Method for purifying phosphoramidite monomers

Abstract

The application discloses a method for purifying phosphoramidite monomers, and comprises the following steps: 1) adding a solvent, tetrazole, N-methyl imidazole and a phosphorus reagent under nitrogen protection into a reaction container and stirring; 2) dissolving raw materials in DMF solvent and adding into a reaction system under nitrogen protection; 3) ending when the peak area ratio of raw materials except the solvent on an HPLC spectrum is less than or equal to 0.5% and the purity is more than 95%; 4) concentrating the organic phase to no distillate and dissolving the solvent; 5) adding water and 3wt% NaCl aqueous solution into the reaction container, stirring, separating, and washing the upper organic phase; 6) concentrating the organic phase, centrifuging and crystallizing; 7) dissolving with methyl tert-butyl ether, adding a mixed solvent of DMF and H2O, separating, and washing the organic phase; 8) drying the organic phase, dropping into a solvent for crystallization; and 9) drying under N2. The method can remove trivalent phosphorus by using common chemical purification methods of water washing and crystallization, can wash pentavalent phosphorus with DMF and water, and can purify products, so that industrial production is realized.

Description

Technical Field

[0001] This invention relates to a method for purifying phosphorus amide monomers, belonging to the field of organic synthesis technology. Background Technology

[0002] Phosphoramide monomers are monomers used in the synthesis of oligonucleotides and DNA. Traditional processes primarily involve synthesizing 2'-F-ibu-dG phosphoramide monomer, followed by prep-HPLC to obtain the purified product. Two patent documents, US2022177888A1 and WO2023039076A1, describe the preparation of the product using a C18 silica column, acetonitrile, and water (0.5% NH4HCO3) as the mobile phase. Existing purification methods all involve prep-HPLC using a C18 silica column, acetonitrile, and water (0.5% NH4HCO3) as the mobile phase. This process is costly and has a relatively low yield of approximately 65%. The high cost is mainly due to the large amount of acetonitrile solvent used, the expensive C18 silica column, the reliance on HPLC equipment, the limited production volume, and the cumbersome production process. Summary of the Invention

[0003] The purpose of this invention is to provide a purification method for phosphoramide monomers, enabling the industrial production of 2'-F-ibu-dG phosphoramide monomers.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] The purification method for phosphoramide monomers includes the following steps:

[0006] (1) Control the temperature at 10±5℃, add solvent, tetrazolium, N-methylimidazole, and phosphorus reagent (bis(diisopropylamino)(2-cyanoethoxy)phosphine, CAS:102691-36-1) to the reaction vessel under nitrogen protection, and stir. The mass ratio of the solvent, tetrazolium, N-methylimidazole and phosphorus reagent is 38-40:0.3-0.4:0.12-0.13:4-4.5. In this step, the solvent is preferably DCM.

[0007] (2) Control the temperature at 10±5℃, dissolve the raw material 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-2'-fluorodeoxyguanosine in DMF solvent, wherein the molar ratio of the raw material to the phosphorus reagent is 1:1.6-1.7, add it to the reaction system of step (1), and the reaction is protected by nitrogen.

[0008] (3) Control the reaction system temperature at 15-20℃ and use HPLC detection. The peak area of ​​the raw materials other than the solvent should account for ≤0.5% on the HPLC spectrum. The reaction is considered complete when the purity of the product is above 95%. In this step, the reaction time is preferably 3-6 hours.

[0009] (4) The organic phase is concentrated until no fraction is produced, and then dissolved in a solvent. In this step, the solvent is methyl tert-butyl ether.

[0010] (5) Control the system temperature to 5-15℃, add water and 3wt% NaCl aqueous solution to the reaction vessel, stir, separate the layers, wash the upper organic phase, and detect it with HPLC until the DMF peak area ratio on the HPLC spectrum is <0.5%. In this step, the preferred mass ratio of water and 3wt% NaCl aqueous solution added to the reaction vessel before stirring is (6-8):(4-5). In this step, the preferred washing agent is water and 3wt% NaCl aqueous solution, with a mass ratio of water and 3wt% NaCl aqueous solution of (6-8):(4-5).

