine pyrophosphate ((Bu)3N)2H4P2O7) Tri-n-butylamine (Bu)3N) and a compound shown as a formula B are subjected to a reaction shown as the following, and water is added for quenching reaction to obtain a compound shown as a formula C;
Figure BDA0003571669580000051
wherein R, R' is as defined above.
In the step (i), the molar ratio of the phosphorus oxychloride to the compound shown in the formula A can be 1-2: 1, e.g. 1.5: 1.
in the step (i), the volume-to-mass ratio of the methyl triphosphate to the compound shown in the formula A can be 10-20mL/g, for example 14.9 mL/g.
In step (i), the reaction temperature of the reaction may be-10 ℃ to 20 ℃, for example 15 ℃.
In step (i), the reaction time of the reaction may be 20min to 40min, for example 30 min.
In the step (ii), the water is preferably ultrapure water.
In the step (ii), the tri-n-butylamine pyrophosphate ((Bu)3N)2H4P2O7) The molar ratio of the compound shown in the formula B to the compound shown in the formula B can be (5-10): 1, e.g. 5: 1.
in the step (ii), the molar ratio of the tri-n-butylamine to the compound shown in the formula B can be (4-8): 1, e.g., 6: 1.
in step (ii), the reaction temperature of the reaction may be from 5 ℃ to 20 ℃, for example 15 ℃.
In step (ii), the reaction time of the reaction may be 5min to 20min, for example 15 min.
The preparation method of the nucleotide triphosphate triethylamine salt can also comprise the following steps: after elution, the purified sample solution was collected, concentrated and lyophilized. The concentration is preferably carried out under reduced pressure. The concentration temperature may be 30-35 ℃. The pressure for the concentration under reduced pressure may be 10 mbar.
The invention also provides a preparation method of the nucleotide triphosphate alkali metal salt, which comprises the following steps: in a solvent, in the presence of perchlorate, carrying out an ion exchange reaction on the prepared nucleotide triphosphate triethylamine salt shown in the formula D to prepare a nucleotide triphosphate alkali metal salt shown in the formula E;
Figure BDA0003571669580000061
wherein M is+Is Li+Or Na+The substituent R, R' is as defined above.
In the ion exchange reaction, the solvent may be acetone or ethanol, such as acetone.
In the ion exchange reaction, the perchlorate can be lithium perchlorate or sodium perchlorate.
In the ion exchange reaction, the molar ratio of the perchlorate to the compound shown in the formula D can be (1000-4000): 1, for example 1500: 1.
in the ion exchange reaction, the volume-to-mass ratio of the solvent to the compound shown in the formula D can be 30-100mL/g, for example 40 mL/g.
The reaction temperature of the ion exchange reaction may be from 0 ℃ to 25 ℃, for example 4 ℃.
The reaction time of the ion exchange reaction may be 20min to 60min, for example 30 min.
The ion exchange reaction may also include the following post-treatment steps: filtering, washing the filter cake with solvent, and drying. The solvent may be acetone, preferably acetone at 4 ℃. The drying is preferably drying under reduced pressure.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: (1) the method realizes large-scale automatic preparation of various modified and non-modified nucleotide triphosphates, has good universality and high separation degree, and can be used for large-scale production of different types of modified nucleotides.
(2) The preparation cost of the nucleotide triphosphate is greatly reduced without using a large amount of organic solvents and expensive preparative liquid chromatography.
Drawings
FIG. 1 is a diagram of compound C13 separated and prepared by using DEAE-Sephadex A250 as a filler.
FIG. 2 is a diagram of compound C14 isolated and prepared using Capto DEAE as a filler.
FIG. 3 is a diagram of compound C14 isolated and prepared using Unigel-DEAE as filler.
FIG. 4 is a diagram of compound C14 isolated using DEAE Sepharose6FF as the filler.
FIG. 5 is a diagram of the isolation preparation of compound C14 using NanoQ as a filler.
FIG. 6 is a spectrum of compound C14 isolated using UniQ as the filler.
FIG. 7 is a diagram of the isolation preparation of compound C14 with Generik MC-Q as filler.
FIG. 8 is a diagram of the isolation preparation of compound C1 with Generik MC-Q as filler.
FIG. 9 is a diagram of the isolation preparation of compound C2 with Generik MC-Q as filler.
FIG. 10 is a diagram of the isolation preparation of compound C3 with Generik MC-Q as filler.
FIG. 11 is a diagram of the isolation preparation of compound C4 with Generik MC-Q as filler.
