N6-Bz-A-(S)-GNA phosphoramidite and preparation method thereof

Abstract

The invention relates to the technical field of phosphoramidite, and discloses N6-Bz-A-(S)-GNA phosphoramidite and a preparation method thereof. The method comprises the following steps: putting dichloromethane, diisopropyl ammonium salt tetrazole, (S)-DMT-glycidol-A (Bz) and bis (diisopropyl amino) (2-cyanoethoxy) phosphine which are prepared in advance into a reaction flask, and carrying out stirring reaction for a preset time; after the stirring reaction, performing HPLC central control reaction to confirm that the reaction is complete; washing the reaction compound for multiple times, removing a water phase and retaining an organic phase; and filtering, concentrating and crystallizing the organic phase subjected to water washing, so as to obtain the N6-Bz-A-(S)-GNA phosphoramidite. The method is simple to operate, low in cost, high in conversion rate and beneficial to large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of phosphoramidites, and particularly relates to an N6-Bz-A-(S)-GNA phosphoramidite and a preparation method thereof. Background Art

[0002] So far, for diseases such as cancer and viral infections, there are still no ideal specific drugs clinically. Fortunately, with the completion of the genome sequencing of humans and important model organisms, and the in-depth research of functional genomics and proteomics, molecular targets related to diseases have been continuously discovered and recognized, providing a premise for gene therapy.

[0003] In the past few decades, artificially synthesized oligonucleotides have been widely used in the research of targeted gene therapy. Oligonucleotides mainly include antisense oligonucleotides (ASODN), small interfering RNA (siRNA), transcription factor decoys, ribozymes, DNAzymes, antigene, CpG oligonucleotides, and aptamers, etc. Among them, ASODN and siRNA are the most commonly used gene regulation tools, which have been widely used and have been developed into gene therapy drugs.

[0004] Currently, oligonucleotides are mainly synthesized by chemical methods, which obtain oligonucleotides by sequentially reacting two active groups on a phosphoramidating reagent with protected nucleosides. The method of using a phosphoramidating reagent to synthesize oligonucleotides is called the phosphoramidite method, which is the most commonly used method in oligonucleotide synthesis, and the phosphoramidating reagent is the key raw material of the phosphoramidite method.

[0005] However, the preparation of traditional phosphoramidites has the characteristics of low yield and / or purity, which significantly increases the cost of oligonucleotide synthesis. For example, in the preparation of traditional N6-BZ-A-(S)-GNA phosphoramidite, mainly (S)-DMT-glycidol-A(Bz) and 2-cyanoethyl N,N-diisopropylphosphorochloridite are used as raw materials, dichloromethane is used as an auxiliary solvent, and column chromatography purification reaction is carried out with the participation of N,N-diisopropylethylamine. In this preparation process, due to the use of N,N-diisopropylethylamine, and the cost of N,N-diisopropylethylamine is relatively high, which further leads to a relatively high cost of the entire preparation process. Moreover, since the existing preparation method uses column chromatography purification process, it also leads to a relatively complex preparation process and low preparation efficiency.

[0006] Therefore, how to provide an efficient preparation method for N6-BZ-A-(S)-GNA phosphoramidite is an urgent problem to be solved at present. Summary of the Invention

[0007] The present invention provides an N6-BZ-A-(S)-GNA phosphoramidite and a preparation method thereof to solve the above technical problems in the prior art.

[0008] According to the first aspect of the present invention, an N6-BZ-A-(S)-GNA phosphoramidite is provided, and the structural formula of the N6-BZ-A-(S)-GNA phosphoramidite is:

[0009]

[0010] Moreover, the N6-BZ-A-(S)-GNA phosphoramidite is made from raw materials with the following weights:

[0011] Methylene chloride 260 - 270, (S)-DMT-glycidol-A(Bz) 20 - 20.1, diisopropylammonium tetrazole 11.1 - 11.2, bis(diisopropylamino)(2-cyanoethoxy)phosphine 11.7 - 11.8.