[0011] (6) The organic phase is concentrated to 15%-20% of the reaction system volume, and then added dropwise to a long-chain alkane solvent at 20℃-30℃ and -0.095Mpa~-0.1Mpa. The solid is obtained by crystallization and centrifugation. In this step, the preferred long-chain alkane solvent is n-heptane.

[0012] (7) Control the system temperature to 5-15℃, dissolve the white solid in methyl tert-butyl ether, add a mixed solvent of DMF and H2O, separate the liquid and wash the organic phase.

[0013] (8) The organic phase is dried and concentrated to 30%-40% of the organic phase solution volume. Then, it is added dropwise to a long-chain alkane solvent to crystallize, and the product is obtained by centrifugation. In this step, the long-chain alkane solvent is n-heptane, and the organic phase is dried with sodium sulfate.

[0014] (9) Drying under N2 protection.

[0015] Purification route diagram of the present invention:

[0016]

[0017] The method of this invention purifies 2'-F-ibu-dG phosphorous amide monomer. It employs common chemical purification methods such as water washing and crystallization to remove trivalent phosphine, and washes pentavalent phosphine with DMF and water. These steps also purify the product. Specifically, residual phosphine reagents from the reaction raw materials are removed by washing and crystallization, while pentavalent phosphine generated in the reaction system is removed by a mixed solvent of DMF and water, followed by crystallization to obtain a white powder product. In this process, both crystallization and washing remove some impurities, resulting in a final product purity >98.5% and a P-spectrum purity of 98%, achieving the goal of industrial-scale production of 2'-F-ibu-dG phosphorous amide monomer. The process of this invention uses common equipment such as reaction vessels, centrifuges, rotary evaporators, and ovens, employing conventional methods and common solvents for product purification. This simplifies factory scale-up, reduces operation, minimizes solvent usage, and saves costs, avoiding the disadvantages of difficult scale-up, large solvent usage, and high costs associated with traditional methods. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 The NMR spectrum of the product was obtained for Example 1.

[0020] Figure 2 The P-spectrum of the product was obtained for Example 1.

[0021] Figure 3 The HPLC chromatogram of the product was obtained for Example 1.

[0022] Figure 4 The NMR spectrum of the product was obtained for Example 2.

[0023] Figure 5 The P-spectrum of the product was obtained for Example 2.

[0024] Figure 6 The HPLC chromatogram of the product was obtained for Example 2.

[0025] Figure 7 The NMR spectrum of the product was obtained for Example 3.

[0026] Figure 8 The P-spectrum of the product was obtained for Example 3.

[0027] Figure 9 The HPLC chromatogram of the product was obtained for Example 3. Detailed Implementation

[0028] Example 1

[0029] Under controlled temperature of 15℃, add 39.75 kg of dichloromethane, 0.32 kg of tetrazolium, and 0.125 kg of 1-methylimidazole to a 150 L reactor. Under nitrogen protection, add 4.13 kg of bis(diisopropylamino)(2-cyanoethoxy)phosphine and stir for 10 min. Under controlled temperature of 15℃, dissolve 1.6 kg of the raw material 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-2'-fluorodeoxyguanosine in DMF (raw material to DMF mass ratio 1:4) and add it dropwise (using a peristaltic pump) to the reaction system. Under nitrogen protection, maintain the system temperature at 15℃ and react for 4 h. Then, begin HPLC sampling and analysis, sampling every hour. The reaction is considered successful if the peak area of ​​the raw material (excluding the solvent) on the HPLC spectrum is ≤0.5% and the product purity is ≥95%. Concentrate the organic phase until no fraction is produced, then dissolve in 75 L of methyl tert-butyl ether. The system temperature was controlled at 15℃. 30 kg of water and 20 kg of 3 wt% NaCl aqueous solution were added to the reactor and stirred for 0.5 h. The mixture was separated, and the upper organic phase was washed three times with 30 kg of water and 20 kg of 3 wt% NaCl aqueous solution. The organic phase was analyzed by HPLC; a DMF peak area ratio <0.5% was considered acceptable. The organic phase was concentrated to 15% of the reaction system (25℃, -0.095 MPa to -0.1 MPa), and then added dropwise to 43.78 kg of n-heptane for crystallization. Centrifugation yielded a white solid. The system temperature was controlled at 10℃. The white solid was dissolved in 49.36 kg of methyl tert-butyl ether, and a DMF / H2O mixed solvent (7.55 kg DMF and 8 kg H2O) was added. The mixture was separated, and the organic phase was washed seven more times. The organic phase was dried with sodium sulfate, concentrated to a solid state, and then added dropwise to 43.78 kg of n-heptane for crystallization. Centrifugation yielded the product. The product was dried in a vacuum drying oven at 25℃ under N2 protection. o The product was obtained by vacuum drying at C. The yield was 81%, purity 98.72%, P-spectrum 99.64%, and NMR characterization confirmed the correct structure. The final product spectrum is shown below. Figure 1-3 As shown.

