CN115215915A - Phosphoramidite monomer and method for purifying oligonucleotide - Google Patents

Abstract

The present invention provides a phosphoramidite monomer having a structure represented by formula I below, or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, or solvate thereof, and a method of purifying an oligonucleotide using the same. In addition, the method of the invention uses a phosphoramidite monomer containing a methacrylamide group capable of carrying out polymerization reaction as a raw material for synthesizing the oligonucleotide, and has the advantages of high synthesis and purification recovery rate, simple equipment and operation, no cross contamination and the like compared with the conventional oligonucleotide purification method.

Description

Phosphoramidite monomer and method for purifying oligonucleotide

Cross Reference to Related Applications

The present application claims priority from chinese patent application No. 202110417554.1 entitled "a phosphoramidite monomer and method of purifying oligonucleotides" filed on 2021, 4, 19, which is incorporated herein by reference in its entirety.

Technical Field

The present invention is in the field of oligonucleotide synthesis, and more particularly, the present invention relates generally to phosphoramidite monomers and methods for purifying oligonucleotides.

Background

Modern oligonucleotide synthesis mainly adopts an automated phosphoramidite solid phase synthesis technology, and the synthesis yield limit of each step can reach 99.6%. Nevertheless, as the length of the oligonucleotide increases, the impurity content increases, for example, in the case of an oligonucleotide chain of 20-mer base length, the yield is usually about 80%, the impurity content is about 20%, and for an oligonucleotide chain of 40-mer base length, the yield and the impurity content are about 65% and 35%, respectively, as calculated by 99% of the synthesis yield. The generated impurities mainly come from small molecules generated in the synthesis and deprotection processes, failure sequences caused by incomplete coupling reaction in each step, missing sequences caused by incomplete capping, insertion sequences caused by multiple coupling and the like, wherein the failure sequences are the most important impurities.

In order to remove impurities and purify the resulting oligonucleotides, various means are currently available for this purpose, such as size exclusion chromatography, polyacrylamide gel electrophoresis, high performance liquid chromatography (including reverse phase HPLC and ion exchange HPLC), column chromatography for purification, and the like, as well as new techniques such as affinity-based purification methods (including biotin-avidin affinity purification and fluorine atom affinity column purification). However, HPLC requires special equipment and generates large amounts of organic waste (e.g., 5mg of oligonucleotide, 500L of solvent is required for purification), and large-scale purification of oligonucleotides is costly; meanwhile, cross contamination is easily caused, if the cross contamination needs to be avoided, each different sequence is provided with a separate HPLC preparative column, so that the purification cost is greatly increased; the polyacrylamide gel electrophoresis operation is complex, and high-throughput purification is difficult to realize; size exclusion chromatography and purification column chromatography can achieve high-throughput purification on a small scale, but also large-scale oligonucleotide purification is difficult to achieve; based on capture and release strategies, cross-contamination could theoretically be solved and large-scale oligonucleotide purification could also be achieved, but the overall recovery of synthetic purification reported to date is only 17.5%.

Therefore, the development of new monomers suitable for this strategy would greatly improve the efficiency of oligonucleotide purification and reduce the cost of purification.

Disclosure of Invention

The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to provide a method for greatly improving the purification efficiency and reducing the purification cost of oligonucleotides, which is capable of improving the purification efficiency of oligonucleotides and reducing the purification cost by using a phosphoramidite monomer containing a methacrylamide group capable of undergoing a polymerization reaction as a raw material for synthesizing oligonucleotides, and which is capable of overcoming the disadvantages of high cost, difficulty in achieving high throughput, low purification recovery rate, and the like in the purification of oligonucleotides and the removal of failure sequences, which are the most important impurities in the oligonucleotides, and which has advantages of high synthesis purification recovery rate, simple equipment and operation, no cross-contamination, and the like.

To achieve the above objects, in one aspect, the present invention provides a phosphoramidite monomer having a structure represented by the following formula I, or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, or solvate thereof:

[ formula I ]

Wherein X is a rigid linker having the structure:

r1 and R2 are both-O (CH) ₂ CH ₂ O) _n CH ₃ And are substituted at two arbitrary different positions on the middle benzene ring, respectively; r3 to R10 are each independently selected from hydrogen, hydroxy, cyano, halogen, or substituted or unsubstituted amino, C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy or C ₃ -C ₆ A cycloalkyl group; m is an integer from 1 to 10; n is an integer of 1 to 10; and Y is a phosphoramidite modified nucleoside, and the amino group on the nucleoside is optionally acylated, wherein the nucleoside can be a ribonucleoside or a deoxyribonucleoside. In a preferred embodiment of the invention, the nucleoside is a ribonucleoside comprising cytidine (C), uridine (U), adenosine (a), thymidine (T) or guanosine (G). In a preferred embodiment of the present invention, the nucleoside is a deoxyribonucleoside including deoxycytidine (C), deoxyuridine (U), deoxyadenosine (a), deoxythymidine (T) or deoxyguanosine (G).

As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Wherein, said pharmaceutically acceptable acid addition salt refers to a salt formed with inorganic or organic acids capable of retaining the biological effectiveness of the free base without other side effects, for example, inorganic acid salts can include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.; the organic acid salts may include, but are not limited to, formates, acetates, trifluoroacetates, propionates, caproates, salicylates, and the like. Pharmaceutically acceptable base addition salts refer to salts with inorganic or organic bases that maintain the bioavailability of the free acid without other side effects, for example, inorganic base salts may include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; organic base salts may include, but are not limited to, salts of primary amines, secondary amines, and tertiary amines. These salts can be prepared by methods known in the art.

As used herein, the term "acylation" (otherwise known as "acylation") refers to a reaction of introducing an aliphatic acyl RCO-or an aromatic acyl ArCO-on an atom of nitrogen, oxygen, carbon or sulfur, etc. in an organic molecule, wherein the acylation suitable for use in the present invention may include, but is not limited to, formylation, acetylation, propionylation, isopropylylation, benzoylation, etc.; and the commonly used acylating agent may include, for example, acid chlorides, acid anhydrides and carboxylic acids, more specifically, acetyl chloride or acetic anhydride, etc.