FIG. 12 is a diagram of the isolation preparation of compound C6 with Generik MC-Q as filler.
FIG. 13 is a diagram of the isolation preparation of compound C7 with Generik MC-Q as filler.
FIG. 14 is a diagram of the isolation preparation of compound C8 with Generik MC-Q as filler.
FIG. 15 is a chart of the isolated preparation of compound C9 using Generik MC-Q as a filler.
FIG. 16 is a diagram of the isolation preparation of compound C10 using Generik MC-Q as a filler.
FIG. 17 is a diagram of the isolation preparation of compound C15 with Generik MC-Q as filler.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Methyl triphosphate, phosphorus oxychloride, tri-n-butylamine pyrophosphate, dichloromethane, acetonitrile, triethylamine, sodium hydroxide and ethanol are purchased from national reagent official website. Nucleoside raw materials A1, A2, A3, A4, A6, A7 and A9 are all purchased from Bigdai medicines. A5, A8 and A10 are self-synthesized raw materials.
The column packing for separation preparation is anion exchange resin packing such as DEAE-Sephadex A250, Capto DEAE series, UniDEAE series, DEAE Sepharose6FF, UniQ series, NanoQ series, Unicore-DEAE series, Generik MC-DEAE, etc. The purification equipment adopts Cytiva AKTA Avant 150, AKTA Pure 150 or other protein chromatography purification equipment.
EXAMPLE 1 preparation of nucleotide triphosphate Compound C
Figure BDA0003571669580000071
(when R is a group 1, compound A is referred to as A1, compound B is referred to as B1, compound C is referred to as C1; and so on, when R is a group n, compound A is referred to as An, compound B is referred to as Bn, compound C is referred to as Cn)
Respectively dissolving different nucleoside compounds A in methyl triphosphate, cooling to 10 ℃, slowly adding 1.5eq of phosphorus oxychloride dropwise, heating the reaction system to 15 ℃, and continuously stirring until reaction raw materials completely disappear and the reaction raw materials are converted into an intermediate product B. Then 5eq of tri-n-butylamine pyrophosphate and 6eq of tri-n-butylamine which are pre-cooled to-10 ℃ are added, the reaction system is heated to 15 ℃ and stirred until the raw material B is completely converted into the target product C. Extracting with ultrapure water cooled to 4 deg.C, adjusting pH to 6, extracting with dichloromethane for three times, vacuum distilling the extracted water phase system, concentrating to obtain crude product C as yellowish green oily substance, and storing at-80 deg.C.
EXAMPLE 2 preparation of 2M TEAB buffer
A number of sample bottles were placed on ice in a fume hood, 1.1L HPLC grade triethylamine was added, 4L ultrapure water was added, carbon dioxide gas bubbled through, pH was measured every 10 minutes until pH 8.5, and carbon dioxide gas was turned off. After 2M TEAB buffer was degassed by filtration with a2 μ M Teflon filter, the vial was sealed and stored in a refrigerator at 4 ℃ and the stock 2M TEAB was diluted to different concentrations of 1M, 50mM, 10mM before use.
0.5M sodium hydroxide aqueous solution and 20% ethanol solution were used for column washing and storage.
Example 3 purification sample preparation
The crude product of product C was prepared as 10-20mg/mL of a10 mM TEAB (pH 8.5) aqueous solution and stored in a refrigerator at 4 ℃.
Example 4 preparation with DEAE-Sephadex A250 as filler
The column diameter is 25mM, the column height is 220mM, the DEAE-Sephadex A250 filler is based on Sephadex, the ligand is diethylamine ethyl (DEAE), the particle size is 40-120 μ M, the A Buffer is 10mM TEAB (pH 8.5), and the B Buffer is 1M TEAB (pH 8.5).
The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using A Buffer with 5 times of column volume; product C (exemplified by compound C13) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: firstly, initially eluting impurities by using a mobile phase with 1 time of column volume, wherein the proportion of B Buffer is 12 percent; the B Buffer ratio was then increased to 30% in a linear gradient over 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; eluting the residual impurities with a mobile phase with 1 time of column volume, wherein the B Buffer ratio is 60% (or 100%); finally the column was washed and equilibrated with 1 column volume mobile phase without addition of B Buffer. This filler loading was calculated to be 0.48mg/mL and the degree of separation R was 1.46 (fig. 1).
