Phosphoramidite cation and synthesis method thereof

Abstract

The structure of the phosphoramidite cation is shown as follows: # imgabs0 #, wherein B is a basic group and is selected from one of A, U, C, G and T; r2 is alkyl, A2 is O or C, n is a natural number of 1-20, and X is selected from one of F, Cl, Br and I.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and particularly to a phosphoramidite cation and a method for synthesizing the same. Background Art

[0002] After the outbreak of the epidemic, the efficient research and development of mRNA vaccines has driven the rapid development of the entire nucleic acid drug field. Domestic and foreign capitals have targeted its huge market space and development potential and have all made layouts. The research and development of nucleic acid drugs has become one of the hottest fields in the world. 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) is the key technology of the solid-phase synthesis process and has a relatively high technical barrier.

[0003] Due to reasons such as the easy degradation of the original small nucleic acid structure by nuclease in the body and poor pharmacokinetic properties, it generally cannot be directly used as a drug and needs to undergo chemical modifications at multiple sites to improve its affinity, stability, metabolic properties, etc. After decades of accumulation, three generations of technologies have been developed. Commonly used chemical modifications are the first-generation phosphorothioate and the second-generation methylphosphonate. Methylphosphonate is uncharged, so it is more lipophilic than natural DNA or RNA and can better penetrate cells. Subsequently, the third-generation PNA, LNA and other technologies have emerged. The unique chemical structure of traditional oligonucleotide drugs shows poor drugability: large molecular weight, strong hydrophilicity, highly negative charge, not following Lipinski's rule, and also having poor pharmacokinetic characteristics, unable to pass through biological membranes, and there will also be off-target effects. These disadvantages will be significantly amplified after using cationic monomers. Summary of the Invention

[0004] Researchers have discovered a series of active substances in the field of phosphoramidite cation monomers, expanding the breadth of this field. The effective delivery of oligonucleotide therapies to various tissues remains a major challenge. Oligonucleotides are usually large hydrophilic polyanions (single-stranded ASO is about 410 kDa, double-stranded siRNA is about 14 kDa), which means they are not easily passed through the plasma membrane. After synthesizing oligonucleic acids using cationic phosphoramidite monomers, due to the positive charge, the physical properties will change significantly, and many disadvantages of traditional oligonucleic acid drugs can be overcome, such as improving permeability, delivery ability, etc.

[0005] The main technical problem to be solved by the present invention is to provide a phosphoramidite cation monomer and a synthesis method. This synthesis method can provide raw materials for synthesizing new RNA nucleoside sequences, and lay a foundation for the low-cost, high-speed and high-throughput research and development of new siRNA drugs and new nucleic acid sequence analysis technologies. Through the synthesis method of this patent, the target product can be obtained efficiently and quickly, and the yields obtained from the substrates listed in the present invention are all relatively high.

[0006] An embodiment of the present invention provides a phosphoramidite cation, and the structure of the phosphoramidite cation is shown as follows:

[0007] Wherein B is a base, selected from one of A, U, C, G, T; R2 is an alkyl group, A2 is O or C, n is a natural number from 1 to 20, and X is selected from one of F, Cl, Br, I; preferably, R2 is an alkyl group with 1 to 18 carbon atoms; more preferably, R2 is an alkyl group with 1 to 8 carbon atoms.

[0008] An embodiment of the present invention also provides a preparation method of the phosphoramidite cation, and the route of the preparation method is as follows:

[0009]

[0010] An embodiment of the present invention provides a preparation method of the phosphoramidite cation, and the route of the preparation method is as follows:

[0011]

[0012] The technical solution of the present invention has the following excellent technical effects:

[0013] 1. The synthesis method of the present invention has mild reaction conditions, simple post-treatment and convenient operation, and is suitable for industrial production;

[0014] 2. After synthesizing oligonucleic acids using the cationic phosphoramidite monomer, due to the positive charge of the cationic phosphoramidite, the physical properties will change significantly, and many disadvantages of traditional oligonucleic acid drugs can be overcome, such as improving permeability and delivery ability. After introducing a structure with such characteristics into oligonucleotide fragments, breakthrough results are expected to be achieved in related research fields (especially in the field of siRNA drug research and development); BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a trend graph of the hepatic microsomal stability of four compounds prepared in the examples of the present invention;

[0016] Figure 2 It is a linear graph of the half-life of four compounds prepared in the examples of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the following further details the present invention with reference to specific embodiments and the accompanying drawings. However, those skilled in the art will understand that the present invention is not limited to the drawings and the following embodiments.