With respect to the specific structure of the rigid linker of the invention, in a preferred embodiment of the invention, R1 and R2 may be ortho, meta or para substituted with respect to each other; r3 to R10 may each independently be selected from hydrogen, hydroxy, cyano, halogen, or substituted or unsubstituted amino, C1-C6 alkyl; m may be an integer from 3 to 8 (e.g., 3, 4, 5, 6, 7, or 8); n may be an integer from 2 to 5 (e.g., 2, 3, 4, or 5); and Y may be a phosphoramidite modified deoxyribonucleoside which may include deoxycytidine (C), deoxyuridine (U), deoxyadenosine (a), deoxythymidine (T) or deoxyguanosine (G), and the amino group on the nucleoside may optionally be acylated (and in preferred cases, the amino group on the nucleoside is acylated).

Further, in a preferred embodiment of the present invention, R1 and R2 may be para-substituted to each other. In a preferred embodiment of the present invention, R3 to R10 may each independently be selected from hydrogen, hydroxy, cyano, halogen, or substituted or unsubstituted amino, methyl, ethyl, propyl, isopropyl.

In a preferred embodiment of the invention, the substitution may be by halogen (e.g. fluorine, chlorine or bromine), hydroxy, amino, cyano, C ₁ -C ₃ Alkyl (e.g. methyl, ethyl, propyl or isopropyl) or C ₁ -C ₃ Haloalkyl (such as halomethyl, haloethyl, or halopropyl) groups, but is not limited thereto. In addition, the substitution may include one or more substitutions, the number of substitutionsThe upper limit of (d) may depend on the amount of hydrogen, i.e. total substitution. Further, in a preferred embodiment of the present invention, R1 and R2 are para-substituted; r3 to R10 are all selected from hydrogen; m is 5; and n is 2.

In a preferred embodiment of the present invention, the modifying group in the phosphoramidite modification has the following structure:

in a preferred embodiment of the present invention, the phosphoramidite monomer may be further represented as a specific compound, for example, which may have one of the structures shown below:

in another aspect, the present invention also provides a method for purifying an oligonucleotide, comprising the steps of: (1) Coupling an oligonucleotide with the phosphoramidite monomer to attach the phosphoramidite monomer to the end of the oligonucleotide to provide a full-length oligonucleotide; (2) polymerizing the full-length oligonucleotide; (3) Removing the failure sequence from the polymerized full-length oligonucleotide; and (4) recovering the full-length oligonucleotide.

In the method for purifying an oligonucleotide according to the present invention, there is no particular requirement for the length of the oligonucleotide, and it can be selected according to actual needs. In one embodiment of the present invention, the oligonucleotide may be 10 to 100nt in length; preferably 20-50nt (e.g., 25nt, 30nt, 35nt, 40nt, etc.).

For step (1), i.e. the coupling step, the time of the coupling reaction may be in the range of 0.2h to 1h (e.g. 0.3h, 0.5h or 0.8h etc.), preferably 0.3h. In another embodiment, the coupling reaction may be carried out in multiple steps. For example, preferably, the coupling may be carried out in 6 portions, each for 0.05h (total 0.3 h). The phosphoramidite monomer can be attached to the 5' end or the 3' end of the oligonucleotide via a coupling reaction, but preferably, the phosphoramidite monomer can be attached to the 5' end of the oligonucleotide.

For step (2), the polymerization may be carried out in the presence of a crosslinking agent, for example, in one embodiment, the polymerization may be carried out by using reagents including N, N-dimethylacrylamide and N, N' -methylenebisacrylamide. Preferably, the concentration of the N, N-dimethylacrylamide may be 2 to 5M (e.g., 2.5M, 3M, 4M, or the like); more preferably 3.7M; and/or the concentration of said N, N' -methylenebisacrylamide may be from 0.1 to 2M (e.g., 0.5M, 1M, or 1.5M, etc.); more preferably 0.18M. In addition, the polymerization may also be carried out in the presence of a promoter, for example, in one embodiment, the reagents used in the polymerization further include ammonium persulfate and tetramethylethylenediamine. Preferably, the mass volume concentration of the ammonium persulfate can be 1-10% (such as 3%, 6% or 8% and the like); more preferably 5%; and/or the concentration of the tetramethylethylenediamine may be from 0.4M to 1.0M (e.g., 0.5M, 0.7M, or 0.9M, etc.); preferably 0.66M.

For step (3), the removal failure sequence may be performed by sequentially performing the steps of chopping the gel of the polymeric oligonucleotide in step (2), soaking in a buffer, and eluting. In a preferred embodiment, the buffer may be pure water or 3% to 10% (e.g., 4%, 6%, 8%, etc.) aqueous triethylamine; more preferably 5% aqueous triethylamine solution.

For the step (4), the recovery may be performed by sequentially performing the steps of acid solution soaking, aqueous solution elution, alkali solution neutralization, and concentration. In a preferred embodiment, the acid solution may be a 70% to 95% (e.g., 75%, 80%, 85%, or 95%, etc.) acetic acid solution; and/or the base solution may be concentrated aqueous ammonia (e.g., an aqueous ammonia solution having a concentration greater than 15%).

It is noted that the method of the present invention uses, as a raw material for synthesizing an oligonucleotide, a phosphoramidite monomer containing a methacrylamide group capable of undergoing polymerization, which methacrylamide structure can be stably linked to an oligonucleotide chain in all steps of solid phase synthesis, carrier cleavage and base deprotection, and covalently links the oligonucleotide chain to a solid high molecular polymer (such as polyacrylamide gel) through polymerization.

In addition, according to the present invention, in the purification of an oligonucleotide using the phosphoramidite monomer provided by the present invention, the phosphoramidite monomer is generally introduced in the last cycle of the oligonucleotide chain synthesis (preferably, at the 5' end of the oligonucleotide chain). Since the hydroxyl group at the 5' -position of the oligonucleotide chain with a failure sequence is blocked by a capping step in solid phase synthesis and cannot react with the monomer in the invention to be linked to the high molecular polymer, the failure sequence can be purified and removed by a simple operation of washing the high molecular polymer, and the full-length oligonucleotide chain can be released by a simple acid treatment, thereby achieving the purpose of purification.

