CN117756850A - N4-AC-C- (S) -GNA phosphoramidite and preparation method thereof - Google Patents
N4-AC-C- (S) -GNA phosphoramidite and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 46
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000012074 organic phase Substances 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 17
- -1 (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine Chemical compound 0.000 claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 15
- 238000003756 stirring Methods 0.000 claims abstract description 15
- RKVHNYJPIXOHRW-UHFFFAOYSA-N 3-bis[di(propan-2-yl)amino]phosphanyloxypropanenitrile Chemical compound CC(C)N(C(C)C)P(N(C(C)C)C(C)C)OCCC#N RKVHNYJPIXOHRW-UHFFFAOYSA-N 0.000 claims abstract description 13
- CIXHNHBHWVDBGQ-UHFFFAOYSA-N n-propan-2-ylpropan-2-amine;2h-tetrazole Chemical compound C1=NN=NN1.CC(C)NC(C)C CIXHNHBHWVDBGQ-UHFFFAOYSA-N 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 239000012071 phase Substances 0.000 claims abstract description 6
- 238000004519 manufacturing process Methods 0.000 claims abstract 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 15
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 14
- 238000002425 crystallisation Methods 0.000 claims description 12
- 230000008025 crystallization Effects 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 9
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 9
- 235000011152 sodium sulphate Nutrition 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 8
- 239000008346 aqueous phase Substances 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 2
- 150000008300 phosphoramidites Chemical class 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 239000000376 reactant Substances 0.000 abstract description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 6
- XGDRLCRGKUCBQL-UHFFFAOYSA-N 1h-imidazole-4,5-dicarbonitrile Chemical compound N#CC=1N=CNC=1C#N XGDRLCRGKUCBQL-UHFFFAOYSA-N 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 238000002515 oligonucleotide synthesis Methods 0.000 description 3
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 2
- 108090000994 Catalytic RNA Proteins 0.000 description 2
- 102000053642 Catalytic RNA Human genes 0.000 description 2
- 108091027757 Deoxyribozyme Proteins 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- UAOMVDZJSHZZME-UHFFFAOYSA-N diisopropylamine Chemical compound CC(C)NC(C)C UAOMVDZJSHZZME-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 150000003536 tetrazoles Chemical class 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-N hydroperoxyl Chemical compound O[O] OUUQCZGPVNCOIJ-UHFFFAOYSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108091008104 nucleic acid aptamers Proteins 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000005731 phosphitylation reaction Methods 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to the technical field of phosphoramidites, and discloses N4-AC-C- (S) -GNA phosphoramidite and a preparation method thereof. The method comprises the following steps: placing pre-configured dichloromethane, diisopropyl ammonium salt tetrazole, (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine and bis (diisopropylamino) (2-cyanoethoxy) phosphine into a reaction bottle, and carrying out stirring reaction for a preset time; after stirring reaction, performing HPLC (high performance liquid chromatography) central control reaction on reactants obtained by the stirring reaction to obtain a reaction compound; washing the reaction compound for a plurality of times to remove the water phase and keep an organic phase; and filtering, concentrating and crystallizing the washed organic phase to obtain the N4-AC-C- (S) -GNA phosphoramidite. The invention has simple operation, low cost and high conversion rate, and is beneficial to mass production.
Description
Technical Field
The invention relates to the technical field of phosphoramidites, in particular to N4-AC-C- (S) -GNA phosphoramidite and a preparation method thereof.
Background
So far, the clinic lack of ideal specific medicines for diseases such as cancer, viral infection and the like. It is appreciated that with the completion of genome sequencing in humans and in important models, and with the intensive research of functional genomics and proteomics, molecular targets associated with disease are continually discovered and recognized, providing a premise for gene therapy.
Artificially synthesized oligonucleotides have been widely used in research on targeted gene therapy in the past decades. Oligonucleotides mainly include antisense oligonucleotides (Antisense oligonucleotides, ASODN), small interfering RNAs (Small interference RNA, siRNA), transcription factor decoys (decoys), ribozymes (ribozymes), deoxyribozymes (DNAzymes), anti-genes (anti-genes), cpG oligonucleotides, and nucleic acid aptamers (Aptamers), etc. Among them, ASODN and siRNA are the most commonly used gene regulation tools, are widely used, and have been developed as gene therapy drugs.