[0018]

[0019] Example 1 (Synthetic Route 1)

[0020] Operate according to the following steps:

[0021] Step 1:

[0022] Add anhydrous DMF solution (100 mL) to 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-C-D-ribofuranose (1.201 g, 0.064 eq.) (Compound A). Add sodium hydride (60% mineral oil w / w, total 2.85 g, 0.075 eq.) at -5°C. Dropwise add benzyl bromide (8.9 mL, 0.075 eq.) and stir at room temperature for 3 hours, then add ice water (50 mL). Extract the reaction solution with ethyl acetate (4 × 100 mL), and dry the combined organic phases (Na2SO4). After concentration, the residue is eluted with petroleum ether / ethyl acetate = 1 / 20 to obtain product Compound B (18.5 g, 71%).

[0023] Step 2:

[0024] Dropwise add acetic anhydride (1.08 mL, 11.4 mmol) to an anhydrous pyridine (4.5 mL) solution of 3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-x-D-ribofuranose (913 mg, 2.28 mmol) (Compound B), and react at room temperature for 3 hours. Quench the reaction by adding ice water (50 mL), and extract with dichloromethane (3 × 50 mL). Wash the combined organic phases with saturated aqueous sodium bicarbonate solution (2 × 50 mL), dry (Na2SO4), filter, concentrate, and purify by silica gel column chromatography using dichloromethane as the eluent to obtain the product as a transparent oily substance Compound C (911 mg, 90%).

[0025] Steps 3 & 4:

[0026] 4-C-Acetoxymethyl-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose (830 mg, 1.88 mmol) (Compound C) was added to 80% acetic acid to form a solution (10 mL), and the solution was stirred at 90 °C for 4 h. After removing the solvent under reduced pressure, the residue was co-evaporated with ethanol (3 x 5 mL), toluene (3 x 5 mL) and anhydrous pyridine (3 x 5 mL), and redissolved in anhydrous pyridine (3.7 mL). Acetic anhydride (2.85 mL) was added and the solution was stirred, and the solution was stirred at room temperature for 72 h. The solution was poured into 20 mL of ice water, and the mixture was extracted with dichloromethane (2 x 20 mL). The combined organic phases were washed with saturated aqueous sodium bicarbonate (2 x 20 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane as the eluent to give Compound E (the ratio of the two configurations β:α ~ 1:3) as a clear oil (789 mg, 86%).

[0027] Step 5:

[0028] Compound E (736 mg, 1.51 mmol) and thymine (381 mg, 3.03 mmol) were dissolved in anhydrous acetonitrile (14.5 mL) to form a solution, and N,O-bis(trimethylsilyl)acetamide (2.61 mL, 10.6 mmol) was added with stirring. The reaction mixture was stirred under reflux for 1 h and then cooled to 0 °C. Trimethylsilyl trifluoromethanesulfonate (0.47 mL, 2.56 mmol) was added dropwise with stirring, and the solution was stirred at 65 °C for 2 h. Saturated aqueous sodium bicarbonate (15 mL) was added and the mixture was extracted with dichloromethane (3 x 10 mL). The combined organic phases were washed with saturated aqueous sodium bicarbonate (2 × 10 mL) and brine (2 × 10 mL), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane / methanol (98:2, v / v) as the eluent to give Compound F as a white solid (639 mg, 76%).