In another aspect, the present invention also provides a kit comprising the above phosphoramidite monomer.

In another aspect, the present invention also provides the use of the above phosphoramidite monomer and the above kit for oligonucleotide purification.

Based on the research of the inventor, the phosphoramidite monomer provided by the invention has at least the following advantages: 1) The synthesis and purification recovery rate is high; 2) The required equipment is simple, and only a glass instrument is needed; 3) The raw materials are cheap and easy to obtain, and the polymerization chemicals are chemicals used by the conventional polyacrylamide gel; 4) The operation is simple, and only simple liquid transfer, filtration and washing operations are needed; 5) The purification time is short, is not longer than 24 hours, and the cycle is short; 6) The purification scale is not limited, and the method is suitable for large-scale oligonucleotide synthesis and purification; 7) No cross contamination, and the brand new copolymer is used for each different sequence and different batches of oligonucleotides, so that no cross contamination exists; 8) The method has the advantages of low energy consumption, environmental protection, small usage amount of solvent and buffer solution required by the purification technology, less generated waste liquid, short concentration time and low energy consumption.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows a synthetic scheme for preparing phosphoramidite monomers according to the method of example 1 of the present invention (wherein, a) NaOH, THF,2h; b) P-diphenol, K ₂ CO ₃ Acetonitrile for 72h; c) I.C. A ₂ ， KIO ₃ ，AcOH，48h；d)Pd(PPh ₃ ) ₄ CuI, 4-ethynylaniline, THF, et ₃ N,24h; e) 6- {4- [ hydroxy- (4-methoxyphenyl) -phenyl-methyl]Phenoxy } hexanoic acid, DIEA, HATU, DCM,12h; f) DIEA, methacryloyl chloride, DCM for 12h; g) Acetyl chloride, dC-Ac, pyridine, 16h; h) 2-cyanoethyl N, N, N ', N' -tetraisopropyl phosphoramidite, BTT, DCM,1h; and i) acetyl chloride, dT, pyridine, 16 h);

FIG. 2 shows an HPLC plot of crude oligonucleotide;

FIG. 3 shows a diagram of the structure of an oligonucleotide modified with monomeric T-8;

FIG. 4 shows an HPLC plot (dC) of an oligonucleotide after 200nmol synthesis scale purification;

FIG. 5 shows a pure mass spectrum (dC) of an oligonucleotide after 200nmol synthesis scale purification;

FIG. 6 shows an HPLC plot (dC) of an oligonucleotide after 1000nmol synthetic scale purification;

FIG. 7 shows the pure quality spectrum (dC) of the oligonucleotide after 1000nmol synthesis scale purification;

FIG. 8 shows an HPLC plot (dT) of the oligonucleotide after 200nmol synthesis scale purification;

FIG. 9 shows a pure mass spectrum (dT) of the oligonucleotide after 200nmol synthesis scale purification;

FIG. 10 shows an HPLC plot (dT) of the oligonucleotide after 1000nmol synthesis scale purification;

FIG. 11 shows a pure mass spectrum (dT) of the oligonucleotide after 1000nmol synthesis scale purification;

FIG. 12 shows a gel of the product of capture and release strategy purification and HPLC purification primers used for PCR (where M: marker; 1: T-8 purified forward primer; 2

Figure 13 shows a plot of capture and release strategy purification versus HPLC purification of primers for qPCR amplification; and

FIG. 14 shows a gel plot of the products of capture and release strategy purification and HPLC purification primers used for qPCR (where M: marker; 1.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to these ranges or values. For numerical ranges, combinations of values between the endpoints of each range, between the endpoints of each range and individual values, and between individual values of points can result in one or more new numerical ranges, which should be considered as specifically disclosed herein.

In the following examples, the sources of some of the chemicals used are shown in table 1 below:

TABLE 1

Preparation of the Compound of example 1

Synthesis of Compound T-1

Adding diethylene glycolMonomethyl ether (58.65mL, 500mmol) was dissolved in 160mL of tetrahydrofuran and ice-cooled to 0 ℃. Sodium hydroxide (40g, 1000mmol) was dissolved in 110mL of water and added slowly. Subsequently, p-toluenesulfonyl chloride (142.74g, 750 mmol) was weighed and dissolved in 110mL of tetrahydrofuran solution, and was slowly dropped in the mixture using a constant pressure dropping funnel. After completion of the dropwise addition, the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC, and after completion of the reaction, 400mL of water was added and the organic layer was separated. Then washed twice with 1M sodium hydroxide solution. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated. 115.08g of T-1 product was obtained with a yield of about 84%. MS (ESI) m/z theoretical value C ₁₂ H ₁₈ O ₅ S[M+Na] ⁺ Is 278.33; measurement, 298.6. ¹ H NMR(400MHz,CDCl ₃ )δ7.79 (d,J＝8.0Hz,2H),7.33(d,J＝8.0Hz,2H),4.16(t,J＝4.9Hz,2H),3.68(t,J＝ 4.9Hz,2H),3.60-3.53(m,2H),3.50-3.44(m,2H),3.34(s,3H),2.43(s,3H)。

Synthesis of Compound T-2

Hydroquinone (14.64g, 133.1 mmol) and potassium carbonate (73.47g, 532.4 mmol) were charged in a 2L round bottom flask, and 1.1L of acetonitrile was added. The mixture was heated to reflux for 30min and then cooled to room temperature. Compound T-1 (72.95g, 266.2mmol) was dissolved in 220mL of acetonitrile and slowly added dropwise to the mixture. Heated under reflux and stirred for 72h. After the reaction is finished, cooling to room temperature, carrying out suction filtration, collecting supernatant and concentrating. Column chromatography purification (petroleum ether/ethyl acetate 90. MS (ESI) m/z theoretical value C ₁₆ H ₂₆ O ₆ [M+Na] ⁺ 337.37; measurement, 337.2. ¹ H NMR(400MHz,CDCl ₃ )δ6.82(s,1H),4.10-4.05(m,1H), 3.83-3.80(m,1H),3.72-3.69(m,1H),3.58-3.55(m,1H),3.38(s,2H)。