At present, oligonucleotides are mainly synthesized by chemical methods, and the oligonucleotides are obtained by sequentially reacting two active groups on a phosphitylation reagent with a protected nucleoside. The method of synthesizing oligonucleotides using a phosphitylating reagent, which is a key raw material for the phosphoramidite method, is called the phosphoramidite method, and is the most commonly used method in oligonucleotide synthesis at present.
However, traditional phosphoramidite preparation has low yield and/or purity characteristics, which significantly increases the cost of oligonucleotide synthesis. Such as: in the preparation process of the traditional N4-AC-C- (S) -GNA phosphoramidite, column chromatography purification reaction is mainly carried out by taking (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine and bis (diisopropylamino) (2-cyanoethoxy) phosphine as raw materials and dichloromethane as an auxiliary solvent in the presence of 4, 5-dicyanoimidazole. In the preparation process, the cost of the whole preparation process is higher due to the adoption of 4, 5-dicyanoimidazole and the higher cost of the 4, 5-dicyanoimidazole, and meanwhile, the preparation process is more complicated due to the adoption of a column chromatography purification process in the existing preparation method, and the preparation efficiency is lower.
Therefore, how to provide a high-efficiency preparation method of N4-AC-C- (S) -GNA phosphoramidite is a problem to be solved at present.
Disclosure of Invention
The invention provides N4-AC-C- (S) -GNA phosphoramidite and a preparation method thereof, which aim to solve the technical problems in the prior art.
According to a first aspect of the present invention, there is provided an N4-AC-C- (S) -GNA phosphoramidite, the N4-AC-C- (S) -GNA phosphoramidite having the structural formula:
and, the N4-AC-C- (S) -GNA phosphoramidite is prepared from the following raw materials by weight:
315-325g of methylene dichloride, 24-24.1g of (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine, 15.5-15.6g of diisopropyl ammonium salt tetrazole and 16.35-16.45g of bis (diisopropylamino) (2-cyanoethoxy) phosphine.
Wherein the structural formula of the diisopropyl ammonium salt tetrazole is as follows:
wherein the structural formula of the (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine is as follows:
wherein the structural formula of the bis (diisopropylamino) (2-cyanoethoxy) phosphine is as follows:
according to a second aspect of the present invention, there is provided a process for the preparation of N4-AC-C- (S) -GNA phosphoramidite.
The synthetic route of the N4-AC-C- (S) -GNA phosphoramidite is as follows:
wherein: the main reaction mechanism formula of (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxy propyl) -N4-acetylcytosine to N4-AC-C- (S) -GNA phosphoramidite is as follows:
specifically, under the catalysis of diisopropyl ammonium salt tetrazole, bis (diisopropylamino) (2-cyanoethoxy) phosphine is decomposed to remove one molecule of diisopropylammonia, and the molecule of diisopropylammonia is combined with tetrazole to obtain (diisopropylamino) (tetrazole) (2-cyanoethoxy) phosphine; the hydroxyl oxygen of (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine attacks the phosphorus atom, and then tetrazole is removed to obtain N4-AC-C- (S) -GNA phosphoramidite.
And, the preparation method steps of the N4-AC-C- (S) -GNA phosphoramidite include:
placing pre-configured dichloromethane, (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine, diisopropyl ammonium salt tetrazole and bis (diisopropylamino) (2-cyanoethoxy) phosphine into a reaction bottle, and carrying out stirring reaction for a preset time;
after stirring reaction, performing HPLC (high performance liquid chromatography) central control reaction, and after confirming that the reaction is complete, performing multiple water washes on the reaction compound to remove an aqueous phase and a remaining organic phase;
the washed organic phase was filtered, concentrated and crystallized to give N4-AC-C- (S) -GNA phosphoramidite.
Alternatively, the predetermined time is 17 to 20 hours, and the temperature at the time of stirring reaction is 20 to 30 ℃.
Optionally, subjecting the reaction compound to a plurality of water washes, removing the aqueous phase remaining organic phase comprises: and (3) washing the reaction compound for multiple times by using sodium chloride with the mass concentration of 10% to remove the water phase and retain the organic phase.
Alternatively, the number of water washes is 2, and the amount of sodium chloride used per water wash is 400-450ml.
Optionally, filtering, concentrating and crystallizing the washed organic phase to obtain N4-AC-C- (S) -GNA phosphoramidite comprises: mixing the washed organic phase with preset sodium sulfate, standing for a set time, and filtering the mixture after the standing time reaches the set time; concentrating the filtered mixture under reduced pressure, adding methyl tertiary butyl ether and N-heptane to dissolve organic matters, and crystallizing to obtain N4-AC-C- (S) -GNA phosphoramidite.