[0029] Step 6:

[0030] Compound F (553 mg, 1.05 mmol) was dissolved in methanol (5.5 mL) to form a solution, and sodium methoxide (287 mg, 5.25 mmol) was added with stirring. The reaction mixture was stirred at room temperature for 10 min and then neutralized with dilute hydrochloric acid. The solvent was partially evaporated and the mixture was extracted with dichloromethane (2 x 20 mL). The combined organic phases were washed with saturated aqueous sodium bicarbonate (3 × 20 mL) and dried over Na2SO4. The solvent was removed under reduced pressure to give Compound G as a white solid (476 mg, 97%).

[0031] Step 7:

[0032] Compound G (225 mg, 0.48 mmol) was added to anhydrous pyridine (1.3 mL) to form a solution, which was stirred, and a small amount of p-toluenesulfonyl chloride (118 mg, 0.62 mmol) was added at 0 °C. The solution was stirred at room temperature for 16 h and additional p-toluenesulfonyl chloride (36 mg, 0.19 mmol) was added. Stirring was continued for 4 h, ice water (15 mL) was added, and the mixture was extracted with dichloromethane (2 × 15 mL). The combined organic phases were washed with saturated aqueous sodium bicarbonate (3 × 15 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel using dichloromethane / methanol (99:1, v / v) as the eluent to give the intermediate (140 mg). This intermediate (102.2 mg) was dissolved in anhydrous DMF (0.8 mL). The solution was added dropwise to a mixture of 60% sodium hydride in mineral oil (w / w, 32 mg, 0.80 mmol) and anhydrous DMF (0.8 mL). The mixture was stirred for 72 h and then concentrated under reduced pressure. The residue was dissolved in dichloromethane (10 mL), washed with saturated aqueous sodium bicarbonate (3 × 5 mL) and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel using dichloromethane / methanol (99:1, v / v) as the eluent to give Compound H as a white solid (65.7 mg, 42%).

[0033] Step 8:

[0034] Compound H (97 mg, 0.215 mmol) was dissolved in ethanol (1.5 mL), and the mixture was stirred at room temperature. 20% Palladium hydroxide on carbon (50 mg) was added. The mixture was purged with argon three times and with hydrogen three times, and then heated to 65 °C under a hydrogen atmosphere. After stirring for 4 h, the mixture was subjected to silica gel column chromatography using dichloromethane-methanol (97:3, v / v) as the eluent to give Compound J as a white solid material (57 mg, 98%).

[0035] Step 9:

[0036] At 0 °C, compound J (1.2 g, 4.44 mmol) was dissolved in anhydrous pyridine (5 mL) to form a solution, and 4,4'-dimethoxytributyl chloride (2.37 g, 7.0 mmol) was added. The solution was stirred at room temperature for 2 hours, then the reaction was quenched with ice water (10 mL) and extracted with dichloromethane (3 x 15 mL). The combined organic phases were washed with saturated aqueous sodium bicarbonate (3 x 10 mL), brine (2 x 10 mL) and dried over Na2SO4. The solvent residue was removed under reduced pressure and purified by silica gel column chromatography using dichloromethane / methanol (98:2, v / v) to give compound K as a white solid (2.35 g, 93%).

[0037] Step 10:

[0038] Bis(N,N-diisopropylamino)chlorophosphine (323 mg, 1.21 mmol) was dissolved in anhydrous dichloromethane (3 mL) to form a solution. The above solution was added dropwise to a stirred solution of compound K (646 mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) in anhydrous dichloromethane (4 mL) while controlling the temperature at -20 °C. The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3 x 50 mL). The extract was washed with brine, dried over anhydrous Na2SO4 and evaporated to an oil. The product was separated by column chromatography on silica gel eluted with a step gradient starting from TEA / ethyl acetate / hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of the fractions gave compound L as a viscous oil, yield 1038 mg (81.2%).