Example 5 preparation with Capto DEAE as filler
The diameter of the chromatographic column is 10mm, and the height of the chromatographic column is 200 mm. The Capto DEAE packing material uses highly cross-linked agarose with dextran surface extender as matrix, and the ligand is diethylamine ethyl (DEAE) with particle size of 90 μm. The a Buffer was 10mM TEAB (pH 8.5) and the B Buffer was 1M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using A Buffer with 5 times of column volume; product C (exemplified by compound C14) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased from 10% to 100% at 20 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio being 100%. This filler loading was calculated to be 0.50mg/mL and the resolution R was 1.60 (fig. 2).
Example 6 preparation with Unigel-DEAE as filler
The diameter of the chromatographic column is 10mm, and the height of the chromatographic column is 200 mm; the Unigel-DEAE filler takes Polyacrylate (PMMA) as a matrix, the ligand is diethylamine ethyl (DEAE), and the particle size is 50 μm. The a Buffer was 10mM TEAB (pH 8.5) and the B Buffer was 1M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C14) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: first, the B Buffer ratio was increased to 100% with 10 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; collecting a sample solution corresponding to a chromatographic peak; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio being 100%. This filler loading was calculated to be 0.62mg/mL and the degree of separation R was 1.33 (fig. 3).
Example 7 preparation with DEAE Sepharose6FF as packing
The diameter of the chromatographic column is 10mm, and the height of the chromatographic column is 200 mm; the DEAE Sepharose6FF filler is prepared from agarose gel as matrix, and diethylamine ethyl (DEAE) as ligand, and has a particle size of 90 μm. A Buffer was 10mM TEAB (pH 8.5) and B Buffer was 1M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C14) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased from 10% to 100% at 10 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio being 100%. This filler loading was calculated to be 0.47mg/mL, with a resolution R of 1.80 (fig. 4).
Example 8 preparation with NanoQ as filler
The diameter of the chromatographic column is 7.7mm, and the height of the chromatographic column is 100 mm; the NanoQ filler is based on monodisperse polystyrene-divinylbenzene (PS/DVB), and the ligand is a Quaternary ammonium group (Quaternary ammonium) with a particle size of 10 μm. A Buffer was ultrapure water, and B Buffer was 1M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C14) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: first, the B Buffer ratio was increased to 80% with 20 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; collecting a sample solution corresponding to a chromatographic peak; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio being 100%. This filler loading was calculated to be 1.01mg/mL and the resolution R was 2.26 (fig. 5).
Example 9 preparation with UniQ as filler
The diameter of the chromatographic column is 7.7mm, and the height of the chromatographic column is 100 mm; the UniQ filler is based on monodisperse polystyrene, the ligand is Quaternary ammonium group (Quaternary ammonium), and the particle size is 15 μm. During the purification, A Buffer was ultrapure water, and B Buffer was 1M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using A Buffer with 5 times of column volume; product C (exemplified by compound C14) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is performed: first, the B Buffer ratio was increased to 80% with 20 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; collecting a sample solution corresponding to a chromatographic peak; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio being 100%. This filler loading was calculated to be 0.91mg/mL, with a resolution R of 2.4 (fig. 6).
Example 10 preparation with Generik MC-Q as Filler
The diameter of the chromatographic column is 10mm, and the height of the chromatographic column is 200 mm; the Generic MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups (Quaternary ammonium) with a particle size of 30 μm. A Buffer was ultrapure water, and B Buffer was 1M TEAB (pH 8.5).
The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C14) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is performed: first, the B Buffer ratio was increased to 80% with 20 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP, nucleotide diphosphate NDP will be eluted sequentially, elute the main peak as the goal product NTP finally; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio being 100%. This filler loading was calculated to be 0.87mg/mL and the degree of separation R was 2.89 (fig. 7).
Example 11 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups (Quaternary ammonium). A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C1) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was found to be 1.90 by calculation (fig. 8).
Example 12 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C2) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: first, the B Buffer ratio was increased to 22.5% at 5.5 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 2 column volumes of mobile phase, with a B Buffer ratio of 50%. The degree of separation R was calculated to be 2.41 (fig. 9).
Example 13 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C3) was then injected and washed with 2.5 column volumes of A Buffer without addition of B Buffer. Then the elution phase is carried out: the initial impurities were first eluted with 0.5 column volumes of mobile phase, with a B Buffer ratio of 20%, then increased to 30% with 6 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, the B Buffer ratio was 100%. The resolution R was found to be 1.98 by calculation (fig. 10).
Example 14 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C4) was then injected and the column was washed with two column volumes of A Buffer without the addition of B Buffer. Then the elution phase is performed: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The resolution R was calculated to be 4.38 (fig. 11).