Synthesis of Compound T-3

Compound T-2 (25g, 79.6mmol), iodine (22.3g, 87.6mmol) and potassium iodate (6.8g, 31.8mmol) were weighed into a 1000mL round-bottomed flask. 250mL of acetic acid, 25mL of water, and 3.3mL of concentrated sulfuric acid were added. Heated to reflux for 48h. After the experiment, the mixture is cooled to room temperature, and 10 percent sodium thiosulfate and dichloromethane are added to wash the mixture to remove muchThe remaining iodine was then washed twice with saturated sodium bicarbonate solution and once with water. The organic phase was collected and dried by adding anhydrous sodium sulfate. The organic phase is concentrated and the filter residue is recrystallized from ethanol. 34.00g of T-3 product was obtained in 42.7% yield. MS (ESI) m/z theoretical value C ₁₆ H ₂₄ I ₂ O ₆ [M+Na] ⁺ Is 589.17; measurement, 589.87. ¹ H NMR(400MHz,CDCl ₃ )δ 7.28(s,2H),4.13(d,J＝5.0Hz,4H),3.90(t,J＝4.9Hz,4H),3.82-3.77(m,4H), 3.63-3.57(m,4H),3.42(s,6H)。

Synthesis of Compound T-4

Compound T-3 (5.66g, 10mmol), tetrakis (triphenylphosphine) palladium (1.2g, 1.0mmol), cuprous iodide (200mg, 1.0mmol) and 4-ethynylaniline (2.34g, 20mmol) were weighed out and placed in a 100mL two-neck flask, and 20mL of tetrahydrofuran and 30mL of triethylamine were added under anhydrous and oxygen-free conditions to dissolve the compound. And reacting for 24 hours at 45 ℃ under the protection of nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solvent was removed by rotary evaporation. Dichloromethane and saturated sodium bicarbonate solution were added for washing. The organic phase was dried using anhydrous sodium sulfate. After concentration, purification by column chromatography was performed to obtain 4.73g of T-4 product in 86.9% yield. MS (ESI) m/z theoretical value C ₃₂ H ₃₆ N ₂ O ₆ [M+Na] ⁺ 567.64; measured value, 568.8. ¹ H NMR (400MHz,CDCl ₃ )δ7.32(d,J＝8.5Hz,4H),6.99(s,2H),6.62(d,J＝8.5Hz, 4H),4.19(t,J＝4.9Hz,4H),3.91(t,J＝4.9Hz,4H),3.86(d,J＝9.9Hz,4H), 3.82-3.79(m,4H),3.57-3.51(m,4H),3.36(s,6H)。

Synthesis of Compound T-5

The compound 6- {4- [ hydroxy- (4-methoxyphenyl) -phenyl-methyl]Phenoxy } hexanoic acid (2.1 g,5 mmol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (2.28 g,6 mmol), N, N-diisopropylethylamine (0.78g, 6 mmol) were dissolved in a certain amount of dichloromethane and stirred at room temperature for 30min. The mixture was added dropwise to a solution of 2.838g (5.0 mmol) of Compound T-4 in methylene chloride. Stir warm overnight. After completion of the reaction, the reaction mixture was washed with a saturated sodium hydrogencarbonate solution, and the organic phase was dried over anhydrous sodium sulfate. Concentrating, purifying by column chromatography to obtain T-5 product3.42g, yield 72.3%. MS (ESI) m/z theoretical value C ₅₈ H ₆₂ N ₂ O ₁₀ [M-H] ^- 947.12; measurement 947.12. ¹ H NMR(400MHz,CDCl ₃ )δ8.03(s,1H),7.55(d,J＝9.1Hz,4H),7.35-7.33 (m,2H),7.31-7.28(m,5H),7.17(d,J＝8.2Hz,4H),7.02(d,J＝2.2Hz,2H), 6.82(d,J＝9.1Hz,4H),6.64(d,J＝8.2Hz,2H),5.32(s,2H),4.22(t,J＝4.9Hz, 4H),3.97-3.92(m,5H),3.82(d,J＝3.9Hz,4H),3.58-3.53(m,3H),3.38(d,J ＝2.9Hz,4H),2.97(s,3H),2.90(s,4H),2.40(dd,J＝8.7,6.2Hz,2H),2.05(d,J ＝3.5Hz,1H),1.84-1.79(m,4H),1.58-1.53(m,2H)。

Synthesis of Compound T-6

Compound T-5 (3.78g, 4mmol), anhydrous dichloromethane (20 mL) and N, N-diisopropylethylamine (1.56g, 12mmol) were added to a dried round-bottomed flask and cooled to 0 ℃. To 2mL of methylene chloride was added a solution of methacryloyl chloride (4 mmol) and the mixture was stirred slowly through the addition funnel. After addition, the reaction flask was disconnected from the nitrogen and connected to air via a desiccant tube. The mixture was stirred at room temperature overnight. The reaction was transferred to a separatory funnel and washed with 10% sodium bicarbonate solution and dichloromethane solution. The organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give 3.40g of T-6 product in 83.7% yield. MS (ESI) m/z theoretical value C ₆₂ H ₆₆ N ₂ O ₁₁ [M-H] ^- 1014.19; measurement, 1014.87. ¹ H NMR(400MHz,CDCl ₃ )δ7.61-7.44(m,9H),7.30(m,4H), 7.16(ddd,J＝8.7,6.0,2.7Hz,4H),7.03(s,2H),6.82(dd,J＝8.9,7.4Hz,4H), 5.81(s,1H),5.50(s,1H),4.21(t,J＝4.8Hz,4H),3.94(m,7H),3.85-3.74(m, 6H),3.54(dt,J＝6.3,2.9Hz,4H),3.36(d,J＝2.7Hz,6H),2.73(s,1H)2.40(t,J ＝7.4Hz,2H),2.08(s,3H),1.81(m,4H),1.57(m,2H)。