[0039] Step 11:

[0040] 1H-Tetrazole (0.4 eq.) was added to a mixture of compound L, choline (1.5 eq.) and dichloromethane (10 mL). The resulting solution was stirred at room temperature for 2 hours. 5% aqueous NaHCO3 (10 mL) was added, the emulsion was diluted with brine (50 mL), and the product was extracted with ethyl acetate (3 x 75 mL). The extract was washed with brine (3 x 50 mL), dried over Na2SO4 and evaporated to dryness. The residue was dissolved in toluene (25 mL), applied to a silica gel column and separated with an eluent gradient of ethyl acetate / hexane / triethylamine 15:80:5 to 80:15:5. The collected fractions were evaporated, co-evaporated with dry MeCN (2 x 50 mL) and dried on a high vacuum pump to give compound M.

[0041] Example 2 (Synthetic Route II)

[0042]

[0043] Operation:

[0044] Step 1:

[0045] Under nitrogen protection, add 3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-C-D-ribofuranose (1 Kg, 3.2 mol, 1 eq.) (Compound A1) to 2.8 L of dichloromethane / pyridine (1:1) for dissolution, cool to 0 - 5 °C, dropwise add MsCl (885 g, 7.73 mol, 2.4 eq.), stir for 2 hours, detect by TLC. After the raw material reaction is completed, add water at 10 °C, add 1.5 L of dichloromethane, wash the aqueous phase with 1N hydrochloric acid (2 * 2.5 L + 1.2 L) and 6% aqueous sodium bicarbonate solution (2.0 L), dry the organic phase with sodium sulfate (600 g), filter, and concentrate the organic phase to obtain 1500 g of product Compound B1 (yield: 98%).

[0046] Step 2:

[0047] Suspend Compound B1 (1000 g) in 3.0 L of acetic acid to obtain an off-white suspension. Dissolve 5.7 mL of sulfuric acid in 100 mL of acetic acid and add it to the above suspension. Dropwise add acetic anhydride (364 mL) at 20 °C for 1 hour. Stir at 25 °C for 16 hours. After detecting by LCMS that the raw material reaction is completed, add 3.0 L of dichloromethane and 3.3 L of water to the reaction solution. Separate the layers, wash the organic phase with 1M KH2PO4 (2 * 2.5 L) and saturated sodium bicarbonate 2.5 L, concentrate the organic phase to obtain 1200 g of product Compound C1. The yield is between 90% - 95%.

[0048] Step 3:

[0049] Under a nitrogen atmosphere, dissolve Compound C1 (1000 g) in anhydrous acetonitrile (2 L), add adenine (291 g), and stir the mixture at 25 °C. Dropwise add SnCl4 (1123 g), with the internal temperature not exceeding 45 °C. Keep the temperature at 40 °C for 2 hours. After LCMS shows that the raw material reaction is completed, cool the reaction solution to 0 °C, adjust the pH to 4.5 - 5.0 with 14M sodium hydroxide, filter with diatomaceous earth, wash the filter cake with ethyl acetate (2 L), wash the organic phase with 1M KH2PO4 (1 * 3 L), and concentrate the organic phase to obtain 780 g of product D1. The yield is between 65% - 71%.

[0050] Step 4:

[0051] Compound D1 (1000 g) was suspended in THF (3.33 L) to obtain a white mixture. 1 M sodium hydroxide (6.38 L) was added, and the mixture was stirred at 20 °C for 1 hour. LCMS showed that the raw material had reacted completely. The solvent was evaporated using a rotary evaporator until 2 L was collected. The mixture was added to 2.5 L of deionized water, filtered on GF-P3, and the filter cake was washed with deionized water (3 * 1.5 L). The product was dried to obtain 688 g of product compound E1. The yield was 90% - 95%.

[0052] Step 5:

[0053] Compound E1 (1000 g) was dissolved in acetonitrile (3 L), concentrated twice, then concentrated twice with 2 L of toluene, and finally dissolved in DMF (9.0 L). Sodium benzoate (805 g) was added, and the mixture was heated to 100 °C and reacted for 2 hours. LCMS showed that the raw material had reacted completely. The reaction solution was cooled to room temperature, 20 L of deionized water was added, and the mixture was filtered on GF-P3. The filter cake was washed with deionized water, and the filter cake compound F1 was directly used for the next step.