Example 15 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups (Quaternary ammonium). A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C6) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was found to be 3.66 by calculation (fig. 12).
Example 16 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C7) was then injected and the column was washed with two column volumes of A Buffer without the addition of B Buffer. Then the elution phase is carried out: first, the B Buffer ratio was increased to 18% with 8 column volumes, the main impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was calculated to be 2.33 (fig. 13).
Example 17 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using A Buffer with 5 times of column volume; product C (exemplified by compound C8) was then injected and the column was washed with two column volumes of A Buffer without the addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was found to be 4.05 by calculation (fig. 14).
Example 18 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups (Quaternary ammonium). A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C9) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was found to be 4.10 by calculation (fig. 15).
Example 19 preparation with Generik MC-Q as filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C10) was then injected and the column was washed with two column volumes of A Buffer without the addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was calculated to be 2.4 (fig. 16).
Example 20 preparation with Generik MC-Q as Filler
The diameter of the chromatographic column is 100mm, and the height of the chromatographic column is 220 mm; the Generik MC-Q filler is based on polymethacrylates and the ligands are Quaternary ammonium groups. A Buffer was ultrapure water, and B Buffer was 2M TEAB (pH 8.5). The preparation process comprises the following steps:
fully disinfecting and cleaning a chromatographic column before use, and balancing by using Buffer which is 5 times of the column volume A; product C (exemplified by compound C15) was then injected and washed with two column volumes A Buffer without addition of B Buffer. Then the elution phase is carried out: the B Buffer ratio was first increased to 22.5% at 8 column volumes, the major impurities generated in the synthesis: nucleotide monophosphate NMP and nucleotide diphosphate NDP are eluted in sequence, and the final elution main peak is the target product NTP; the remaining impurities were then eluted with 1 column volume of mobile phase, B Buffer ratio 50%. The degree of separation R was found to be 1.67 by calculation (fig. 17).
EXAMPLE 21 preparation of nucleotide triphosphate sodium salt
Figure BDA0003571669580000131
After the purified sample solution is collected, the solution is concentrated to 200mL at 30-35 ℃ under reduced pressure by rotary evaporation and 10 mBar. And freeze-drying the product concentrated solution to obtain triethylamine salts of different nucleotide triphosphates.
The triethylamine salt of nucleotide triphosphate obtained by freeze-drying was added to a 5% (m/v) sodium perchlorate/lithium perchlorate acetone solution at a ratio of 1:100(m/v), and the mixture was vigorously stirred at 4 ℃ for 1 hour until the solution was converted into a uniform white suspension. After filtration, the filter cake was washed with 200 times the volume of a4 ℃ acetone solution and dried under reduced pressure. Sodium/lithium triphosphate salts can be obtained.
Quality identification of Compound E
The quality determination apparatus used was Shimadzu i-Serious UPLC, Shimadzu LCMS 2040, Shim-pack GIST 5uM C184.6X250mm, BRUKER 400 MHz.
Compound E1: HPLC Rt＝2.030min,99.39％.1H NMR(400MHz,D2O):δ8.57(s,1H),8.40(s,1H),6.09(d,J＝5.2Hz,1H),4.66(t,J＝5.2Hz,1H),4.53(t,J＝4.5Hz,1H),4.48–4.24(m,3H).31P NMR(400MHz,D2O):δ-15.34(d,J＝18.6Hz),-15.89(d,J＝19.5Hz),-27.49(t,J＝18.9Hz).ESI-MS m/z calcd for C10H15N5O13P3[M-H]-506.0；found.506.0
Compound E2: HPLC Rt＝2.046min，100％.1H NMR(400MHz,D2O):δ8.24(s,1H),5.92(d,J＝5.3Hz,1H),4.71(t,J＝5.2Hz,1H),4.53(h,J＝4.6Hz,1H),4.36(p,J＝3.1Hz,1H),4.33–4.18(m,2H).31P NMR(400MHz,D2O):δ-10.63(d,J＝19.0Hz),-11.15(d,J＝19.6Hz),-22.73(t,J＝19.1Hz).ESI-MS m/z calcd for C10H15N5O13P3[M-H]-522.0；found.522.0.
Compound E3: HPLC Rt＝2.052min,98.12％.1H NMR(400MHz,D2O):δ8.17(d,J＝7.9Hz,2H),6.30(d,J＝7.9Hz,2H),5.94(d,J＝3.7Hz,2H),4.41–4.19(m,9H),3.64(q,J＝7.1Hz,1H),1.17(t,J＝7.1Hz,2H).31P NMR(400MHz,D2O):δ-10.70(d,J＝18.9Hz),-11.32(d,J＝19.5Hz),-22.88(t,J＝19.1Hz).ESI-MS m/z calcd for C9H15N3O14P3[M-H]-482.0；found.482.0.