[0012] Among them, the structural formula of the (S)-DMT-glycidol-A(Bz) is:

[0013]

[0014] Among them, the structural formula of the diisopropylammonium tetrazole is:

[0015]

[0016] Among them, the structural formula of the bis(diisopropylamino)(2-cyanoethoxy)phosphine is:

[0017]

[0018] According to the second aspect of the present invention, a preparation method for the N6-BZ-A-(S)-GNA phosphoramidite is provided.

[0019] The synthesis route of the N6-BZ-A-(S)-GNA phosphoramidite is:

[0020]

[0021] Among them: The main reaction mechanism formula from (S)-DMT-glycidol-A(Bz) to N6-BZ-A-(S)-GNA phosphoramidite is:

[0022]

[0023] Specifically, bis(diisopropylamino)(2-cyanoethoxy)phosphine decomposes under the catalysis of diisopropylammonium salt tetrazole, losing one molecule of diisopropylamine and combining with tetrazole to obtain (diisopropylamino)(tetrazole)(2-cyanoethoxy)phosphine; the hydroxyl oxygen of (S)-DMT-glycidol-A(Bz) attacks the phosphorus atom, and then tetrazole is removed to obtain N6-BZ-A-(S)-GNA phosphoramidite.

[0024] Moreover, the preparation method steps of the N6-BZ-A-(S)-GNA phosphoramidite include:

[0025] Put the pre-prepared dichloromethane, diisopropylammonium salt tetrazole, (S)-DMT-glycidol-A(Bz), and bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask and carry out a stirring reaction for a predetermined time.

[0026] After the stirring reaction, perform HPLC in-process control of the reaction. After confirming that the reaction is complete, wash the reaction compound with water multiple times to remove the aqueous phase and retain the organic phase.

[0027] Filter, concentrate, and crystallize the washed organic phase to obtain N6-BZ-A-(S)-GNA phosphoramidite.

[0028] Optionally, the predetermined time is 17 - 20 hours, and the temperature during the stirring reaction is 20 - 30 °C.

[0029] Optionally, washing the reaction compound with water multiple times to remove the aqueous phase and retain the organic phase includes: washing the reaction compound with water multiple times using sodium chloride with a mass concentration of 10% to remove the aqueous phase and retain the organic phase.

[0030] Optionally, the number of water washings is 2 times, and the amount of sodium chloride used for each water washing is 400 - 500 ml.

[0031] Optionally, filtering, concentrating, and crystallizing the washed organic phase to obtain N6-BZ-A-(S)-GNA phosphoramidite includes: mixing the washed organic phase with pre-set sodium sulfate and standing for a set time. After the standing time reaches the set time, filter the mixture; perform vacuum concentration on the filtered mixture. After vacuum concentration, add methyl tert-butyl ether and n-heptane to dissolve the organic matter and carry out crystallization treatment to obtain N6-BZ-A-(S)-GNA phosphoramidite.

[0032] Optionally, the amount of sodium sulfate used is 30 - 40 g, and the set time is 0.5 - 1 hour.

[0033] Optionally, the temperature during reduced-pressure concentration is 20 - 30°C.

[0034] Optionally, the content of methyl tert-butyl ether is 300 - 330 ml, the content of n-heptane is 600 - 660 ml, and during crystallization, the ambient temperature is 20 - 30°C and the stirring time is 2 - 3 hours.

[0035] Optionally, the number of crystallization treatments is 2 times. The first crystallization treatment yields solid N6-BZ-A-(S)-GNA phosphoramidite, and the second crystallization treatment is to crystallize the filtered aqueous solution after the first crystallization treatment to obtain powdered N6-BZ-A-(S)-GNA phosphoramidite.

[0036] The technical solution provided by the present invention may include the following beneficial effects:

[0037] The invention process has simple operation and low raw material cost. At the same time, the product conversion rate obtained by the process of the present invention is high, which is conducive to large-scale production, provides a high-purity and low-cost raw material guarantee for subsequent oligonucleotide synthesis, and thus facilitates the technological progress of the entire field.

[0038] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention.