[0054] Step 6:

[0055] Compound F1 (1000 g) was dissolved in THF (6.0 L) to form a solution and stirred. Water (5.0 L) and sodium hydroxide (110 g) were added, and the mixture was stirred at room temperature for 1 hour. LCMS showed that the raw material had reacted completely. The mixture was concentrated by rotary evaporation until 4 L was collected. The mixture was added to 4.5 L of deionized water, cooled to 10 °C, filtered on GF-P3, and the filter cake was washed with deionized water (3 L). The white crystals were dried to obtain 750 g of product compound G1. The yield was 90% - 95%.

[0056] Step 7:

[0057] Compound G1 (97 mg, 0.215 mmol) was dissolved in methanol (1.5 mL), and the mixture was stirred at room temperature. 20% palladium on carbon (50 mg) was added. The mixture was purged with argon three times and with hydrogen three times, and then heated to 65 °C under a hydrogen atmosphere. After stirring for 4 hours, the mixture was subjected to silica gel column chromatography using dichloromethane - methanol (97:3, v / v) as the eluent to obtain 57 mg (98%) of white solid material compound J.

[0058] Step 8:

[0059] At 0 °C, compound J (1.2 g, 4.44 mmol) was added to anhydrous pyridine (5 mL) to form a solution. To the above solution was added 4,4'-dimethoxytributyl chloride (2.37 g, 7.0 mmol). The solution was stirred at room temperature for 2 hours, then quenched with ice water (10 mL) and extracted with dichloromethane (3 x 15 mL). The combined organic phases were washed with saturated aqueous sodium hydrogen carbonate, carbonate (3 x 10 mL), brine (2 x 10 mL) and dried over Na2SO4. The solvent residue was removed under reduced pressure and purified by silica gel column chromatography using dichloromethane / methanol (98:2, v / v) to give compound K in the form of a white solid (2.35 g, 93%).

[0060] Step 9:

[0061] Bis(N,N-diisopropylamino)chlorophosphine (323 mg, 1.21 mmol) was dissolved in anhydrous dichloromethane (3 mL) to form a solution. The above solution was added dropwise to a stirred solution of compound K (646 mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) in anhydrous dichloromethane (4 mL) while controlling the temperature at -20 °C. The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3 x 50 mL). The extract was washed with brine, dried over anhydrous Na2SO4 and evaporated to an oil. The product was separated by column chromatography on silica gel, which was eluted with a step gradient starting from TEA / ethyl acetate / hexane (5∶20∶75) to a mixture of TEA in ethyl acetate (5∶95). Evaporation of the fractions gave compound L as a viscous oil, yield 1038 mg (81.2%).

[0062] Step 10:

[0063] 1H-tetrazole (0.4 eq.) was added to a mixture of compound L, choline (1.5 eq.) and dichloromethane (10 mL). The resulting solution was stirred at room temperature for 2 hours. Aqueous NaHCO3 (5%, 10 mL) was added, the emulsion was diluted with brine (50 mL), and the product was extracted with ethyl acetate (3 x 75 mL). The extract was washed with brine (3 x 50 mL), dried over Na2SO4 and evaporated to dryness. The residue was dissolved in toluene (25 mL), applied to a silica gel column and separated with an eluent gradient of ethyl acetate / hexane / triethylamine 15:80:5 to 80:15:5. The collected fractions were evaporated, co-evaporated with dry MeCN (2 x 50 mL) and dried on an oil pump to give compound M.

[0064] Differences between Synthetic Route 1 and Synthetic Route 2:

[0065] Synthetic route 1 has relatively longer steps, but the reagents used are inexpensive and easily available, which is suitable for small-scale research; synthetic route 2 has relatively shorter steps, with good applicability for various types of substrates, ideal yields, suitable for kilogram-scale amplification, and there are research data on kilogram-scale amplification processes.

