A method for purifying phosphoramidite monomer

Abstract

The name of the present invention is a method for purifying a phosphoramidite monomer. A method for purifying a phosphoramidite monomer is provided, which comprises adding a first extractant to a reaction solution containing the phosphoramidite monomer for extraction, collecting the reaction solution after extraction; adding an alkaline aqueous solution to the reaction solution after extraction for water washing; adding a second extractant to the reaction solution after water washing for extraction, collecting the upper organic phase; adding an alkaline adsorbent to the collected organic phase and stirring, then removing the alkaline adsorbent and collecting the clarified organic phase; and removing the organic solvent of the collected clarified organic phase under reduced pressure to obtain a purified phosphoramidite monomer product. The above method has a simple overall process, low cost, is suitable for large-scale industrial applications, and has high production efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of organic synthesis. Specifically, the present invention relates to a method for purifying phosphoramidite monomers. Background Art

[0002] In recent years, with the efficient development and launch of mRNA vaccines, the entire field of nucleic acid drugs has developed rapidly. Nucleic acid drugs are one of the most promising frontiers in the future of biopharmaceuticals. As a representative of nucleic acid drugs, small nucleic acid drugs have shown great potential. Small nucleic acid drugs, namely oligonucleotide drugs, are short-chain nucleic acids composed of a dozen to dozens of nucleotides in series. At present, most oligonucleotide drugs are synthesized by solid-phase phosphoramidite chemistry. Among them, the preparation of the core material nucleoside phosphoramidite monomer (nucleoside monomer), especially its purification technology, is the key technology of the synthesis process.

[0003] The existing purification methods mainly include column chromatography, new purification methods (such as ion exchange resin purification method) and crystallization method. Column chromatography is a purification method that is widely used. For example, in the patent application with publication number US2022177888A, the use of C18 silica gel column is introduced, acetonitrile and water (0.5% ammonium bicarbonate) are used as mobile phases, and the product is prepared by high performance liquid preparative chromatography. However, the cost of column chromatography remains high, mainly due to the large amount of acetonitrile solvent, C18 silica gel is relatively expensive, and because it also needs to rely on special liquid phase equipment, the yield is low, and the production process is cumbersome and cannot be used for large-scale industrial purification of phosphoramidite monomers. A new purification method is introduced in the patent application with publication number CN101580528A. The method uses ion exchange resin for purification, which is equivalent to using ion exchange resin instead of silica gel for separation and purification. However, there are unstable factors in the concentration process of this method, which can easily destroy the product structure and generate new impurities in the scale-up production. In the crystallization method, due to the co-precipitation of impurities and the target product, it is difficult for the crystallization method to completely remove certain impurities generated during the reaction, and these impurities have a great impact on the subsequent application in the medical field. Although in the prior art, there have been studies on improving the crystallization method and obtained good results, for example, in the patent application with the publication number of CN117567259A, a method for purifying phosphoramidite monomers is introduced, which adopts a method of washing with water first and then crystallizing and obtains the effect that the purity of the final product reaches ≥98.5% and the P spectrum reaches 98%. However, the method is still complicated due to the need for washing with water and multi-step concentration and crystallization.

[0004] Therefore, there is still a need for a method for purifying phosphoramidite monomers with a simple process, low cost and high efficiency to solve the above-mentioned deficiencies in the prior art. Summary of the invention

[0005] The object of the present invention is to provide a method for purifying phosphoramidite monomers to solve the above problems.

[0006] In order to solve the above problems, the present invention provides a method for purifying phosphoramidite monomers, which comprises the following steps:

[0007] Step 1: adding a first extractant to a reaction solution containing a phosphoramidite monomer for extraction, and collecting the reaction solution after extraction;

[0008] Step 2: adding an alkaline aqueous solution to the reaction solution after extraction for washing;

[0009] Step 3: adding a second extractant to the reaction solution after washing with water for extraction, and collecting the upper organic phase;

[0010] Step 4: adding a basic adsorbent to the organic phase collected in step 3 and stirring, then removing the basic adsorbent and collecting the clarified organic phase; and

[0011] Step 5: Under reduced pressure, remove the organic solvent from the clear organic phase collected in step 4 to obtain a purified phosphoramidite monomer product.