Compound E4: HPLC Rt＝2.012min,100％.1H NMR(400MHz,D2O):δ7.97(d,J＝8.1Hz,1H),6.00(t,J＝6.7Hz,2H),4.41(t,J＝4.5Hz,2H),4.30(t,J＝3.0Hz,1H),4.29–4.19(m,2H),3.66(q,J＝7.1Hz,1H),1.19(t,J＝7.1Hz,1H).31P NMR(400MHz,D2O):δ0.11,-10.55(d,J＝19.0Hz),-11.31(d,J＝19.9Hz),-22.87(t,J＝19.4Hz).ESI-MS m/z calcd for C9H15N2O15P3[M-H]-483.0；found.482.9.
Compound E6: HPLC Rt＝2.112min,100％.1H NMR(400MHz,D2O):δ2.92(s,3H),4.05(m,2H),4.21(m,1H),4.41(m,1H),4.60(m,1H),5.95(d,1H,J＝6.0Hz),8.09(s,1H),8.29(s,1H).31P NMR(400MHz,D2O):δ–21.47(t),–10.22(d),7.47(d).ESI-MS m/z calcd for C11H18N5O13P3[M-H]-520.0；found.519.9.
Compound E7: HPLC Rt＝2.173min,99.38％.1H NMR(400MHz,D2O):δ9.26(s,1H),6.11(d,J＝3.6Hz,1H),4.73(d,J＝4.3Hz,1H),4.59(t,J＝5.1Hz,1H),4.42(d,J＝23.2Hz,2H),4.31(dd,J＝12.6,5.2Hz,1H),4.17(s,3H).31P NMR(400MHz,D2O):δ-10.44(d,J＝19.8Hz),-11.39(d,J＝20.1Hz),-22.91(t,J＝19.7Hz).ESI-MS m/z calcd for C11H17N2O15P3[M-H]-536.0；found.535.8.
Compound E8: HPLC Rt＝2.166min,99.38％.1H NMR(400MHz,D2O):δ7.81(s,1H),6.02(d,J＝4.7Hz,1H),4.46(t,J＝4.9Hz,1H),4.40–4.21(m,4H),2.02(s,3H).31P NMR(400MHz,D2O):δ-6.39(d,J＝19.8Hz),-11.28(d,J＝19.0Hz),-21.77(t,J＝19.8Hz).ESI-MS m/z calcd for C11H17N2O15P3[M-H]-496.0；found.495.9.
Compound E9: HPLC Rt＝2.142min,99.73％.1H NMR(400MHz,D2O):δ7.42(s,1H),6.06(d,J＝6.0Hz,1H),4.54–4.46(m,2H),4.35–4.28(m,2H),4.21(dt,J＝12.3,3.2Hz,1H),3.83(s,3H).31P NMR(400MHz,D2O):δ-6.09(d,J＝19.4Hz),-11.35(d,J＝19.8Hz),-21.70(t,J＝19.7Hz).ESI-MS m/z calcd for C11H17N2O15P3[M-H]-514.0；found.513.9.
Compound E10: HPLC Rt＝2.122min,100％.1H NMR(400MHz,MeOD):δ4.26(m,2H),4.28(m,1H),4.34(m,1H),4.41(m,1H),6.25(d,1H,J＝8.19Hz),6.68(d,1H,J＝2.83Hz),8.23(d,1H,J＝8.51Hz).31P NMR(400MHz,MeOD):-23.74(t,J＝17.22Hz),-12.31(d,J＝19.68Hz),-10.83(d,J＝17.22Hz).ESI-MS m/z calcd for C9H15N2O14P3S[M-H]-499.0；found.499.0.
Compound E13: HPLC Rt＝2.152min,99.86％.1H NMR(400MHz,D2O):δ8.44(d,J＝7.6Hz,1H),7.35(d,J＝7.6Hz,1H),5.99(d,J＝3.0Hz,1H),4.44–4.28(m,5H),3.36(s,2H).31P NMR(400MHz,D2O):δ-8.16–-8.49(m),-11.23(d,J＝19.7Hz),-22.32(t,J＝19.7Hz).ESI-MS m/z calcd for C11H17N2O15P3[M-H]-524.0；found.523.9.
