(S)-GNA-U phosphoramidite and preparation method thereof

Abstract

The invention relates to the technical field of phosphoramidite, and discloses (S)-GNA-U phosphoramidite and a preparation method thereof. The method comprises the following steps: putting dichloromethane, diisopropyl ammonium salt tetrazole, (S)-DMT-glycidol-urracil and bis (diisopropyl amino) (2-cyanoethoxy) phosphine which are prepared in advance into a reaction flask, and carrying out a stirring reaction for a preset time; after the stirring reaction, performing HPLC central control reaction to determine that the reaction is complete; washing the reaction liquid for multiple times, removing a water phase and retaining an organic phase; and filtering, concentrating and crystallizing the washed organic phase to obtain the (S)-GNA-U 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 (S)-GNA-U phosphoramidite and a preparation method thereof. Background Art

[0002] So far, for diseases such as cancer and viral infections, there is still a lack of ideal specific drugs clinically. Fortunately, with the completion of the genome sequencing of humans and important model organisms, as well as 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 at present, 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 process of traditional (S)-GNA-U phosphoramidite, mainly (S)-DMT-glycidol-uracil, bis(diisopropylamino)(2-cyanoethoxy)phosphine, and tetrazole are used as raw materials, with acetonitrile as an auxiliary solvent, and the reaction is carried out under a column chromatography purification process to obtain the product. In this preparation process, due to the use of tetrazole and acetonitrile, and the high cost of tetrazole and acetonitrile, the cost of the entire preparation process is high, and due to the existing preparation method using a column chromatography purification process, the preparation process steps are also complex and the preparation efficiency is low.

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

[0007] The present invention provides an (S)-GNA-U 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, there is provided an (S)-GNA-U phosphoramidite, and the structural formula of the (S)-GNA-U phosphoramidite is:

[0009]

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

[0011] 260 - 270 g of dichloromethane, 14 - 14.1 g of diisopropylammonium salt tetrazole, 20 - 20.1 g of (S)-DMT-glycidol-uracil, 14.8 - 14.9 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine.

[0012] Among them, the structural formula of the diisopropylammonium salt tetrazole is:

[0013]

[0014] Among them, the structural formula of the (S)-DMT-glycidol-uracil 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, there is provided a preparation method of an (S)-GNA-U phosphoramidite. The synthesis route of the (S)-GNA-U phosphoramidite is:

[0019]

[0020] Among them: The main reaction mechanism formula from (S)-DMT-glycidol-uracil to (S)-GNA-U phosphoramidite is:

[0021]

[0022] Specifically, bis(diisopropylamino)(2-cyanoethoxy)phosphine decomposes under the action of diisopropylammonium salt tetrazole, eliminating one molecule of diisopropylamine and combining with tetrazole to obtain (diisopropylamino)(tetrazole)(2-cyanoethoxy)phosphine; the hydroxyl oxygen of (S)-DMT-glycidol-uracil attacks the phosphorus atom, and then tetrazole is eliminated to obtain (S)-GNA-U phosphoramidite.

[0023] Moreover, the preparation method steps of the (S)-GNA-U phosphoramidite include:

[0024] Put the pre-prepared dichloromethane, diisopropylammonium salt tetrazole, (S)-DMT-glycidol-uracil, and bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask and carry out a stirring reaction for a predetermined time;

[0025] After the stirring reaction, perform HPLC in-process control of the reaction, and 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;

[0026] Filter, concentrate, and crystallize the washed organic phase to obtain (S)-GNA-U phosphoramidite.

[0027] Optionally, the predetermined time is 17 - 20 hours, and the temperature during the stirring reaction is 20 - 30 °C.

[0028] 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.

[0029] Optionally, the number of water washings is 2 times, and the amount of sodium chloride used each time is 400 - 500 ml.

[0030] Optionally, filtering, concentrating, and crystallizing the washed organic phase to obtain (S)-GNA-U phosphoramidite includes: mixing the washed organic phase with pre-set sodium sulfate and standing for a set time, and after the standing time reaches the set time, filtering the mixture; performing vacuum concentration on the filtered mixture, and after vacuum concentration, adding methyl tert-butyl ether and n-heptane to dissolve the organic matter and performing crystallization treatment to obtain (S)-GNA-U phosphoramidite.