Synthesis of Compound T-7

T-6 (3.045g, 3mmol) and N-4-acetyl-2' -deoxycytidine (0.81g, 3mmol) were placed in two round-bottom flasks and dried overnight under vacuum, acetyl chloride (4 mL) was added to the flask containing compound T-6, and the mixture was stirred under nitrogen at room temperature for two hours. Vacuum pumping acetyl chloride, washing with dry n-hexane, and vacuum dryingDrying for 1h. N-4-acetyl-2' -deoxycytidine was dissolved in 5mL of pyridine and added thereto, followed by stirring at room temperature for 16 hours. After the reaction was completed, pyridine was removed by rotary evaporation. The concentrate was washed with dichloromethane and saturated sodium bicarbonate solution, the organic phase was dried over anhydrous sodium sulfate, and after concentration, purification by column chromatography gave 1.50g of T-7 product in 39.4% yield. MS (ESI) m/z theoretical value C ₇₃ H ₇₉ N ₅ O ₁₅ [M+H] ⁺ 1267.43; measured value 1267.80. ¹ H NMR(400MHz,CDCl ₃ )δ8.67(s,1H),8.16 (dd,J＝7.5,2.6Hz,1H),7.97(s,1H),7.64(t,J＝8.0Hz,3H),7.53-7.32(m, 6H),7.25(m,3H),7.24-7.08(m,4H),7.04-6.92(m,2H),6.80(ddd,J＝13.5, 8.7,1.9Hz,4H),6.21(t,J＝5.8Hz,1H),5.85(s,1H),5.47(s,1H),4.47(q,J＝ 4.8Hz,1H),4.16(m,5H),3.97-3.84(m,6H),3.77(m,7H),3.51(m,4H),3.48- 3.39(m,2H),3.33(m,6H),2.66(dt,J＝12.5,5.7Hz,1H),2.45(t,J＝7.5Hz, 2H),2.24(s,3H),2.18(m,1H),2.05(s,3H),1.77(h,J＝6.3,5.6Hz,4H),1.49 (m,2H)。

Synthesis of Compound T-8

Compound T-7 (1.26g, 1.0 mmol) was dissolved in 6mL of anhydrous dichloromethane, and 5-benzylthiotetrazole (307.2mg, 1.6 mmol) and 2-cyanoethyl N, N, N ', N' -tetraisopropylphosphorodiamidite (600. Mu.L, 1.5 mmol) were added. After the reaction was stirred at room temperature for 1h, it was washed three times with saturated sodium bicarbonate solution, the organic phase was removed by rotary evaporation and precipitated with n-hexane to give 1.34g of T-8 product in 91.2% yield. MS (ESI) m/z theoretical value C ₈₂ H ₉₆ N ₇ O ₁₆ P[M+H] ⁺ 1489.65; measured value, 1489.01. ¹ H NMR(400MHz,CDCl ₃ )δ8.32-8.14(m,1H),7.66-7.42(m,9H),7.39(t,J ＝6.9Hz,2H),7.32-7.23(m,6H),7.20-7.05(m,2H),7.02(d,J＝2.3Hz,2H), 6.83(ddd,J＝10.1,7.5,5.1Hz,4H),6.24(q,J＝6.2Hz,1H),5.81(s,1H),5.49 (s,1H),4.67-4.50(m,1H),4.20(d,J＝4.8Hz,4H),4.04-3.89(m,6H),3.79-85 (m,7H),3.66-3.48(m,8H),3.36(d,J＝2.5Hz,6H),2.72(ddd,J＝26.0,13.0, 6.6Hz,1H),2.54(q,J＝7.2Hz,4H),2.47-2.33(m,2H),2.20(s,3H),2.07(s, 3H),1.80(p,J＝7.3Hz,4H),1.65-1.56(m,2H),1.19-1.12(m,12H)。 ³¹ P NMR (400MHz,CDCl3)δ149.41,149.38,148.76,148.73。

Synthesis of Compound T-9

T-6 (3.045g, 3 mmol) and 2' -deoxythymidine (0.73g, 3 mmol) were placed in two round-bottomed flasks, dried overnight under vacuum, acetyl chloride (4 mL) was added to the flask containing compound T-6, and the mixture was stirred under nitrogen at room temperature for two hours. Acetyl chloride was vacuum dried, washed with dry n-hexane and vacuum dried for 1h. 2' -deoxythymidine was dissolved in 5mL of pyridine and added thereto, and stirred at room temperature for 16 hours. After the reaction was completed, pyridine was removed by rotary evaporation. The concentrate was washed with dichloromethane and saturated sodium bicarbonate solution, and the organic phase was dried over anhydrous sodium sulfate, concentrated and purified by column chromatography to give 2.58g of T-9 product in 69.0% yield. MS (ESI) m/z theoretical value C ₇₂ H ₇₈ N ₄ NaO ₁₅ [M+Na] ⁺ 1261.54; measured value 1261.60. ¹ H NMR(400MHz,CDCl ₃ )δ7.80(s,1H),7.63(d,J＝3.7Hz,2H),7.61(d,J＝4.1 Hz,2H),7.50(d,J＝8.2Hz,2H),7.45(d,J＝8.3Hz,2H),7.39(t,J＝4.8Hz, 3H),7.30-7.24(m,9H),7.02(s,2H),6.84-6.79(m,4H),6.41(t,J＝6.8Hz, 1H),5.84(s,1H),5.50(s,1H),4.59(d,J＝9.0Hz,1H),4.20(d,J＝4.8Hz,4H), 4.08(d,J＝3.1Hz,1H),3.97-3.90(m,7H),3.80(t,J＝3.3Hz,7H),3.57-3.53 (m,4H),3.44(d,J＝6.8Hz,1H),3.37(d,J＝3.1Hz,6H),2.44(q,J＝7.2,6.5Hz, 4H),2.08(s,3H),1.79(s,4H),1.56-1.51(m,2H),1.41(s,4H)。