[0066] Detection and comparison of activity data:

[0067]

[0068] Six representative phosphoramidite monomers of the present invention were synthesized (B = A, R2 = Me, A2 = O, n = 2, X are F, Cl, Br, I, SO4 2- , PO4 3- ). Oligonucleic acids were synthesized using Cytiva OP100 to detect hepatic microsomal stability. The hepatic microsomal stability data and half-life chart activity chart are shown in the attached figure: (Among them, for SO4 2- , PO4 3- The synthesis of oligonucleic acids from two monomers failed, possibly due to the too strong electronegativity of the anionic group. For compounds 1, 2, 3, and 4, X are F, Cl, Br, and I respectively)

[0069] Half-life and clearance rate data:

[0070] Compound 1

[0071]

[0072]

[0073] Compound 2

[0074]

[0075] Compound 3

[0076]

[0077] Compound 4

[0078]

[0079]

[0080] From the above data, it can be seen that the half-life and clearance rate data of Compound 4 are relatively good, and the trends of liver microsome stability and half-life are also relatively gentle, with little difference overall except for Compound 2. The half-life of Compound 4 of the present invention is relatively short, and the metabolism rate of the drug is relatively fast, indicating that the metabolism rate of the drug in the body is also relatively fast, and it can be metabolized out of the body in a timely manner, generally not causing too much impact on physical health. Moreover, the clearance rate data can meet the ideal human requirements. For drugs with too high clearance rate, the half-life in the body is short, the oral bioavailability is poor, and the blood drug concentration is difficult to maintain above the effective concentration, resulting in poor drug efficacy in the body. While drugs with too low clearance rate are prone to accumulate in the body and produce toxicity. For drugs with a small safety window, special attention needs to be paid to the changes in their blood drug concentration. The compounds of the present invention avoid the above adverse effects.

Claims

1. A phosphoramidite cation, characterized in that, The phosphoramidite cation structure is shown as follows: Wherein B is a base selected from one of A, U, C, G, and T; R2 is an alkyl group, A2 is O or C, n is a natural number from 1 to 20, and X is selected from one of F, Cl, Br, and I; preferably, R2 is an alkyl group having 1 to 18 carbon atoms; more preferably, R2 is an alkyl group having 1 to 8 carbon atoms.

2. The preparation method of the phosphoramidite cation according to claim 1, characterized in that, The preparation method route is as follows:

3. The preparation method of the phosphoramidite cation according to claim 2, characterized in that, The preparation method comprises the following steps: Step 1: Add compound A and anhydrous DMF into a reaction flask, protect with nitrogen, stir, add sodium hydride portionwise at -5 °C, add benzyl bromide dropwise and stir at room temperature for 3 hours, then add ice water; wash the reaction solution with EtOAc, and dry the combined organic phases; after concentration, elute the residue with petroleum ether / EtOAc = 1 / 20 to obtain compound B; Step 2: Dropwise add acetic anhydride to the anhydrous pyridine solution of compound B, and react at room temperature for 3 hours; quench the reaction by adding ice water, and extract with dichloromethane; wash the combined organic phases with saturated aqueous sodium bicarbonate solution, dry, filter, concentrate, and purify by silica gel column chromatography using dichloromethane as the eluent to obtain the product as a transparent oily compound C; Steps 3 & 4: Add compound C to 80% acetic acid to form a solution, stir at 90 °C for 4 hours; after removing the solvent under reduced pressure, co-evaporate the residue with ethanol, toluene, and anhydrous pyridine, and redissolve in anhydrous pyridine; add acetic anhydride and stir the solution, let stand at room temperature for 72 hours; pour the solution into ice water, and extract the mixture with dichloromethane; wash the combined organic phases with saturated aqueous sodium bicarbonate solution, dry, and concentrate under reduced pressure; purify the residue by silica gel column chromatography using dichloromethane as the eluent to obtain compound E; Step 5: Dissolve compound E and thymine in anhydrous acetonitrile to form a solution, add N,O-bis(trimethylsilyl)acetamide with stirring; reflux and stir the reaction mixture, then cool; Dropwise add trimethylsilyl trifluoromethanesulfonate with stirring, and stir the solution at 65 °C, extract with saturated aqueous sodium bicarbonate solution and dichloromethane; wash the combined organic phases with saturated aqueous sodium bicarbonate solution and brine, dry; remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography to obtain compound F; preferably, the silica gel column chromatography uses dichloromethane / methanol (98:2, v / v) as the eluent; Step 6: Dissolve compound F in methanol to form a solution, add sodium methoxide with stirring; stir the reaction mixture at room temperature, then neutralize with dilute hydrochloric acid; partially evaporate the solvent, and extract; wash the combined organic phases with saturated aqueous sodium bicarbonate solution, and dry; remove the solvent under reduced pressure to obtain compound G; Step 7: Add compound G to anhydrous pyridine to form a solution and stir, add p-toluenesulfonyl chloride at 0 °C; stir the solution at room temperature for reaction and add p-toluenesulfonyl chloride; continue to stir, add ice water, and extract; wash the combined organic phases with saturated aqueous sodium bicarbonate solution and dry; remove the solvent under reduced pressure, purify the residue with silica gel to obtain an intermediate; dissolve this intermediate in anhydrous DMF; The solution was added dropwise to a mixed solution of 60% sodium hydride in mineral oil and anhydrous DMF; the mixture was stirred and then concentrated under reduced pressure; the residue was dissolved in dichloromethane, washed with saturated aqueous sodium bicarbonate and dried; the solvent was removed under reduced pressure, and the residue was purified by silica gel and separated by column chromatography to obtain compound H; Step 8: Compound H was dissolved in ethanol, and the mixture was stirred at room temperature. 20% palladium hydroxide on carbon was added; the mixture was purged with argon and then with hydrogen, and heated to 65 °C under a hydrogen atmosphere; after stirring for 4 hours, the mixture was separated by silica gel column chromatography to obtain compound J; Step 9: At 0 °C, compound J was dissolved in anhydrous pyridine to form a solution, and 4,4'-dimethoxytributyl chloride was added; the solution was stirred at room temperature, then quenched with ice water, and extracted with dichloromethane; the combined organic phases were washed with saturated aqueous sodium bicarbonate and brine and dried; the solvent residue was removed under reduced pressure and purified by silica gel column chromatography to obtain compound K; Step 10: Bis(N,N-diisopropylamino)chlorophosphine was dissolved in anhydrous dichloromethane to form a solution, and the above solution was added dropwise to a stirred anhydrous dichloromethane solution of compound K and N-ethyl-N,N-diisopropylamine while controlling the temperature at -20 °C; The solution was stirred at room temperature for 2 hours; the reaction solution was diluted with 5% aqueous NaHCO3, and the product was extracted with ethyl acetate; the extract was washed with brine, dried, and evaporated to an oil; the product was separated by column chromatography on silica gel, and the fractions were evaporated to obtain compound L; Step 11: 1H-tetrazole was added to a mixture of compound L, choline, and CH2Cl2; the resulting solution was stirred at room temperature; aqueous NaHCO3 was added, the emulsion was diluted with brine, and the product was extracted with ethyl acetate; the extract was washed with brine, dried, and evaporated to dryness; the residue was dissolved in toluene, applied to a silica gel column, the collected fractions were evaporated, co-evaporated with dry MeCN, and dried on an oil pump to obtain compound M, the target product.