Compound E14: HPLC Rt＝2.091min,100％.1H NMR(400MHz,D2O):δ7.85(s,2H),7.60(s,1H),5.07(s,1H),4.52(s,1H),4.40(s,1H),4.34(t,J＝5.8Hz,2H),4.31–4.11(m,11H).31P NMR(400MHz,D2O):δ-5.63(d,J＝19.4Hz),-10.55(dd,J＝18.7,11.3Hz),-21.34(t,J＝18.6Hz).ESI-MS m/z calcd for C9H14N2O15P3[M-H]-482.96；found.482.87.
Compound E15: HPLC Rt＝2.090min，99.45％.1H NMR(400MHz,D2O):δ7.86(s,3H),4.85(s,6H),4.51(s,1H),4.33(d,J＝5.8Hz,3H),4.25(s,5H),4.20(s,3H),4.15(s,4H),3.42(d,J＝3.7Hz,10H).31P NMR(400MHz,D2O):δ-7.96(d,J＝23.6Hz),-10.89(dd,J＝43.6,19.3Hz),-21.32–-22.47(m).ESI-MS m/z calcd for C10H16N2O15P3[M-H]-496.95；found.497.15.
Example 4-example 10 comparison of filler loading and separation preparation:
compared with anion exchange resin using Diethylaminoethyl (DEAE) as a ligand, the anion exchange resin using Quaternary ammonium groups (Quaternary ammonium) as the ligand has the advantages that the particle size of the Quaternary ammonium groups (Quaternary ammonium) resin is 10-30 mu m, the particle size of the Quaternary ammonium groups (Quaternary ammonium) resin is smaller than that of diethylamine ethyl (DEAE) filler with the particle size of more than 40 mu m, the filler loading capacity of the Quaternary ammonium groups (Quaternary ammonium) resin is 0.87-1.01mg/mL, and the specific loading capacity of the diethylamine ethyl (DEAE) resin is about 79% higher than that of the diethylamine ethyl (DEAE) filler with the loading capacity of 0.47-0.62 mg/mL. Meanwhile, the Quaternary ammonium group (Quaternary ammonium) resin has good pressure bearing performance, higher purification flow rate and better separation effect, and the separation degree of the Quaternary ammonium group (Quaternary ammonium) resin is 2.26-2.89, which is about 63% higher than that of diethyl aminoethyl (DEAE) resin by 1.33-1.80%. Table 1 compares the results of different filler loading tests.
TABLE 1
Figure BDA0003571669580000151
The data in the table are compared by taking compound C14 as an example, according to the formula of chromatographic resolution in Chinese pharmacopoeia: r is 2 (t)NTP-tNDP)/(WNTP+WNDP) And calculating the total separation efficiency index of the product nucleotide triphosphate NTP and the adjacent main impurity NDP.

Claims (10)

1. A preparation method of nucleotide triphosphate triethylamine salt is characterized by comprising the following steps: separating and preparing a substance to be purified with the active ingredient of the nucleotide triphosphate shown as the formula C by adopting a chromatography method to obtain the nucleotide triphosphate triethylamine salt shown as the formula D;
Figure FDA0003571669570000011
wherein R is a basic group, R' is-OH, -OCH3Or H;
the chromatographic column is an anion exchange column, the ligand of the anion exchange column packing is a quaternary ammonium group or a diethylaminoethyl group, and the mobile phase comprises triethylamine-carbonic acid buffer solution.
2. The method of claim 1, wherein the method satisfies one or more of the following conditions:
(a) the separation preparation comprises the following scheme one or scheme two;
the first scheme is as follows: the ligand of the anion exchange column filler is a quaternary ammonium group; separating and preparing by taking water as Buffer A and triethylamine-carbonic acid Buffer solution as Buffer B;
scheme II: the ligand of the anion exchange column packing is diethylamine ethyl; separating and preparing by taking 10mM triethylamine-carbonic acid Buffer solution as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B;
(b) the base is a natural base or a modified base;
(c) the matrix of the anion exchange column filler is cross-linked dextran, highly cross-linked agarose with dextran surface extender, polyacrylate, agarose gel, monodisperse polystyrene-divinylbenzene, monodisperse polystyrene or polymethacrylate;
(d) the diameter of the anion exchange column is 5-100 mm;
(e) the height of the anion exchange column is 50-400 mm;
(f) the column temperature of the anion exchange column is 4-20 ℃;
(g) the initial concentration of the mobile phase is less than the concentration at which the triethylamine triphosphate salt of nucleotide triphosphate is eluted.