Synthesis of Compound T-10

T-9 (1.24g, 1.0 mmol) was dissolved in 6mL of anhydrous dichloromethane, and 5-benzylthiotetrazole (307.2mg, 1.6 mmol) and 2-cyanoethyl N, N, N ', N' -tetraisopropylphosphoramidite (600. Mu.L, 1.5 mmol) were added. After the reaction was stirred at room temperature for 1h, it was washed three times with saturated sodium bicarbonate solution, the organic phase was removed by rotary evaporation and precipitated with n-hexane to give 1.15g of T-10 product in 79.8% yield. MS (ESI) m/z theoretical value C ₈₁ H ₉₅ N ₆ NaO ₁₆ P[M+Na] ⁺ Is 1461.64; measurement, 1462.13. ¹ H NMR(400MHz,CDCl ₃ )δ7.78(d,J＝5.4Hz,1H),7.68(d,J＝3.6 Hz,2H),7.61-7.54(m,5H),7.48(t,J＝7.7Hz,4H),7.41(d,J＝7.7Hz,3H), 7.30-7.26(m,6H),7.04(d,J＝1.9Hz,2H),6.86-6.82(m,4H),6.45-6.39(m, 1H),5.83(s,1H),5.51(s,1H),4.69(dq,J＝10.6,7.2,5.2Hz,1H),4.22(t,J＝ 4.9Hz,5H),3.99(d,J＝6.7Hz,2H),3.95-3.92(m,4H),3.81(d,J＝2.2Hz, 7H),3.60-3.55(m,7H),3.38(d,J＝2.0Hz,8H),2.78(dt,J＝6.3,3.2Hz,1H), 2.64(t,J＝6.2Hz,1H),2.44-2.38(m,4H),2.07(d,J＝11.9Hz,4H),1.81(t,J＝ 7.3Hz,5H),1.40(d,J＝3.6Hz,4H),1.28(s,12H)； ³¹ P NMR(400MHz,CDCl ₃ ) δ149.37,149.03,148.97,148.45。

Specifically, the overall synthetic route of example 1 is shown in fig. 1, wherein the conditions of each step are as follows: a) NaOH, THF,2h; b) P-diphenol, K ₂ CO ₃ Acetonitrile for 72h; c) I is ₂ ，KIO ₃ ， AcOH，48h；d)Pd(PPh ₃ ) ₄ CuI, 4-ethynylaniline, THF, et ₃ N,24h; e) 6- {4- [ hydroxy- (4-methoxyphenyl) -phenyl-methyl]Phenoxy } hexanoic acid, DIEA, HATU, DCM,12h; f) DIEA, methacryloyl chloride, DCM for 12h; g) Acetyl chloride, dC-Ac, pyridine, 16h; h) 2-cyanoethyl N, N, N ', N' -tetraisopropyl phosphoramidite, BTT, DCM,1h; and i) acetyl chloride, dT, pyridine, 16h.

Example 2 detection of purification recovery

A stretch of 25 base oligonucleotide (5 '-HO-CACACACCACTTTCCCTACACGACGCCTC-OH-3', SEQ ID NO: 1) was used to verify the application of the capture and release strategy of the invention in oligonucleotide purification. The position information of the oligonucleotide sequence (cacactctttccctacacacgucctc) was edited and then uploaded to an automated dr. Dissolving the monomers A, C, G and T phosphoramidite monomers in dichloromethane, wherein the concentration is 0.06M; after being respectively arranged in the synthesis instrument independent corresponding synthesis channels, the synthesis scale is 200nmol or 1000nmol, the mode is DMTr on, and the solid phase synthesis and the cleavage deprotection method are the same as the conventional oligonucleotide solid phase synthesis. The crude product after cutting and removing the base protecting group is analyzed by mass spectrometry and HPLC, and the HPLC adopts a Waters Xbridge Oligonucleotide BEH C18 chromatographic column with the model number of 186003953. The gradient elution condition of crude product analysis HPLC is 0-6min, and the acetonitrile concentration is 5% -50%;6.01-8min, the concentration of acetonitrile is 88%;8.01-10min, and the concentration of acetonitrile is 5%. As shown in FIG. 2, the retention time of the oligonucleotide was 4.842min, the purity of the unpurified oligonucleotide was 83.86%, and the condensation efficiency of each step was shown to be 99.27% on average.

As an experimental group, the monomer T-8 was used in place of the ordinary C monomer, the compound T-8 was dissolved in acetonitrile to give a concentration of 0.10M, and the T-8 monomer was ligated to the 5' -end of an oligonucleotide of 24 bases in length (5 ' -HO-ACACTTTCCCTACACGACCTC-OH-3 ', SEQ ID NO:2, the target structure is shown in FIG. 3). The coupling time of the compound T-8 is 180s multiplied by 6 times, and the total coupling time is 18min. After the synthesis is finished, 1.5mL of ammonia water is added, ammonolysis is carried out for 15h at 55 ℃, and then the crude product is obtained by concentration and pumping. 200nmol of the crude oligonucleotide was dissolved in 50. Mu.L of water and shaken to dissolve it sufficiently. Polymerization reagents (24. Mu.L, 3.7M N, N-dimethylacrylamide and 0.18M N, N' -methylenebisacrylamide), 5. Mu.L of 5% ammonium persulfate, and 5. Mu.L of 0.66M tetramethylethylenediamine were added. Shaking to mix them evenly. Standing at normal temperature for 15min to form gel, and finishing the polymerization reaction after 1h. The gel-like polymer was cut into pieces, 250. Mu.L of 5% aqueous triethylamine solution was added, and after waiting for 3min, the solution was removed, and this step was repeated 5 times. Finally, 250. Mu.L of ultrapure water was added to wash off the residual triethylamine solution. Then 80% acetic acid solution (about 100. Mu.L) was added to the gel in an amount that covered the gel to a minimum, and after 5min, 80% acetic acid solution (AcOH) was collected and repeated 3 times. Then 100. Mu.L of water was added to the gel, and after 3min the aqueous solution was collected and repeated 5 times. The collected acetic acid solution and aqueous solution were combined and concentrated in vacuo. Add 100. Mu.L of concentrated ammonia to the concentrated oligonucleotide, shake briefly, and then place at 85 ℃ for 15min. After cooling to room temperature, 900. Mu.L of n-butanol was added, and after shaking for 1min, the mixture was centrifuged in a high-speed centrifuge at 14000rpm for 15min. The supernatant was carefully removed by pipette and concentrated to remove excess n-butanol. The white solid left behind was the purified oligonucleotide. The product was analyzed by HPLC using Shim-pack GIST Shijin silica gel column 227-30011-03. The gradient elution condition of pure product analytical HPLC is 0-6min, and the concentration of acetonitrile is 5% -50%;6.01-8min, the concentration of acetonitrile is 88%;8.01-10min, and the concentration of acetonitrile is 5%. As shown in fig. 4, the retention time of the product obtained after purification is 2.477min; the molecular weight was confirmed by ESI mass spectrometry (FIG. 5), MS (ESI) m/z theoretical 7458.87, and the measurement was 7458. The recovery was 53.37nmol with a recovery of 26.7%, 53% higher than the reported reagent recovery based on capture and release strategies, and the oligonucleotide purity was equivalent to 99.40%.