[0040] Figure 1 is the 1H NMR spectrum of N6-BZ-A-(S)-GNA phosphoramidite shown according to an exemplary embodiment 1 ;

[0041] Figure 2 is the 31P NMR spectrum of N6-BZ-A-(S)-GNA phosphoramidite shown according to an exemplary embodiment 31 ;

[0042] Figure 3 is the HPLC purity spectrum of N6-BZ-A-(S)-GNA phosphoramidite shown according to an exemplary embodiment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art shall fall within the protection scope of the present invention.

[0044] In the following examples, dichloromethane is the dichloromethane produced by Shanghai Lingfeng Chemical Reagent Co., Ltd. with the batch number 20230427; (S)-DMT-glycidol-A(Bz) is the (S)-DMT-glycidol-A(Bz) produced by Suzhou Novicon Biotechnology Co., Ltd. with the batch number NVC0024-40; diisopropylammonium salt tetrazole is the diisopropylammonium salt tetrazole produced by Suzhou Novicon Biotechnology Co., Ltd. with the batch number 230433; bis(diisopropylamino)(2-cyanoethoxy)phosphine is the bis(diisopropylamino)(2-cyanoethoxy)phosphine produced by Xinxiang Runyu New Materials Technology Co., Ltd. with the batch number RV1361230412-RP172; 10% sodium chloride is the 10% sodium chloride produced by Jiangsu Qiangsheng Functional Chemical Co., Ltd. with the batch number 20230208; sodium sulfate is the sodium sulfate produced by Jiangsu Qiangsheng Functional Chemical Co., Ltd. with the batch number 20230225; methyl tert-butyl ether is the methyl tert-butyl ether produced by Shanghai Lingfeng Chemical Reagent Co., Ltd. with the batch number 20230421; n-heptane is the n-heptane produced by Shanghai Lingfeng Chemical Reagent Co., Ltd. with the batch number 20230405.

[0045] Example 1

[0046] A method for preparing N6-BZ-A-(S)-GNA phosphoramidite according to an embodiment of the present invention includes the following steps:

[0047] Put 265 g of pre-prepared dichloromethane, 11.13 g of diisopropylammonium salt tetrazole, 20 g of (S)-DMT-glycidol-A(Bz), and 11.75 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask and stir at 20 °C for 17 hours;

[0048] After the stirring reaction, perform HPLC in-process control of the reaction to confirm that the reaction is complete;

[0049] Wash the reaction compound once with 400 ml of 10% sodium chloride by mass, remove the aqueous phase and retain the organic phase; after the first water wash, wash it again with 400 ml of 10% sodium chloride by mass for a second water wash, remove the aqueous phase and retain the organic phase;

[0050] Mix the washed organic phase with 30 g of sodium sulfate, let it stand for 0.5 hour, then filter the mixture; and perform vacuum concentration at 20 °C;

[0051] After concentration under reduced pressure, 300 ml of methyl tert-butyl ether and 600 ml of n-heptane were added to dissolve the organic matter, and the mixture was stirred at 20 °C for 2 hours. Then, solid N6-BZ-A-(S)-GNA phosphoramidite was obtained by filtration. The solid N6-BZ-A-(S)-GNA phosphoramidite was added again to 300 ml of methyl tert-butyl ether and 600 ml of n-heptane to dissolve, and the mixture was stirred at 20 °C for 2 hours. Then, crystallization and filtration were carried out to obtain 21.5 g of powdery N6-BZ-A-(S)-GNA phosphoramidite with a purity greater than 98% and a yield of 82.5%.