3. The production method according to claim 1 or 2, wherein the production method satisfies one or more of the following conditions:
(a) in the first scheme, the packing of the anion exchange column is UniQ, NanoQ or Generick MC-Q;
(b) in the second scheme, the anion exchange column filler is DEAE-Sephadex A250, Capto DEAE, UniDEAE, Unicore-DEAE, DEAE Sepharose6FF or Generik MC-DEAE;
(c) the diameter of the anion exchange column is 7.7-10 mm;
(d) the height of the anion exchange column is 100-300 mm;
(e) in the first scheme, the particle size of the anion exchange column packing is 10-30 μm;
(f) in the second scheme, the particle size of the anion exchange column packing is 40-120 μm;
(g) the temperature of the anion exchange column is 4 ℃;
(h) in the first scheme, the Buffer A is ultrapure water;
(i) in the first scheme, the Buffer B is 1-2M triethylamine-carbonic acid Buffer solution;
(j) in the first embodiment, the separation preparation comprises the following steps: (1) balancing by A Buffer with 3-5 times of column volume; (2) washing impurities by using Buffer in the column volume of 1-3 times; (3) eluting by 5-20 times of column volume, wherein the final proportion of B Buffer is 18% -80%; (4) eluting residual impurities by 1-2 times of the column volume, wherein the proportion of B Buffer is 50-100%;
(k) in scheme two, the separation preparation comprises the following steps: (1) balancing by A Buffer with 3-5 times of column volume; (2) washing impurities by using A Buffer in 0.5-2 times of column volume; (3) eluting by 8-20 times of column volume, wherein the final proportion of B Buffer is 12% -100%; (4) eluting residual impurities by 1-2 times of the column volume, wherein the proportion of B Buffer is 60-100%;
(l) The base is one of the following structures:
Figure FDA0003571669570000021
4. the method of claim 3, wherein the method satisfies one or more of the following conditions:
(a) in the second scheme, the anion exchange column filler is DEAE-Sephadex A250, Capto DEAE, Unigel-DEAE or DEAE Sepharose6 FF;
(b) the height of the anion exchange column is 200-220 mm;
(c) in the first scheme, the particle size of the anion exchange column packing is 15 microns;
(d) in the second scheme, the particle size of the anion exchange column packing is 50-90 μm;
(e) in the first embodiment, the separation preparation comprises the following steps: (1) balancing by A Buffer with 3-5 times of column volume; (2) washing impurities by using Buffer in a column volume of 2-2.5 times; (3) eluting by 5.5-8 times of column volume, wherein the final proportion of B Buffer is 18% -30%; (4) eluting residual impurities by 1-2 times of the column volume, wherein the proportion of B Buffer is 50-100%.
5. The method of claim 4, wherein in the first embodiment, the isolation comprises the steps of: (1) balancing by A Buffer with 3-5 times of column volume; (2) washing impurities by using Buffer in a column volume of 2-2.5 times; (3) eluting by 6.5 times of column volume, wherein the final proportion of B Buffer is 20-30%; (4) eluting residual impurities by 1-2 times of the column volume, wherein the proportion of B Buffer is 50-100%.
6. The method of claim 3, wherein the elution comprises a linear gradient elution and/or a step gradient elution.
7. The method of claim 1, wherein the isolation is performed by any one of the following methods:
the method 101 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking NanoQ as a filler, ultrapure water as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 80%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 102 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by using UniQ as a filler, using ultrapure water as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 80%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 103 comprises the following steps: the method comprises the following steps of (1) performing separation preparation by taking Generic MC-Q as a filler, ultrapure water as Buffer A and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution is carried out by 20 times of column volume, and the final proportion of B Buffer is 80%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 104 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) performing linear gradient elution by 8 times of column volume, wherein the final proportion of B Buffer is 22.5%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 105 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution is carried out by 5.5 times of column volume, and the final proportion of B Buffer is 22.5%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 106 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by Buffer in 2.5 times of column volume A; (3) performing step gradient elution by 0.5 times of column volume, wherein the proportion of B Buffer is 20 percent, performing linear gradient elution by 6 times of column volume, and the final proportion of B Buffer is 30 percent; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 107 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume; (3) performing linear gradient elution by 8 times of column volume, wherein the final proportion of B Buffer is 22.5%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 108 comprises the following steps: the method comprises the following steps of (1) carrying out separation preparation by taking Generik MC-Q as a filler, ultrapure water as Buffer A and 2M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing A Buffer with 5 times of column volume; (2) washing impurities by using Buffer in 2 times of column volume A; (3) performing linear gradient elution by 8 times of column volume, wherein the final proportion of B Buffer is 18 percent; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 50 percent;