Optionally, the dosage of the sodium sulfate is 30-50g, and the set time is 0.5-1 hour.
Optionally, the temperature at the time of vacuum concentration is 20-30 ℃.
Optionally, the content of the methyl tertiary butyl ether is 300-330ml, the content of the n-heptane is 600-660ml, and when crystallization is carried out, the ambient temperature is 20-30 ℃, and the stirring time is 2-3 hours.
Optionally, the times of the crystallization treatment are 2 times, the first crystallization treatment is to obtain solid N4-AC-C- (S) -GNA phosphoramidite, and the second crystallization treatment is to crystallize the filtered water solution after the first crystallization treatment to obtain powdery N4-AC-C- (S) -GNA phosphoramidite.
The technical scheme provided by the invention can comprise the following beneficial effects:
the invention has simple process operation and low cost of raw materials, and meanwhile, the product obtained by the process has high conversion rate, is beneficial to large-scale production, provides raw material guarantee with high purity and low price for subsequent oligonucleotide synthesis, and is beneficial to technical progress in the whole field.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 is a schematic illustration of N4-AC-C- (S) -GNA phosphoramidite according to an exemplary embodiment 1 H NMR spectrum;
FIG. 2 is a schematic illustration of N4-AC-C- (S) -GNA phosphoramidite according to an exemplary embodiment 31 PNMR spectrogram;
FIG. 3 is an HPLC purity profile of N4-AC-C- (S) -GNA phosphoramidite according to an exemplary embodiment.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
In the following examples, dichloromethane was used as the lot number 20230427 of Shanghai Lingfeng chemical company, inc.; (S) -1- (3- (4, 4 '-Dimethoxytrityl) -2-hydroxypropyl) -N4-acetylcytosine was prepared from NVC0024-54 (S) -1- (3- (4, 4' -Dimethoxytrityl) -2-hydroxypropyl) -N4-acetylcytosine by Norvigneaux Biotechnology Co., ltd. In Suzhou; the diisopropylammonium salt tetrazole is obtained by using diisopropylammonium salt tetrazole with lot number 230433 produced by Norvigneaux Biotechnology Co., ltd. In Suzhou; bis (diisopropylamino) (2-cyanoethoxy) phosphine was used as bis (diisopropylamino) (2-cyanoethoxy) phosphine of lot RV1361230412-RP172, manufactured by Runyu New Material technology Co., ltd; 10% sodium chloride with lot number 20230208 produced by Jiangsu Qiangsheng functional chemical Co., ltd; the sodium sulfate is sodium sulfate with a lot number of 20230225 produced by Jiangsu Qiangsheng functional chemical Co., ltd; methyl tertiary butyl ether is methyl tertiary butyl ether with batch number 20230421 produced by Shanghai Lingfeng chemical reagent Co., ltd; n-heptane, batch number 20230405, manufactured by Shanghai Lingfeng chemical Co., ltd.
Example 1
The preparation method of the N4-AC-C- (S) -GNA phosphoramidite comprises the following steps of:
318g of preconfigured dichloromethane, 15.52g of diisopropyl ammonium salt tetrazole, 24g of (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine and 16.39g of bis (diisopropylamino) (2-cyanoethoxy) phosphine are placed in a reaction bottle, and stirred at 20 ℃ for 17 hours;
after stirring reaction, performing HPLC (high performance liquid chromatography) central control reaction, and confirming that the reaction is complete;
washing the reaction compound with 400ml of sodium chloride with the mass concentration of 10% for one time to remove an aqueous phase and a remaining organic phase; after the primary water washing, 400ml of sodium chloride with the mass concentration of 10% is used for secondary water washing, and the water phase is removed to keep an organic phase;
mixing the washed organic phase with 30g of sodium sulfate, standing for 0.5 hour, and filtering the mixture; concentrating under reduced pressure at 20deg.C;
after concentrating under reduced pressure, 300ml of methyl tertiary butyl ether and 600ml of N-heptane are added to dissolve the organic matters, and the mixture is stirred for 2 hours at 20 ℃ to carry out crystallization treatment to obtain solid N4-AC-C- (S) -GNA phosphoramidite; the solid N4-AC-C- (S) -GNA phosphoramidite is added with 300ml of methyl tertiary butyl ether and 600ml of N-heptane again to be dissolved, stirred for 2 hours at 20 ℃ and recrystallized to obtain 23.5g of N4-AC-C- (S) -GNA phosphoramidite with purity more than 98 percent and yield: 71.1%.