[0012] In some embodiments, the extractant used in step 1 is selected from alkanes having 4 to 14 carbon atoms or mixtures thereof; more preferably, the extractant may be selected from petroleum ether, n-hexane, and n-heptane. Most preferably, the extractant may be n-heptane.

[0013] In some embodiments, the extraction is performed two or more times using the extractant in step 1.

[0014] In some embodiments, the alkaline aqueous solution used in step 2 is selected from sodium carbonate solution, sodium bicarbonate solution, potassium carbonate solution, potassium bicarbonate solution or any combination thereof.

[0015] In some embodiments, the concentration of the alkaline aqueous solution used in step 2 may be about 0.05-0.15 mol / L, preferably, about 0.07-0.13 mol / L, more preferably, about 0.09-0.11 mol / L, and most preferably, about 0.10 mol / L. In some embodiments, the concentration of the alkaline aqueous solution used in step 2 may be about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, or about 0.15 mol / L, or any value therebetween.

[0016] In some embodiments, the second extractant may be selected from toluene or ethyl acetate.

[0017] In some embodiments, step 2 and step 3 can be performed sequentially or simultaneously.

[0018] In some embodiments, the alkaline adsorbent used in step 4 can be selected from alkaline activated carbon, alkaline molecular sieve or a combination thereof. Preferably, the alkaline adsorbent used in step 4 can be alkaline activated carbon; preferably, the pH value of the alkaline adsorbent is optimally in the range of 9-11.

[0019] In some embodiments, in step 4, the alkaline adsorbent in the organic phase can be removed by suction filtration.

[0020] In some embodiments, in step 5, the solvent of the organic phase may be removed by distillation under reduced pressure or rotary evaporation.

[0021] In some embodiments, the phosphoramidite monomer purification method of the present invention optionally includes between step 3 and step 4:

[0022] Step 3a: adding sodium chloride solution to the organic phase collected in step 3 for washing, and then collecting the upper organic phase; and

[0023] Step 3b: Add a desiccant to the organic phase collected in step 3a for drying, and then remove the desiccant.

[0024] In some embodiments, the concentration of the sodium chloride solution added in step 3a is 10 wt % or more, for example, 10 wt %, 11 wt %, 12 wt %, 13 wt %, 14 wt %, 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 26.5 wt % or any value therebetween, preferably, about 20 wt %. In some embodiments, the sodium chloride solution added in step 3a is a saturated sodium chloride solution.

[0025] In some embodiments, the ratio of the amount of sodium chloride solution added in step 3a to the amount of the second extractant added in step 3 is about 1:3 to about 1:1; preferably, about 1:2.

[0026] In some embodiments, the desiccant added in step 3b is anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous copper phosphate, anhydrous silica gel; preferably, anhydrous sodium sulfate.

[0027] In some embodiments, the reaction solution containing the phosphoramidite monomer in step 1 is obtained by the following method:

[0028] At room temperature, preferably at about 25° C., a phosphorus reagent and a catalyst are added to a reaction solvent containing a precursor of a phosphoramidite monomer (e.g., TBS-rG (iBu), TBS-rA (Bz), TBS-rC (Ac), TBS-rU), and then reacted to completion at an elevated temperature (e.g., about 35-40° C.) under an inert atmosphere. The reaction can be monitored, for example, by HPLC, and the reaction is completed when the precursor content in the reaction solution is lower than a predetermined value.

[0029] In some embodiments, the reaction solvent is dimethylformamide (DMF).

[0030] In some embodiments, the total amount of the first extractant used in step 1 is about 0.5-2.5 times the amount of the reaction solvent, preferably, about 0.75-1.25 times, and more preferably, the total amount of the extractant used in step 1 is equal to the amount of the reaction solvent.

[0031] In some embodiments, the amount of the alkaline aqueous solution used in step 2 or the amount of toluene used in step 3 is about 1.5 to 2.5 times the amount of the reaction solvent.