the method 201: using DEAE-Sephadex A250 as a filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A, and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1)5 times of column volume of A Buffer balance; (2) washing impurities by using Buffer in 2 times of column volume A; (3) gradient elution with 1 column volume step, B Buffer ratio is 12%; then linear gradient elution is carried out by 8 times of column volume, and the final proportion of B Buffer is 30 percent; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 60 percent or 100 percent;
the method 202 comprises the following steps: the separation preparation method comprises the following steps of (1) balancing an A Buffer with 5 times of column volume by taking Capto DEAE as a filler, taking 10mM triethylamine-carbonic acid Buffer solution as a Buffer A and taking 1M triethylamine-carbonic acid Buffer solution as a Buffer B; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the initial proportion of B Buffer is 10%, and the final proportion is 100%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 203 comprises the following steps: using Unigel-DEAE as filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A, and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1) balancing 5 times column volume of A Buffer; (2) washing impurities by using Buffer in 2 times of column volume A; (3) linear gradient elution with 20 times column volume, the final proportion of B Buffer is 100%; (4) eluting residual impurities by 1 time of column volume, wherein the proportion of B Buffer is 100 percent;
the method 204 comprises the following steps: using DEAE Sepharose6FF as a filler, 10mM triethylamine-carbonic acid Buffer solution as Buffer A, and 1M triethylamine-carbonic acid Buffer solution as Buffer B, wherein the separation preparation comprises the following steps of (1)5 times of column volume of A Buffer balance; (2) washing impurities by using Buffer in 2 times of column volume; (3) performing linear gradient elution by 10 times of column volume, wherein the initial proportion of B Buffer is 10 percent, and the final proportion is 100 percent; (4) the remaining impurities were eluted at 1 column volume, the B Buffer ratio was 100%.
8. The method of any one of claims 1-7, wherein the method satisfies one or more of the following conditions:
(1) the material to be purified is pretreated before elution, and the pretreatment comprises the following steps: dissolving the substance to be purified in a TEAB solution to obtain a sample;
(2) the synthesis of the compound to be purified comprises the following steps: (i) in methyl triphosphate, carrying out phosphorylation reaction on a compound shown as a formula A and phosphorus oxychloride as shown in the specification to obtain a compound shown as a formula B;
(ii) will (Bu)3N)2H4P2O7Carrying out the following reaction on the tri-n-butylamine and the compound shown as the formula B, and adding water for quenching to obtain a compound shown as a formula C;
Figure FDA0003571669570000041
wherein R, R' is defined as set forth in claim 1;
(3) the preparation method of the nucleotide triphosphate triethylamine salt further comprises the following steps: after elution, the purified sample solution was collected, concentrated and lyophilized.
9. The method of claim 8, wherein the method satisfies one or more of the following conditions:
(1) the concentration of the substance to be purified in the TEAB solution is 0-100 mg/mL;
(2) in the step (i), the molar ratio of the phosphorus oxychloride to the compound shown in the formula A is 1-2: 1;
(3) in the step (i), the volume-to-mass ratio of the methyl triphosphate to the compound shown in the formula A is 10-20 mL/g;
(4) in the step (i), the reaction temperature of the reaction is-10-20 ℃;
(5) in the step (ii), the water is ultrapure water;
(6) in step (ii), said (Bu)3N)2H4P2O7The mol ratio of the compound shown in the formula B to the compound shown in the formula B is (5-10): 1;
(7) in the step (ii), the molar ratio of the tri-n-butylamine to the compound shown in the formula B is (4-8): 1;
(8) in step (ii), the reaction temperature of the reaction is 5 ℃ to 20 ℃.
10. The method of claim 9, wherein the method satisfies one or more of the following conditions:
(1) the concentration of the substance to be purified in the TEAB solution is 10-20 mg/mL;
(2) in the step (i), the molar ratio of the phosphorus oxychloride to the compound shown in the formula A is 1.5: 1;
(3) in the step (i), the volume-to-mass ratio of the methyl triphosphate to the compound shown in the formula A is 14.9 mL/g;
(4) in step (i), the reaction temperature of the reaction is 15 ℃;
(5) in step (ii), said (Bu)3N)2H4P2O7With the compound of formula BThe molar ratio is 5: 1;
(6) in the step (ii), the molar ratio of the tri-n-butylamine to the compound shown in the formula B is 6: 1;
(7) in step (ii), the reaction temperature of the reaction is 15 ℃.
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