[0052] Example 2

[0053] A preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to the embodiment of the present invention includes the following steps:

[0054] 265 g of dichloromethane, 11.13 g of diisopropylammonium salt tetrazole, 20 g of (S)-DMT-glycidol-A(Bz), and 11.75 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine which were pre-prepared were put into a reaction flask and stirred at 24 °C for 19 hours;

[0055] After the stirring reaction, HPLC was used for in-process control of the reaction to confirm that the reaction was complete;

[0056] The reaction compound was washed once with 400 ml of 10% sodium chloride by mass to remove the aqueous phase and retain the organic phase. After the first water wash, it was washed twice with 400 ml of 10% sodium chloride by mass to remove the aqueous phase and retain the organic phase;

[0057] The washed organic phase was mixed with 35 g of sodium sulfate, and after standing for 0.5 hour, the mixture was filtered; and concentration under reduced pressure was carried out at 25 °C;

[0058] After concentration under reduced pressure, 300 ml of methyl tert-butyl ether and 600 ml of n-heptane were added to dissolve the organic matter, and the mixture was stirred at 25 °C for 2.5 hours. Solid N6-BZ-A-(S)-GNA phosphoramidite was obtained by filtration. The solid N6-BZ-A-(S)-GNA phosphoramidite was added again to 300 ml of methyl tert-butyl ether and 600 ml of n-heptane to dissolve, and the mixture was stirred at 25 °C for 2.5 hours. Crystallization and filtration were carried out to obtain 21.1 g of powdery N6-BZ-A-(S)-GNA phosphoramidite with a purity greater than 98% and a yield of 81%.

[0059] Example 3

[0060] A preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to the embodiment of the present invention includes the following steps:

[0061] Put 265 g of pre-configured dichloromethane, 11.13 g of diisopropylammonium salt tetrazole, 20 g of (S)-DMT-glycidol-A(Bz), and 11.75 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask, and stir at 30 °C for 20 hours;

[0062] After the stirring reaction, conduct HPLC in-process control of the reaction to confirm that the reaction is complete;

[0063] Wash the reaction compound once with 400 ml of 10% sodium chloride by mass, remove the aqueous phase and retain the organic phase; after the first water wash, wash it again with 400 ml of 10% sodium chloride by mass for a second water wash, remove the aqueous phase and retain the organic phase;

[0064] Mix the washed organic phase with 40 g of sodium sulfate, let it stand for 0.5 hour, and then filter the mixture; and conduct vacuum concentration at 30 °C;

[0065] After vacuum concentration, add 300 ml of methyl tert-butyl ether and 600 ml of n-heptane to dissolve the organic matter, stir at 30 °C for 3 hours, and filter to obtain solid N6-BZ-A-(S)-GNA phosphoramidite; add the solid N6-BZ-A-(S)-GNA phosphoramidite again to 300 ml of methyl tert-butyl ether and 600 ml of n-heptane to dissolve it, stir at 30 °C for 3 hours, crystallize and filter to obtain 21.8 g of powdery N6-BZ-A-(S)-GNA phosphoramidite with a purity greater than 98% and a yield of 83.7%.

[0066] In specific applications, conduct 1 1H NMR analysis on the N6-BZ-A-(S)-GNA phosphoramidite prepared by the present invention, and the results are as Figure 1 shown:

[0067] 11H NMR (400 MHz, CDCl3): δ = 8.99 - 9.00 (br, 2H), 8.79 (s, 1H), 8.78 (s, 1H), 8.02 - 8.07 (m, 6H), 7.58 - 7.63 (m, 2H), 7.51 - 7.55 (m, 4H), 7.43 - 7.66 (m, 4H), 7.28–7.33 (m, 12H), 7.21 - 7.24 (m, 2H), 6.80 - 6.83 (m, 8H), 4.49 - 4.58 (m, 4H), 4.37 (m, 2H), 3.79 (s, 6H), 3.78 (s, 6H), 3.45–3.70 (m, 8H), 3.21 - 3.32 (m, 4H), 3.13 - 3.17 (m, 1H), 2.45 - 2.49 (q, J = 6.4 Hz, 2H), 2.37 (t, J = 6.4 Hz, 2H), 1.72 (s, 6H), 1.10 - 1.11 (d, J = 6.8 Hz, 12H), 1.05 - 1.07 (d, J = 6.8 Hz, 6H), 1.00 - 1.01 (d, J = 6.8 Hz, 6H).