[0032] In some embodiments, the weight ratio of the amount of the alkaline adsorbent added in step 4 to the amount of the phosphorus reagent added is about 1:10 to about 1:1, about 1:5 to about 1:1, about 1:5 to about 1:1.25, about 1:3 to about 1:1.5, preferably, about 1:3 to about 1:2.

[0033] In some embodiments, the added catalyst is 5-ethylthiotetrazolyl.

[0034] Additionally, the present invention provides a composition comprising a phosphoramidite monomer, the composition being obtained by the method of the present invention.

[0035] In the above method, the unreacted phosphorus reagent and impurities related to the phosphorus reagent can be removed by directly adding the first extractant to the phosphoramidite monomer solution. Then, impurities soluble in water and weak alkaline solution and soluble in the reaction solvent are removed by a water washing step. Finally, the remaining impurities in the reaction solution can be further removed using alkaline activated carbon without destroying the structure of the product. Optionally, the purity of the final phosphoramidite monomer can be further improved by washing the reaction solution with sodium chloride solution and drying. The method of the present invention has a simple overall process, low cost, is suitable for large-scale industrial applications, and has high production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1-2 HPLC chromatogram and P spectrum of the phosphoramidite monomer in Example 2;

[0037] Figure 3-4HPLC chromatogram and P spectrum of the phosphoramidite monomer in Example 3;

[0038] Figure 5-6 HPLC chromatogram and P spectrum of the phosphoramidite monomer in Example 4;

[0039] Figure 7-8 HPLC chromatogram and P spectrum of the phosphoramidite monomer in Example 5. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0041] The present invention provides a method for purifying a phosphoramidite monomer, which comprises adding an extractant to a phosphoramidite monomer solution for extraction twice or more, washing the reaction solution after extraction with a mixture of toluene and a sodium bicarbonate solution, and finally removing the remaining impurities with an alkaline adsorbent. The method removes impurities in the synthetic phosphoramidite monomer solution by combining extraction, water washing and adsorption purification, and obtains a final product with a relatively high purity (e.g., 99%).

[0042] Additionally, by adding a sodium chloride washing step, the purity of the final product can be further increased to more than 99%.

[0043] As used herein, "phosphoramidite monomer" refers to a nucleoside phosphoramide, which is a compound used to synthesize oligonucleotides.

[0044] The extractant that can be used in the present invention can be an alkane having 4-14 carbon atoms or a mixture thereof, such as an alkane having 5-12 carbon atoms, such as an alkane having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms, or a compound comprising such an alkane. Examples of the extractant include, but are not limited to, hexane (e.g., n-hexane), heptane (e.g., n-heptane), octane (e.g., n-octane) and petroleum ether, etc.

[0045] Example

[0046] The reagents and materials used in this example are all commonly used in the art and can be obtained through commercial purchase. The detection instruments and detection conditions are conventional instruments and general detection conditions in the art.

[0047] The meanings of some abbreviations in the following embodiments are as follows:

[0048] TBS-rG(iBu): 5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-O-[(tert-butyl)dimethylsilyl]-N-(2-methyl-1-oxopropyl)guanosine (CAS No.81279-39-2);

[0049] TBS-rA(Bz): 5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-N6-benzoyladenosine (CAS No. 81265-93-2);

[0050] TBS-rC(Ac): N-(1-((2r,3r,4r,5r)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)acetamide (CAS No.121058-85-3);

[0051] TBS-rU: 5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyluridine (CAS No. 81246-80-2);

[0052] DMF: N,N-dimethylformamide.

[0053] The pH value of alkaline activated carbon is around 10; the pH value of acidic activated carbon is between 3-5.

[0054] Example 1

[0055] 10g TBS-rG(iBu) and 50mL DMF were added to a 250mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7g phosphorus reagent (2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, CAS No.: 102691-36-1) and 1.69g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC.

[0056] After the reaction was completed, n-heptane was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0057] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0058] 2.0 g of 200 mesh alkaline activated carbon was added to the collected organic phase, stirred at 40°C for 1 hour, then filtered and the filtrate was collected, and distilled under reduced pressure at 40°C to obtain 11.73 g of the product, with a product yield of 90%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 99.0% and phosphorus spectrum purity of 99.0%.