For purification on a 1000nmol synthesis scale, 1000nmol of the crude oligonucleotide was dissolved in 100. Mu.L of water, shaken to dissolve it thoroughly, and polymerization reagents (60. Mu.L, 3.7M N, N-dimethylacrylamide and 0.18M N, N' -methylenebisacrylamide), 5. Mu.L of 5% ammonium persulfate and 5. Mu.L of 0.66M tetramethylethylenediamine were added and mixed well by shaking. Standing at normal temperature for 15min to obtain gel state, and finishing polymerization reaction after 1h. The gel-like polymer was cut into pieces, 500. Mu.L of 5% triethylamine solution was added, and after waiting for 3min, the solution was removed, and this step was repeated 5 times. Finally, 500 ultrapure water is added to wash away residual triethylamine solution. Then 80% acetic acid solution (about 300. Mu.L) was added to the gel in an amount to cover the minimum amount of the gel, and after 5min, the 80% acetic acid solution was collected and repeated 3 times. Then 300. Mu.L of water was added to the gel, and the aqueous solution was collected after 3min and repeated 5 times. The collected acetic acid solution and aqueous solution were combined and concentrated in vacuo. 200 μ L of concentrated ammonia water was added to the concentrated residue, and the mixture was shaken briefly and then allowed to stand at 85 ℃ for 15min. After cooling to room temperature, 1800. Mu.L of n-butanol was added, and after shaking for 1min, the mixture was centrifuged in a high-speed centrifuge at 14000rpm for 15min. The supernatant was carefully removed by pipette and concentrated to remove excess n-butanol. The white solid left behind was the purified oligonucleotide. The product was analyzed by HPLC using Shim-pack GIST Shijin high purity silica gel column, model 227-30011-03. The gradient elution condition of pure product analytical HPLC is 0-6min, and the concentration of acetonitrile is 5% -50%;6.01-8min, the concentration of acetonitrile is 88%;8.01-10min, the concentration of the acetonitrile is 5%. As shown in fig. 6, a clean product was obtained after purification with a retention time of 2.462min; the molecular weight was confirmed by ESI mass spectrometry without error (FIG. 7). MS (ESI) m/z theoretical 7458.87 with measured value 7458. The recovery amount is 227.7nmol, the recovery rate is 22.77 percent, the recovery rate is improved by 30 percent compared with the existing reagent based on the capture and release strategy, and the purity of the oligonucleotide is 99.78 percent.

As a test group, T-10 was used as a purification agent for purifying 25-base long oligonucleotides (5 '-HO-TACACATCTTCCCTACGACGCGCCTC-OH-3', SEQ ID NO: 3) at a synthesis scale of 200nmol, a recovery of 53.8nmol, a recovery of 26.90%, an improvement of 54% over the existing reagent recovery based on the capture and release strategy, an oligonucleotide purity of 99.26% (FIG. 8), a molecular weight confirmed by ESI mass spectrometry (FIG. 9), MS (ESI) m/z theoretical value 7472.87, a measurement of 7473; the recovery of 1000nmol was 262nmol, the recovery was 26.20%, which is 50% higher than the existing reagent recovery based on capture and release strategies, the oligonucleotide purity was 99.13% (FIG. 10), the molecular weight was confirmed by ESI mass spectrometry without errors (FIG. 11), MS (ESI) m/z theory 7472.87, the measurement was 7473.

Example 3 validation of downstream application of oligonucleotides

To verify that the oligonucleotides purified using the purification method of the invention had no effect on downstream applications, both Polymerase Chain Reaction (PCR) and instant polymerase chain reaction (qPCR) applications were selected for testing. The oligonucleotides purified by the present invention and the oligonucleotides purified by HPLC were used for PCR and qPCR, respectively.

The concentrations of the forward oligonucleotide primer purified by the present invention and the reverse oligonucleotide primer purified by HPLC were set to 10. Mu.M, respectively, and the template concentration was 1.5 pg/. Mu.L. mu.L of forward primer, 2. Mu.L of reverse primer, 1. Mu.L of template (primer and template sequences are shown in Table 2 below), 25. Mu.L of KAPA SYBR FAST qPCR Master Mix (2X) and 20. Mu.L of water were added to a PCR tube and amplified for 30 cycles in a PCR instrument in the set program (95 ℃ 3min,98 ℃ 20s,60 ℃ 30s,72 ℃ 5 min). After the experiment, 4. Mu.L of each PCR tube was analyzed by using 2.5% agarose gel, and the results are shown in FIG. 12, which shows that there is no difference in the results after the forward primers purified by the two methods were used in the PCR reaction.

TABLE 2

For the qPCR reaction, the concentrations of the forward primer and the reverse primer were set to 10. Mu.M, respectively, and the template concentration was 1.5 pg/. Mu.L. mu.L of forward primer, 0.6. Mu.L of reverse primer, 1. Mu.L of template, 10. Mu.L of KAPA SYBR FAST qPCR Master Mix (2X), 0.6. Mu.L of ROX Low solution and 7.4. Mu.L of water were added to each PCR tube and placed in a qPCR apparatus for 40 cycles of amplification in the set program (95 ℃ 3min,98 ℃ 20s,60 ℃ 30s,72 ℃ 30s,95 ℃ 15s,60 ℃ 1min,95 ℃ 1 s) in parallel with three sets. The amplification plot is shown in FIG. 13, and the Ct values are shown in Table 3. mu.L of each PCR tube was analyzed using 2.5% agarose gel. Shows that there is no difference in the Ct values after the two purified forward primers were used in the qPCR reaction, and the glue plots (figure 14) both show the correct oligonucleotide sizes.