[0030] Example 2

[0031] Under nitrogen protection, add 38 kg of dichloromethane, 0.30 kg of tetrazolium, and 0.12 kg of 1-methylimidazole to a 150 L reactor at a controlled temperature of 10 °C. Then add 4.5 kg of bis(diisopropylamino)(2-cyanoethoxy)phosphine and stir for 10 min. At a controlled temperature of 15 °C, dissolve 1.7 kg of the raw material 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-2'-fluorodeoxyguanosine in DMF (raw material to DMF mass ratio 1:4.5) dropwise into the reaction system (using a peristaltic pump). Maintain nitrogen protection during the reaction. After reacting for 4 h at a controlled temperature of 15 °C, begin HPLC sampling and analysis, sampling every hour. The reaction is considered successful if the area of ​​the raw material peaks (excluding the solvent) on the HPLC chromatogram is ≤0.5% and the product purity is ≥95%. Concentrate the organic phase until no fraction is produced, then dissolve in 75 L of methyl tert-butyl ether. The system temperature was controlled at 10℃. 40 kg of water and 25 kg of 3wt% NaCl aqueous solution were added to the reactor and stirred for 0.5 h. The mixture was separated, and the upper organic phase was washed three times with 40 kg of water and 25 kg of 3wt% NaCl aqueous solution. The organic phase was analyzed by HPLC; a DMF peak area ratio <0.5% was considered acceptable. The organic phase was concentrated to 20% of the reaction system (20℃, -0.095 MPa to -0.1 MPa), and then added dropwise to 46.51 kg of n-heptane for crystallization. Centrifugation yielded a white solid. The system temperature was controlled at 15℃. The white solid was dissolved in 50.32 kg of methyl tert-butyl ether, and a DMF / H2O mixed solvent (8.02 kg DMF and 8.5 kg H2O) was added. The mixture was separated, and the organic phase was washed seven more times. The organic phase was dried with sodium sulfate, concentrated to a solid state, and then added dropwise to 46.51 kg of n-heptane for crystallization. Centrifugation yielded the product. The product was dried in a vacuum drying oven at 25℃ under N2 protection. o The product was obtained by vacuum drying at C. The yield was 83%, purity 98.52%, P-spectrum 99.5%, and NMR characterization confirmed the correct structure. The final product spectrum is shown below. Figure 4-6 As shown.

[0032] Example 3

[0033] Under controlled temperature of 15℃, add 40 kg of dichloromethane, 0.40 kg of tetrazolium, and 0.13 kg of 1-methylimidazole to a 150 L reactor. Under nitrogen protection, add 4 kg of bis(diisopropylamino)(2-cyanoethoxy)phosphine and stir for 10 min. Under controlled temperature of 15℃, dissolve 1.6 kg of the raw material 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-2'-fluorodeoxyguanosine in DMF (raw material to DMF mass ratio 1:4) and add it dropwise (using a peristaltic pump) to the reaction system. Under nitrogen protection, maintain the system temperature at 15℃ and react for 4 h. Then, begin HPLC sampling and analysis, sampling every hour. The reaction is considered successful if the peak area of ​​the raw material (excluding the solvent) on the HPLC chromatogram is ≤0.5% and the product purity is ≥95%. Concentrate the organic phase until no fraction is produced, and dissolve in 100 L of methyl tert-butyl ether. The system temperature was controlled at 10℃. 30 kg of water and 20 kg of 3wt% NaCl aqueous solution were added to the reactor and stirred for 0.5 h. The mixture was separated, and the upper organic phase was washed three times with 30 kg of water and 20 kg of 3wt% NaCl aqueous solution. The organic phase was analyzed by HPLC; a DMF peak area ratio <0.5% on the HPLC chromatogram was considered acceptable. The organic phase was concentrated to 15% of the reaction system (30℃, -0.095 MPa to -0.1 MPa), and then added dropwise to 43.78 kg of n-heptane for crystallization. Centrifugation yielded a white solid. The system temperature was controlled at 5℃. The white solid was dissolved in 47.36 kg of methyl tert-butyl ether, and a DMF / H2O mixed solvent (7.55 kg DMF and 8 kg H2O) was added. The mixture was separated, and the organic phase was washed seven more times. The organic phase was dried with sodium sulfate, concentrated to a solid state, and then added dropwise to 43.78 kg of n-heptane for crystallization. Centrifugation yielded the product. The product was dried in a vacuum drying oven at 25℃ under N2 protection. o The product was obtained by vacuum drying at C. The yield was 82.1%, purity 98.56%, P-spectrum 98.91%, and NMR characterization confirmed the correct structure. The final product spectrum is shown below. Figure 7-9 As shown.