[0059] Example 2

[0060] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0061] After the reaction was completed, n-heptane was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0062] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0063] Add 50 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, remove the lower aqueous layer and collect the upper organic phase.

[0064] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0065] 2.0 g of 200 mesh alkaline activated carbon was added to the filtrate, stirred at 40°C for 1 hour, then filtered and the filtrate was collected, and distilled under reduced pressure at 40°C to obtain 11.73 g of the product with a product yield of 90%. The product was confirmed to be a phosphoramidite monomer (MS: [M+H] + 968.9), HPLC purity: 99.3%, phosphorus spectrum purity: 99.0%. Spectral data are as follows Figure 1-2 shown.

[0066] Example 3

[0067] 10 g TBS-rA(Bz) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 5.47 g phosphorus reagent and 1.97 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0068] After the reaction was completed, n-heptane was added to the reaction solution for extraction (100 mL, × 2), and the reaction solutions after extraction were combined.

[0069] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers, and the lower aqueous layer was removed.

[0070] Add 50 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, and remove the lower aqueous layer.

[0071] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0072] Add 2.0 g of 200 mesh alkaline activated carbon to the filtrate, stir at 40°C for 1 hour, then filter and collect the filtrate and concentrate under reduced pressure at 40°C to obtain 11.30 g of the product with a product yield of 90%. The product was confirmed to be phosphoramidite (MS: [M+H] + 987.4), HPLC purity: 99.3%, phosphorus spectrum purity: 99.0%. Spectral data are as follows Figure 3-4 shown.

[0073] Example 4

[0074] 10 g TBS-rU and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 5.47 g phosphorus reagent and 1.97 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0075] After the reaction was completed, n-heptane was added to the reaction solution for extraction (100 mL, × 2), and the reaction solutions after extraction were combined.

[0076] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers, and the lower aqueous layer was removed.

[0077] Add 50 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, and remove the lower aqueous layer.

[0078] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0079] Add 2.0 g of 200 mesh alkaline activated carbon to the filtrate, stir at 40°C for 1 hour, then filter and collect the filtrate and concentrate under reduced pressure at 40°C to obtain 11.39 g of the product with a product yield of 90%. The product was confirmed to be phosphoramidite (MS: [M+H] + 860.3), HPLC purity: 99.3%, phosphorus spectrum purity: 99.0%. Spectral data are as follows Figure 5-6 shown.

[0080] Example 5

[0081] 10 g TBS-rC(Ac) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 5.15 g phosphorus reagent and 1.85 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0082] After the reaction was completed, n-heptane was added to the reaction solution for extraction (100 mL, × 2), and the reaction solutions after extraction were combined.

[0083] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was stirred for 30 minutes and then allowed to stand to separate the layers, and the lower aqueous layer was removed.

[0084] Add 100 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, and remove the lower aqueous layer.

[0085] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0086] Add 2.0 g of 200 mesh alkaline activated carbon to the filtrate, stir at 40°C for 1 hour, then filter and collect the filtrate and concentrate under reduced pressure at 40°C to obtain 11.13 g of the product with a product yield of 90%. The product was confirmed to be phosphoramidite (MS: [M+H] + 901.4), HPLC purity: 99.3%, phosphorus spectrum purity: 99.0%. Spectral data as shown Figure 7-8 shown.

[0087] Example 6

[0088] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0089] After the reaction was completed, n-heptane was added to the reaction solution for extraction (50 mL, × 1), and the reaction solutions after extraction were combined.

[0090] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0091] Add 50 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, remove the lower aqueous layer and collect the upper organic phase.

[0092] Anhydrous copper phosphate was added to the organic phase washed with the sodium chloride solution, dried for 1 hour, and then filtered to obtain a filtrate.

[0093] Add 2.8g of 200-mesh alkaline activated carbon to the filtrate, stir at 40°C for 1 hour, then filter and collect the filtrate, and distill under reduced pressure at 40°C to obtain 11.85g of the product, with a product yield of 91%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 99.3% and phosphorus spectrum purity of 99.0%.

[0094] Example 7

[0095] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0096] After the reaction was completed, n-hexane was added to the reaction solution for extraction (25 mL, ×2), and the reaction solutions after extraction were combined.