[0031] Optionally, the amount of sodium sulfate used is 30 - 40 g, and the set time is 0.5 - 1 hour.

[0032] Optionally, the temperature during the vacuum concentration is 20 - 30 °C.

[0033] 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.

[0034] Optionally, the number of crystallization treatments is 2 times. The first crystallization treatment yields solid (S)-GNA-U phosphoramidite, and the second crystallization treatment is to crystallize the filtered aqueous solution after the first crystallization treatment to obtain powdered (S)-GNA-U phosphoramidite.

[0035] The technical solution provided by the present invention may include the following beneficial effects:

[0036] 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.

[0037] 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

[0038] 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.

[0039] Figure 1 is the 1H NMR spectrum of (S)-GNA-U phosphoramidite shown according to an exemplary embodiment 1 ;

[0040] Figure 2 is the 31P NMR spectrum of (S)-GNA-U phosphoramidite shown according to an exemplary embodiment 31 ;

[0041] Figure 3 is the HPLC purity spectrum of (S)-GNA-U phosphoramidite shown according to an exemplary embodiment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all 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.

[0043] In the following examples, dichloromethane is the dichloromethane produced by Shanghai Lingfeng Chemical Reagent Co., Ltd. with the batch number 20230427; diisopropylammonium salt tetrazole is the diisopropylammonium salt tetrazole produced by Suzhou NoviCare Biotechnology Co., Ltd. with the batch number 230433; (S)-DMT-glycidol-uracil is the (S)-DMT-glycidol-uracil produced by Suzhou NoviCare Biotechnology Co., Ltd. with the batch number NVC0024-62; 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 20230521; n-heptane is the n-heptane produced by Shanghai Lingfeng Chemical Reagent Co., Ltd. with the batch number 20230605.

[0044] Example 1

[0045] A method for preparing (S)-GNA-U phosphoramidite according to an embodiment of the present invention includes the following steps:

[0046] Put 265 g of pre-prepared dichloromethane, 14.02 g of diisopropylammonium salt tetrazole, 20 g of (S)-DMT-glycidol-uracil, and 14.81 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask and stir at 20°C for 17 hours;

[0047] After the stirring reaction, perform HPLC in-process control of the reaction to determine that the reaction is complete;

[0048] 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, and retain the organic phase of the aqueous phase;

[0049] Mix the washed organic phase with 40 g of sodium sulfate, let it stand for 0.5 hour, then filter the mixture; and perform vacuum concentration at 20°C;

[0050] After vacuum concentration, 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. The solid (S)-GNA-U phosphoramidite was obtained by filtration; the solid (S)-GNA-U phosphoramidite was dissolved again with 300 ml of methyl tert-butyl ether and 600 ml of n-heptane, stirred at 20 °C for 2 hours, and filtered to obtain 16.9 g of powdered (S)-GNA-U phosphoramidite with a purity greater than 98% and a yield of 60%.

[0051] Example 2

[0052] A method for preparing (S)-GNA-U phosphoramidite according to the embodiment of the present invention includes the following steps:

[0053] 265 g of dichloromethane, 14.02 g of diisopropylammonium salt tetrazole, 20 g of (S)-DMT-glycidol-uracil, and 14.81 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine prepared in advance were placed in a reaction flask and stirred at 25 °C for 18 hours;

[0054] After the stirring reaction, HPLC was used for in-process control of the reaction to determine that the reaction was complete;

[0055] 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, and the aqueous phase was retained and the organic phase was retained;

[0056] The washed organic phase was mixed with 40 g of sodium sulfate, allowed to stand for 0.5 hour, and then the mixture was filtered; and vacuum concentration was carried out at 25 °C;

[0057] After vacuum concentration, 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. The solid (S)-GNA-U phosphoramidite was obtained by filtration; the solid (S)-GNA-U phosphoramidite was added again with 300 ml of methyl tert-butyl ether and 600 ml of n-heptane to dissolve, stirred at 25 °C for 2.5 hours, and crystals were precipitated and filtered to obtain 16.81 g of powdered (S)-GNA-U phosphoramidite with a purity greater than 98% and a yield of 59.7%.