Example two
The preparation method of the N4-AC-C- (S) -GNA phosphoramidite comprises the following steps of:
265g of preconfigured dichloromethane, 14.02g of diisopropylammonium tetrazole, 20g of (S) -1- (3- (4, 4' -dimethoxytrityl) -2-hydroxypropyl) -N4-acetylcytosine and 14.81g of bis (diisopropylamino) (2-cyanoethoxy) phosphine are placed in a reaction bottle, and stirred at 24 ℃ for 19 hours;
after stirring reaction, performing HPLC (high performance liquid chromatography) central control reaction on reactants obtained by the stirring reaction to obtain a reaction compound;
washing the reaction compound with 400ml of sodium chloride with the mass concentration of 10% for one time to remove an aqueous phase and a remaining organic phase; after the primary water washing, 400ml of sodium chloride with the mass concentration of 10% is used for secondary water washing, and the water phase is removed to keep an organic phase;
mixing the washed organic phase with 40g of sodium sulfate, standing for 0.5 hour, and filtering the mixture; concentrating under reduced pressure at 25deg.C;
after concentrating under reduced pressure, 300ml of methyl tertiary butyl ether and 600ml of N-heptane are added to dissolve the organic matters, and the mixture is stirred at 25 ℃ for 2.5 hours and subjected to crystallization treatment to obtain solid N4-AC-C- (S) -GNA phosphoramidite; the solid N4-AC-C- (S) -GNA phosphoramidite is added with 300ml of methyl tertiary butyl ether and 600ml of N-heptane again to be dissolved, stirred for 2.5 hours at 25 ℃ and recrystallized to obtain 24.0g of N4-AC-C- (S) -GNA phosphoramidite with purity more than 98 percent and yield: 72.6%.
Example III
The preparation method of the N4-AC-C- (S) -GNA phosphoramidite comprises the following steps of:
265g of preconfigured dichloromethane, 14.02g of diisopropyl ammonium tetrazole, 20g of (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine and 14.81g of bis (diisopropylamino) (2-cyanoethoxy) phosphine are placed in a reaction bottle, and stirred at 30 ℃ for 20 hours;
after stirring reaction, performing HPLC (high performance liquid chromatography) central control reaction, and confirming that the reaction is complete;
washing the reaction compound with 400ml of sodium chloride with the mass concentration of 10% for one time to remove an aqueous phase and a remaining organic phase; after the primary water washing, 400ml of sodium chloride with the mass concentration of 10% is used for secondary water washing, and the water phase is reserved for an organic phase;
mixing the washed organic phase with 50g of sodium sulfate, standing for 0.5 hour, and filtering the mixture; concentrating under reduced pressure at 30deg.C;
after concentrating under reduced pressure, 300ml of methyl tert-butyl ether and 600ml of N-heptane were added to dissolve the organics, stirred at 30 ℃ for 3 hours, filtered to give N4-AC-C- (S) -GNA phosphoramidite as a solid; the solid N4-AC-C- (S) -GNA phosphoramidite is added with 300ml of methyl tertiary butyl ether and 600ml of N-heptane again to be dissolved, stirred for 3 hours at 30 ℃, and the mixture is filtered to obtain 23.8g of N4-AC-C- (S) -GNA phosphoramidite in a powder form, the purity is more than 98 percent, and the yield is: 72%.
In specific application, the N4-AC-C- (S) -GNA phosphoramidite prepared by the invention is subjected to 1 H NMR analysis, results are shown in fig. 1:
1 H NMR(400MHz,CDCl 3 ):d＝7.53-7.58(m,2H),7.44-7.47(m,4H),7.31-7.35(m,8H),7.24-7.28(m,4H),7.20-7.23(m,4H),6.80-6.84(m,8H),4.28-4.41(m,4H),3.50-3.86(m,10H),3.79(s,6H),3.78(s,6H),3.37-3.40(dd,J＝4.6,10.0Hz,1H),3.23-3.26(dd,J＝4.5,9.9Hz,1H),3.17-3.21(dd,J＝2.8,10.0Hz,1H),3.10-3.13(dd,J＝4.4,10.0Hz,1H),2.51-2.68(dt,J＝6.8,16.8Hz,1H),2.39-2.42(t,J＝6.5Hz,2H),2.23(s,3H),2.24(s,3H),1.99(s,1H),1.10-1.15(m,24H)。
the N4-AC-C- (S) -GNA phosphoramidite prepared by the invention is subjected to 31 PNMR analysis, results are shown in fig. 2:
31 PNMR(162MHz,CDCl 3 ):d＝149.52,149.64。
the HPLC purity of the N4-AC-C- (S) -GNA phosphoramidite prepared by the method is shown in figure 3, and the HPLC purity is more than 95%.