[0068] The N6 - BZ - A - (S) - GNA phosphoramidite prepared by the present invention was 31 analyzed by 31P NMR, and the results are as Figure 2 shown below:

[0069] 31 31P NMR (162 MHz, CDCl3): δ = 148.08, 149.14.

[0070] The N6 - BZ - A - (S) - GNA phosphoramidite prepared by the present invention was analyzed for HPLC purity, and the results are as Figure 3 shown below: The HPLC purity is greater than 95%.

[0071] Thus, it can be seen that the N6 - BZ - A - (S) - GNA phosphoramidite prepared by the preparation method of the present invention can be directly used for the next reaction without purification. Compared with the column chromatography purification process, the purity and yield are improved. In addition, in the present invention, diisopropylammonium salt tetrazole is used instead of N,N - diisopropylethylamine, thereby greatly reducing the cost; choosing 10% aqueous sodium chloride solution instead of saturated aqueous sodium bicarbonate solution improves the stability.

[0072] The present invention is not limited to the structures that have been described above and shown in the drawings, and various modifications and changes can be made without departing from its scope. The scope of the present invention is only limited by the appended claims.

Claims

1. An N6-BZ-A-(S)-GNA phosphoramidite, characterized in that, Made from raw materials with the following weights: 260 - 270 g of dichloromethane, 20 - 20.1 g of (S)-DMT-glycidol-A(Bz), 11.1 - 11.2 g of diisopropylammonium salt tetrazole, 11.7 - 11.8 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine.

2. A method for preparing N6-BZ-A-(S)-GNA phosphoramidite, characterized in that, For the preparation of the N6-BZ-A-(S)-GNA phosphoramidite described in claim 1, including: Put the pre-prepared dichloromethane, diisopropylammonium salt tetrazole, (S)-DMT-glycidol-A(Bz), and bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask and carry out a stirring reaction for a predetermined time; After the stirring reaction, conduct an in-process HPLC reaction control. After confirming that the reaction is complete, wash the reaction compound with water multiple times to remove the aqueous phase and retain the organic phase; Filter, concentrate, and crystallize the washed organic phase to obtain N6-BZ-A-(S)-GNA phosphoramidite.

3. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 1, characterized in that The predetermined time is 17 - 20 hours, and the temperature during the stirring reaction is 20 - 30 °C.

4. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 1, characterized in that, Washing the reaction compound with water multiple times to remove the aqueous phase and retain the organic phase includes: Washing the reaction compound with water multiple times using sodium chloride with a mass concentration of 10% to remove the aqueous phase and retain the organic phase.

5. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 4, characterized in that, The number of water washings is 2 times, and the amount of sodium chloride used for each water washing is 400 - 500 ml.

6. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 2, characterized in that, Filtering, concentrating, and crystallizing the washed organic phase to obtain N6-BZ-A-(S)-GNA phosphoramidite includes: Mix the washed organic phase with the pre-set sodium sulfate and let it stand for a set time. After the standing time reaches the set time, filter the mixture; Conduct vacuum concentration on the filtered mixture. After vacuum concentration, add methyl tert-butyl ether and n-heptane to dissolve the organic matter and carry out crystallization treatment to obtain N6-BZ-A-(S)-GNA phosphoramidite.

7. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 6, characterized in that, The amount of sodium sulfate used is 30 - 40 g, and the set time is 0.5 - 1 hour.

8. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 6, characterized in that, The temperature during the vacuum concentration is 20 - 30 °C.

9. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 6, characterized in that, The content of methyl tert-butyl ether is 300 - 330 ml, the content of n-heptane is 600 - 660 ml, and during crystallization, the ambient temperature is 20 - 30 °C and the stirring time is 2 - 3 hours.

10. The preparation method of N6-BZ-A-(S)-GNA phosphoramidite according to claim 6, characterized in that, The number of crystallization treatments is 2 times. The first crystallization treatment yields solid N6-BZ-A-(S)-GNA phosphoramidite, and the second crystallization treatment is to crystallize the filtered aqueous solution after the first crystallization treatment to obtain powdered N6-BZ-A-(S)-GNA phosphoramidite.

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