[0058] Example 3

[0059] A method for preparing (S)-GNA-U phosphoramidite according to the embodiment of the present invention includes the following steps:

[0060] Put 265 g of pre-configured dichloromethane, 14.02 g of diisopropylammonium salt tetrazole, 20 g of (S)-DMT-glycidol-uracil, and 14.81 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine into a reaction flask and stir at 30 °C for 20 hours;

[0061] After the stirring reaction, conduct HPLC in-process control of the reaction to determine that the reaction is complete;

[0062] 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, and retain the organic phase from the aqueous phase;

[0063] Mix the washed organic phase with 40 g of sodium sulfate, let it stand for 0.5 hours, then filter the mixture; and conduct vacuum concentration at 30 °C;

[0064] 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 (S)-GNA-U phosphoramidite; add the solid (S)-GNA-U phosphoramidite again to 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, filter the precipitated crystals to obtain 16.93 g of powdered (S)-GNA-U phosphoramidite with a purity greater than 98% and a yield of 60.1%.

[0065] In specific applications, conduct 1 1H NMR analysis on the (S)-GNA-U phosphoramidite prepared by the present invention, and the results are as Figure 1 shown:

[0066] 1H NMR (400 MHz, CDCl3): δ = 7.43 - 7.46 (m, 4H), 7.27 - 7.35 (m, 12H), 7.21 - 7.24 (tt, J = 1.9, 7.0 Hz, 2H), 7.17 - 7.18 (d, J = 7.9 Hz, 1H), 7.14 - 7.16 (d, J = 7.9 Hz, 1H), 6.18 - 6.85 (m, 8H), 5.54 - 5.56 (d, J = 7.3 Hz, 1H), 5.52 - 5.54 (d, J = 7.2 Hz, 1H), 4.11 - 4.26 (m, 4H), 3.54 - 3.84 (m, 10H), 3.79 (s, 6H), 3.78 (s, 6H), 3.30 - 3.33 (dd, J = 5.2, 9.8 Hz, 1H), 3.23 - 3.26 (dd, J = 3.4, 9.9 Hz, 1H), 3.19 - 3.21 (dd, J = 5.2, 10.0 Hz, 1H), 3.13 - 3.16 (dd, J = 4.5, 10.0 Hz, 1H), 2.58 - 2.62 (m, 2H), 2.40 - 2.43 (t, J = 7.5 Hz, 2H), 1.11 - 1.17 (m, 24H).

[0067] The (S)-GNA-U phosphoramidite prepared by the present invention was 31 analyzed by 31P NMR, and the results are as Figure 2 shown below: 31 31P NMR (162 MHz, CDCl3): δ = 149.35, 149.70.

[0068] The (S)-GNA-U 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%.

[0069] Thus, the (S)-GNA-U 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, dichloromethane is used instead of acetonitrile as the solvent, and diisopropylammonium salt tetrazole is used instead of tetrazole, thereby greatly reducing the cost; choosing 10% aqueous sodium chloride solution instead of saturated aqueous sodium bicarbonate solution achieves improved stability.

[0070] The present invention is not limited to the structures 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 (S)-GNA phosphoramidite, characterized in that, Made from raw materials with the following weights: 260 - 270 g of dichloromethane, 14 - 14.1 g of diisopropylammonium salt tetrazole, 20 - 20.1 g of (S)-DMT-glycidol-uracil, 14.8 - 14.9 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine.

2. A method for preparing (S)-GNA phosphoramidite, characterized in that, For the preparation of the (S)-GNA-U phosphoramidite according to claim 1, comprising: Put the pre-prepared dichloromethane, diisopropylammonium salt tetrazole, (S)-DMT-glycidol-uracil, 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 HPLC in-process control of the reaction. After confirming that the reaction is complete, wash the reaction compound with water multiple times, removing the aqueous phase and retaining the organic phase; Filter, concentrate, and crystallize the washed organic phase to obtain (S)-GNA-U phosphoramidite.

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

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

5. The preparation method of (S)-GNA-U 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 (S)-GNA-U phosphoramidite according to claim 2, characterized in that, Filtering, concentrating, and crystallizing the washed organic phase to obtain (S)-GNA-U 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; Vacuum concentrate 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 (S)-GNA-U phosphoramidite.

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

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

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