TABLE 3

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

<110> Nanjing Kinsrui Biotechnology Ltd

<120> a phosphoramidite monomer and method for purifying oligonucleotide

<130> PD220033N

<150> CN202110417554.1

<151> 2021-04-19

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 1

<400> 1

cacactcttt ccctacacga cgctc 25

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 2

<400> 2

acactctttc cctacacgac gctc 24

<210> 3

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> SEQ ID NO: 3

<400> 3

tacactcttt ccctacacga cgctc 25

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer (SEQ ID NO: 4)

<400> 4

cacactcttt ccctacacga cgctc 25

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer (SEQ ID NO: 5)

<400> 5

tgactggagt tcagacgtgt gctc 24

<210> 6

<211> 165

<212> DNA

<213> Artificial sequence

<220>

<223> template (SEQ ID NO: 6)

<400> 6

cacactcttt ccctacacga cgctcttccg atcttcaaga tgatgctcgt tatggtttcg 60

aatcgattcc gttgctgcca tctcaaaaac atttgacgcc ggactgctcc gcttcctcct 120

gagacgcaga gttgatcgga agagcacacg tctgaactcc agtca 165

Claims (23)

1. A phosphoramidite monomer having a structure represented by formula I below, or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, or solvate thereof:

[ formula I ]

Wherein, the first and the second end of the pipe are connected with each other,

x is a rigid linker having the structure:

r1 and R2 are both-O (CH) ₂ CH ₂ O) _n CH ₃ And are substituted at two arbitrary different positions on the middle benzene ring, respectively;

r3 to R10 are each independently selected from hydrogen, hydroxy, cyano, halogen, or substituted or unsubstituted amino, C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy or C ₃ -C ₆ A cycloalkyl group;

m is an integer of 1 to 10;

n is an integer of 1 to 10; and is

Y is a phosphoramidite modified nucleoside, and the amino group on the nucleoside is optionally acylated.

2. The phosphoramidite monomer of claim 1 wherein,

r1 and R2 are ortho, meta or para substituted to each other;

r3 to R10 are each independently selected from hydrogen, hydroxy, cyano, halogen, or substituted or unsubstituted amino, C ₁ -C ₆ An alkyl group;

m is an integer of 3 to 8;

n is an integer of 2 to 5; and is

Y is a phosphoramidite modified deoxyribonucleoside comprising deoxyadenosine, deoxyguanosine, deoxythymidine, deoxyuridine or deoxycytidine.

3. The phosphoramidite monomer of claim 1 or 2 wherein R1 and R2 are para-substituted with respect to each other.

4. The phosphoramidite monomer of claim 1 or 2, wherein R3 to R10 are each independently selected from hydrogen, hydroxy, cyano, halogen, or substituted or unsubstituted amino, methyl, ethyl, propyl, isopropyl.

5. The phosphoramidite monomer of any of claims 1-2 and 4, wherein the substitution is by halogen, hydroxy, amino, cyano, C ₁ -C ₃ Alkyl or C ₁ -C ₃ Haloalkyl substitution.

6. The phosphoramidite monomer of claim 1, wherein the modifying group in the phosphoramidite modification has the structure:

7. the phosphoramidite monomer of claim 1 having one of the structures shown below:

8. a method of purifying an oligonucleotide, comprising the steps of:

(1) Coupling an oligonucleotide with the phosphoramidite monomer of any of claims 1-7 to ligate the phosphoramidite monomer to a terminus of the oligonucleotide to provide a full-length oligonucleotide;

(2) Polymerizing the full-length oligonucleotide;

(3) Removing the failure sequence from the polymerized full-length oligonucleotide; and

(4) Recovering the full-length oligonucleotide.

9. The method of claim 8, wherein the oligonucleotide is 10-100nt in length; preferably 20-50nt.

10. The process according to claim 8, wherein in step (1), the coupling reaction time is between 0.2h and 1h, preferably 0.3h; more preferably, the coupling reaction is carried out in 6 portions, each for 0.05h.

11. The method of any one of claims 8-10, wherein, in step (1), the phosphoramidite monomer is linked at the 5' end of the oligonucleotide.

12. The method according to claim 8, wherein, in step (2), the polymerization is carried out by using a reagent comprising N, N-dimethylacrylamide and N, N' -methylenebisacrylamide.

13. The method of claim 12, wherein the concentration of N, N-dimethylacrylamide is 2-5M; preferably 3.7M.

14. The method of claim 12, wherein the concentration of N, N' -methylenebisacrylamide is 0.1 to 2M; preferably 0.18M.

15. The method of claim 12, wherein the reagents further comprise ammonium persulfate and tetramethylethylenediamine.

16. The method of claim 15, wherein the mass volume concentration of ammonium persulfate is 1% -10%; preferably 5%.

17. The method of claim 15, wherein the concentration of tetramethylethylenediamine is from 0.4M to 1.0M; preferably 0.66M.

18. The method according to claim 8, wherein, in step (3), the removal failure sequence is performed by cutting up the gel of the polymeric oligonucleotide in step (2), soaking in a buffer, and eluting.

19. The method of claim 18, wherein the buffer is pure water or a 3% -10% aqueous triethylamine solution; preferably 5% aqueous triethylamine.

20. The method of claim 8, wherein, in step (4), the recovering comprises the steps of acid solution soaking, aqueous solution elution, alkali solution neutralization and concentration.

21. The method of claim 20, the acid solution is a 70% -95% acetic acid solution; the alkali solution is strong ammonia water.

22. A kit comprising the phosphoramidite monomer of any of claims 1-7.

23. Use of the phosphoramidite monomer according to any one of claims 1-7 or the kit according to claim 22 in oligonucleotide purification.

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