[0097] To the reaction solution after extraction, 125 mL of toluene and 125 mL of 0.1 mol / L potassium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0098] Add 50 mL of 10% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, remove the lower aqueous layer and collect the upper organic phase.

[0099] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0100] Add 2.0 g of 200 mesh basic molecular sieve to the filtrate, stir at 40°C for 1 hour, then filter and collect the filtrate, and distill under reduced pressure at 40°C to obtain 11.80 g of the product, with a product yield of 90%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 99.3% and phosphorus spectrum purity of 99.0%.

[0101] Example 8

[0102] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0103] After the reaction was completed, petroleum ether was added to the reaction solution for extraction (75 mL, ×2), and the reaction solutions after extraction were combined.

[0104] To the reaction solution after extraction, 100 mL of ethyl acetate and 100 mL of 0.05 mol / L sodium carbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0105] Add 100 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, remove the lower aqueous layer and collect the upper organic phase.

[0106] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0107] 2.3 g of 200 mesh alkaline activated carbon was added to the filtrate, stirred at 40°C for 1 hour, then filtered and the filtrate was collected, and distilled under reduced pressure at 40°C to obtain 11.90 g of the product, with a product yield of 91%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 99.3% and phosphorus spectrum purity of 99.1%.

[0108] Example 9

[0109] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0110] After the reaction was completed, n-hexane was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0111] To the reaction solution after extraction, 75 mL of toluene and 75 mL of 0.15 mol / L potassium carbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0112] Add 50 mL of saturated sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, remove the lower aqueous layer and collect the upper organic phase.

[0113] Anhydrous sulfuric acid was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0114] 1.5 g of 200 mesh alkaline activated carbon was added to the filtrate, stirred at 40°C for 1 hour, then filtered and the filtrate was collected, and distilled under reduced pressure at 40°C to obtain 11.93 g of the product, with a product yield of 91%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 99.3% and phosphorus spectrum purity of 99.0%.

[0115] Example 10

[0116] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0117] After the reaction was completed, petroleum ether was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0118] To the reaction solution after extraction, 100 mL of toluene and 75 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers. The lower aqueous layer was removed, and the upper organic phase was collected.

[0119] Add 50 mL of 15% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, remove the lower aqueous layer and collect the upper organic phase.

[0120] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0121] 1.8 g of 200 mesh alkaline activated carbon was added to the filtrate, stirred at 40°C for 1 hour, then filtered and the filtrate was collected, and distilled under reduced pressure at 40°C to obtain 11.68 g of the product, with a product yield of 90%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 99.3% and phosphorus spectrum purity of 99.1%.

[0122] Comparative Example 1

[0123] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0124] After the reaction was completed, n-heptane was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0125] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers, and the lower aqueous layer was removed.

[0126] Add 50 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, and remove the lower aqueous layer.

[0127] Anhydrous sodium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0128] Add 2.0 g of 200 mesh acid activated carbon to the filtrate, stir at 40°C for 1 hour, then filter and collect the filtrate, and distill under reduced pressure at 40°C to obtain 11.96 g of the product, with a product yield of 90%. The product was confirmed to be a phosphoramidite monomer, with HPLC purity of 98.0% and phosphorus spectrum purity of 98.0%.

[0129] Comparative Example 2

[0130] 10g TBS-rG(iBu) and 50mL DMF were added to a 250mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7g phosphorus reagent (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, CAS No.: 102691-36-1) and 1.69g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC.

[0131] After the reaction was completed, n-heptane was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0132] Add 2.0 g of 200 mesh alkaline activated carbon to the reaction solution after extraction, stir at below 40°C for 1 hour, then filter and collect the filtrate.

[0133] Add 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate to the collected filtrate, stir for 30 minutes and let stand to separate the layers, remove the lower aqueous layer, and collect the upper organic phase.

[0134] The product was obtained by vacuum distillation at a temperature below 40°C with a yield of 89%. The product was confirmed to be a phosphoramidite monomer with a HPLC purity of 97.9% and a phosphorus spectrum purity of 98.1%.