4. The preparation method of the phosphoramidite cation according to claim 1, characterized in that, The preparation method route is as follows:

5. The preparation method of the phosphoramidite cation according to claim 4, characterized in that, The preparation method includes the following steps: Step 1: Under nitrogen protection, compound A1 was added to a mixed solvent of dichloromethane and pyridine and dissolved, cooled, MsCl was added dropwise, stirred, and detected by TLC. After the raw material reaction was completed, water was added, dichloromethane was added, the aqueous phase was washed, the organic phase was dried with sodium sulfate, filtered, and the organic phase was concentrated to obtain compound B1; Step 2: Compound B1 was suspended in acetic acid to obtain an off-white suspension; sulfuric acid was dissolved in acetic acid and added to the above suspension; acetic anhydride was added dropwise; stirred; after detecting that the raw material reaction was completed, dichloromethane and water were added to the reaction solution;The layers were separated, the organic phase was washed, and the organic phase was concentrated to obtain product compound C1; Step 3: Under a nitrogen atmosphere, compound C1 was dissolved in anhydrous acetonitrile, adenine was added, and the mixture was stirred; SnCl4 was added dropwise with the internal temperature not exceeding 45 °C; the temperature was maintained at 40 °C for 2 hours. After the raw materials reacted completely, the reaction solution was cooled, the pH was adjusted to 4.5 - 5.0, filtered through diatomaceous earth, the filter cake was washed with ethyl acetate, the organic phase was washed with 1 M KH2PO4, and the organic phase was concentrated to obtain compound D1; Step 4: Compound D1 was suspended in THF to obtain a white mixture, 1 M sodium hydroxide was added, and the mixture was stirred. After the raw materials reacted completely; the solvent was evaporated using a rotary evaporator, the mixture was added to deionized water, filtered, the filter cake was washed with deionized water, and the product was dried to obtain product compound E1; Step 5: Compound E1 was dissolved in acetonitrile, concentrated twice, then concentrated twice with toluene, and finally dissolved in DMF. Sodium benzoate was added, and the mixture was heated to 100 °C and reacted for 2 hours. After the raw materials reacted completely; the reaction solution was cooled, deionized water was added, filtered, the filter cake was washed with deionized water, and the filter cake compound F1 was directly used for the next step; Step 6: Compound F1 was dissolved in THF to form a solution and stirred, water and sodium hydroxide were added, and the mixture was stirred at room temperature. After the raw materials reacted completely, it was concentrated by rotary evaporation; the mixture was added to deionized water, cooled, filtered, the filter cake was washed with deionized water, and the white crystals were dried to obtain compound G1; Step 7: Compound G1 was dissolved in methanol, and the mixture was stirred at room temperature. 20% palladium on carbon hydroxide was added; the mixture was purged with argon and then with hydrogen. Under a hydrogen atmosphere, it was heated to 65 °C; after stirring for 4 hours, the mixture was separated by silica gel column chromatography to obtain compound J; Step 8: At 0 °C, compound J was added to anhydrous pyridine to form a solution, and 4,4'-dimethoxytributyl chloride was added to the above solution; the solution was stirred at room temperature for 2 hours, then quenched with ice water and extracted with dichloromethane; the combined organic phases were washed with saturated aqueous sodium bicarbonate and brine and dried; the solvent residue was removed under reduced pressure and purified by silica gel column chromatography to obtain compound K; Step 9: Bis(N,N-diisopropylamino)chlorophosphine was dissolved in anhydrous dichloromethane to form a solution. The above solution was added dropwise to a stirred anhydrous dichloromethane solution of compound K and N-ethyl-N,N-diisopropylamine while controlling the temperature at -20 °C; The solution was stirred at room temperature for 2 hours; the reaction solution was diluted with 5% aqueous NaHCO3, and the product was extracted with ethyl acetate; the extract was washed with brine, dried, and evaporated to an oil; the product was separated by column chromatography on silica gel, and the fractions were evaporated to obtain compound L; Step 10: 1H-tetrazole was added to a mixture of compound L, choline, and CH2Cl2; the resulting solution was stirred at room temperature for 2 hours; aqueous NaHCO3 was added, the emulsion was diluted with brine, and the product was extracted with ethyl acetate; The extract was washed with brine, dried, and evaporated to dryness; the residue was dissolved in toluene, applied to a silica gel column for separation; the collected fractions were evaporated, co-evaporated with dry MeCN, and dried on an oil pump to obtain compound M, which is the target product.

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