Therefore, the N4-AC-C- (S) -GNA phosphoramidite prepared by the preparation method can be directly used for the next reaction without purification, and compared with a column chromatography purification process, the purity and the yield are improved. In addition, in the invention, the diisopropyl ammonium salt tetrazole is used for replacing 4, 5-dicyanoimidazole, thereby greatly reducing the cost; the stability is improved by selecting 10% sodium chloride aqueous solution instead of saturated sodium bicarbonate aqueous solution.
The present invention is not limited to the structure that has been described above and shown in the drawings, and various modifications and changes can be made without departing from the scope thereof. The scope of the invention is limited only by the appended claims.
Claims (10)
1. The N4-AC-C- (S) -GNA phosphoramidite is characterized by being prepared from the following raw materials in parts by weight:
315-325g of methylene dichloride, 24-24.1g of (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine, 15.5-15.6g of diisopropyl ammonium salt tetrazole and 16.35-16.45g of bis (diisopropylamino) (2-cyanoethoxy) phosphine.
2. A process for the preparation of N4-AC-C- (S) -GNA phosphoramidite according to claim 1, comprising:
placing pre-configured dichloromethane, (S) -1- (3- (4, 4' -dimethoxy trityl) -2-hydroxypropyl) -N4-acetylcytosine, diisopropyl ammonium salt tetrazole and bis (diisopropylamino) (2-cyanoethoxy) phosphine into a reaction bottle, and carrying out stirring reaction for a preset time;
after stirring reaction, performing HPLC (high performance liquid chromatography) central control reaction, and after confirming that the reaction is complete, performing multiple water washes on the reaction compound to remove an aqueous phase and a remaining organic phase;
the washed organic phase was filtered, concentrated and crystallized to give N4-AC-C- (S) -GNA phosphoramidite.
3. The process for producing N4-AC-C- (S) -GNA phosphoramidite according to claim 1, wherein the predetermined time is 17 to 20 hours and the temperature at the time of stirring reaction is 20 to 30 ℃.
4. The process for the preparation of N4-AC-C- (S) -GNA phosphoramidite according to claim 1, wherein the reaction compound is subjected to a plurality of water washes to remove the aqueous phase remaining organic phase comprising:
and (3) washing the reaction compound for multiple times by using sodium chloride with the mass concentration of 10% to remove the water phase and retain the organic phase.
5. The process for producing N4-AC-C- (S) -GNA phosphoramidite according to claim 4, wherein the number of water washes is 2 and the amount of sodium chloride used per water wash is 400 to 450ml.
6. The process for the preparation of N4-AC-C- (S) -GNA phosphoramidite according to claim 2, wherein the filtering, concentrating and crystallizing of the washed organic phase to obtain N4-AC-C- (S) -GNA phosphoramidite comprises:
mixing the washed organic phase with preset sodium sulfate, standing for a set time, and filtering the mixture after the standing time reaches the set time;
concentrating the filtered mixture under reduced pressure, adding methyl tertiary butyl ether and N-heptane to dissolve organic matters, and crystallizing to obtain N4-AC-C- (S) -GNA phosphoramidite.
7. The process for producing N4-AC-C- (S) -GNA phosphoramidite according to claim 6, wherein the amount of sodium sulfate is 30 to 50g and the time is set to 0.5 to 1 hour.
8. The process for producing N4-AC-C- (S) -GNA phosphoramidite according to claim 6, wherein the concentration under reduced pressure is carried out at a temperature of 20 to 30 ℃.
9. The process for producing N4-AC-C- (S) -GNA phosphoramidite according to claim 6, wherein the methyl tert-butyl ether is contained in an amount of 300 to 330ml, the N-heptane is contained in an amount of 600 to 660ml, and the ambient temperature is 20 to 30℃and the stirring time is 2 to 3 hours when the crystallization is carried out.
10. The method for producing N4-AC-C- (S) -GNA phosphoramidite according to claim 6, wherein the number of times of the crystallization is 2, the first crystallization gives N4-AC-C- (S) -GNA phosphoramidite as a solid, and the second crystallization gives N4-AC-C- (S) -GNA phosphoramidite as a powder by crystallizing the aqueous solution after the first crystallization.
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