[0135] Comparative Example 3

[0136] 10 g TBS-rG (iBu) and 50 mL DMF were added to a 250 mL four-necked bottle and stirred at 25°C for about 20 minutes until completely dissolved. Then 4.7 g phosphorus reagent and 1.69 g 5-ethylthiotetrazolyl were added to the four-necked bottle, and the air in the bottle was replaced with nitrogen three times at 25°C, and then the temperature was raised to 40°C for reaction. The reaction was monitored by HPLC to completion.

[0137] After the reaction was completed, n-heptane was added to the reaction solution for extraction (50 mL, × 2), and the reaction solutions after extraction were combined.

[0138] To the reaction solution after extraction, 100 mL of toluene and 100 mL of 0.1 mol / L sodium bicarbonate were added, and the mixture was allowed to stand for 30 minutes to separate the layers, and the lower aqueous layer was removed.

[0139] Add 50 mL of 20% sodium chloride solution to the organic phase obtained in the previous step, wash with water for 30 minutes, let stand to separate the layers, and remove the lower aqueous layer.

[0140] Anhydrous magnesium sulfate was added to the organic phase washed with the sodium chloride solution, and the mixture was dried for 1 hour, and then suction filtered to obtain a filtrate.

[0141] 12.09 g of the product was obtained by distillation under reduced pressure below 40°C, with a product yield of 90%. The product was confirmed to be phosphoramidite, with an HPLC purity of 97.7% and a phosphorus spectrum purity of 97.9%.

[0142] It can be seen from the above examples that the purity of the phosphoramidite monomers purified by the method of the present invention is greater than 99%, and the phosphorus spectrum data are greater than or equal to 99%.

[0143] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for purifying phosphoramidite monomers, characterized in that: The method comprises the following steps: Step 1: adding a first extractant to a reaction solution containing a phosphoramidite monomer for extraction, and collecting the reaction solution after extraction, wherein the reaction solution containing the phosphoramidite monomer is obtained by adding a phosphorus reagent and a catalyst to a reaction solvent containing any one selected from TBS-rG (iBu), TBS-rA (Bz), TBS-rC (Ac), TBS-rU at room temperature, and then reacting at an elevated temperature under an inert atmosphere, and the first extractant is selected from petroleum ether, n-hexane, and n-heptane; Step 2: adding an alkaline aqueous solution to the reaction solution after extraction for washing; Step 3: adding a second extractant to the reaction solution after washing with water for extraction, and collecting the upper organic phase, wherein the second extractant is selected from toluene or ethyl acetate; Step 4: adding a basic adsorbent to the organic phase collected in step 3 and stirring, then removing the basic adsorbent and collecting a clarified organic phase, wherein the basic adsorbent is selected from basic activated carbon, basic molecular sieves or a combination thereof; and Step 5: Under reduced pressure, remove the organic solvent from the clear organic phase collected in step 4 to obtain a purified phosphoramidite monomer product.

2. The method according to claim 1, characterized in that The alkaline aqueous solution is selected from sodium carbonate solution, sodium bicarbonate solution, potassium carbonate solution, potassium bicarbonate solution or any combination thereof, and / or, The concentration of the alkaline aqueous solution is 0.05-0.15 mol / L.

3. The method according to claim 1, characterized in that Step 2 and step 3 are performed sequentially or simultaneously.

4. The method according to claim 1, characterized in that The method further comprises between step 3 and step 4: Step 3a: adding sodium chloride solution to the organic phase collected in step 3 for washing, and then collecting the upper organic phase; and Step 3b: Add a desiccant to the organic phase collected in step 3a for drying, and then remove the desiccant.

5. The method according to claim 1, characterized in that The total amount of the first extractant used in step 1 is 0.5-2.5 times the amount of the reaction solvent, and the amount of the alkaline aqueous solution used in step 2 and the second extractant used in step 3 are respectively 1.5 to 2.5 times the amount of the reaction solvent.

6. The method according to claim 1, characterized in that The weight ratio of the amount of the alkaline adsorbent added in step 4 to the amount of the phosphorus reagent added is 1:10 to 1:

1.

7. The method according to claim 1, characterized in that The catalyst is 5-ethylthiotetrazolyl, and / or the reaction solvent is DMF.

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