[0034] The above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for purifying phosphoramidite monomers, characterized in that Comprising the following steps: (1) Control temperature 10±5℃, add solvent, tetrazole, N-methyl imidazole to the reaction vessel, under nitrogen protection, add bis (diisopropylamino) (2-cyanoethoxy) phosphine, stir, the mass ratio of the solvent, tetrazole, N-methyl imidazole, phosphine reagent is 38-40:0.3-0.4:0.12-0.13:4-4.5, and the phosphine reagent is bis (diisopropylamino) (2-cyanoethoxy) phosphine; (2) Control temperature 10±5℃, dissolve the raw material 5'-O-(4,4'-dimethoxytriphenylmethyl)-N2-isobutyryl-2'-fluorodeoxyguanosine in DMF solvent, the molar mass ratio of the raw material to the phosphine reagent is 1:1.6-1.7, add the reaction system of step (1), and react under nitrogen protection; (3) Control the temperature of the reaction system at 15-20℃, detect by HPLC, and the peak area ratio of the raw material other than the solvent on the HPLC spectrum is ≤0.5%, the purity of the product in the reaction system is more than 95%, and the reaction is completed; (4) Concentrate the organic phase to no distillate, and dissolve by adding a solvent; (5) Control the system temperature at 5-15℃, add water and 3wt% NaCl aqueous solution to the reaction vessel, stir, separate, wash the upper organic phase, and detect by HPLC until the peak area ratio of DMF on the HPLC spectrum is <0.5%; (6) Concentrate the organic phase to 15%-20% of the volume of the reaction system, add long-chain alkane solvent dropwise at 20℃-30℃ and under -0.095Mpa~-0.1Mpa, centrifuge to obtain white solid; (7) Control the system temperature at 5-15℃, dissolve the white solid with methyl tert-butyl ether, add a mixed solvent of DMF and H2O, separate, and wash the organic phase; (8) Dry the organic phase, concentrate to 30%-40% of the volume of the organic phase solution, then drop into long-chain alkane solvent to crystallize, and centrifuge to obtain the product; (9) Dry under N2 protection; The long-chain alkane solvent is n-heptane.

2. The method of purifying a phosphoramidite monomer according to claim 1, wherein: In step (1), the solvent is DCM.

3. The method of purifying a phosphoramidite monomer according to claim 1, wherein: In step (3), the reaction time is 3-6 hours.

4. The method of purifying a phosphoramidite monomer of claim 1, wherein: In step (4), the solvent is methyl tert-butyl ether.

5. The method of purifying a phosphoramidite monomer according to claim 1, wherein: In step (5), the mass ratio of water and 3wt% NaCl aqueous solution added to the reaction vessel before stirring is (6-8):(4-5).

6. The method of purifying a phosphoramidite monomer of claim 1, wherein: In step (5), the washing agent is water and 3wt% NaCl aqueous solution, and the mass ratio of water and 3wt% NaCl aqueous solution is (6-8):(4-5).

7. The method of purifying a phosphoramidite monomer of claim 1, wherein: In step (8), the organic phase is dried with sodium sulfate.

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