Synthesis and application of N-acetyl protected guanosine RNA monomer

Abstract

The invention relates to synthesis and application of an N-acetyl protected guanosine RNA monomer, and belongs to the technical field of chemical synthesis. In the prior art for synthesizing RNA, RNA is cut off from a carrier through ammonolysis, the time for synchronously removing an i-Bu protecting group is long, the required reaction temperature is high, RNA degradation, crude product purity reduction, purification difficulty and yield reduction are caused, and long-time ammonolysis causes breakage of fluorescent groups and temperature-sensitive modification groups in an RNA chain structure, so that the RNA is difficult to purify. In addition, the price of isobutyryl chloride or isobutyric anhydride is still high from the perspective of large-scale production, and isobutyric acid generated by removing i-Bu has certain toxicity and is poor in environmental friendliness. Therefore, the invention aims to develop the N-protected guanosine RNA monomer which is simple, easy to obtain, environment-friendly, good in group compatibility, mild in ammonolysis condition, good in stability and easy to remove, so that the problems are solved, and large-scale synthesis of RNA drugs is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of chemical synthesis, and in particular to the synthesis and application of an N-acetyl-protected guanosine RNA monomer. Background Art

[0002] With the successful completion of the Human Genome Project, the identification of functional genes and the research on the causes and diagnosis of diseases have continued to progress. Combined with improved oligonucleotide synthesis methods and administration methods, genes have gradually become new drug targets. Compared with traditional protein-targeted drugs, gene-targeted drugs are simpler in design, lower in cost, require smaller doses, and have milder side effects. Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA), as two important tools for gene expression regulation, have been widely used in gene function research and drug development. However, although phosphoramidite chemistry is widely used in DNA synthesis, traditional RNA synthesis methods face challenges due to low monomer preparation efficiency and high cost. In addition, compared with DNA synthesis, the low coupling efficiency of RNA monomers limits the length of RNA synthesis, and the deprotection process of RNA products is cumbersome and time-consuming, making it difficult to achieve large-scale production.

[0003] RNA monomers are the basis of RNA synthesis, and their preparation is more difficult than DNA monomers. In addition to the need for additional protecting group screening for the 2'-hydroxyl group, the compatibility of the protecting group of the hydroxyl group on the sugar ring with the protecting group of the amino group on the base is also crucial. The stability of the protecting group directly affects the efficiency of solid-phase synthesis, and the ease of its removal also affects the degradation of RNA during post-processing. In conventional solid-phase synthesis processes based on phosphoramidite chemistry, the synthesized RNA needs to be cut off from the solid phase support and the protecting group removed under alkaline conditions (such as ammonia water, methylamine aqueous solution, etc.). Prolonged alkaline treatment may lead to RNA degradation, increased impurities and reduced yield. N-protected guanosine RNA monomer is one of the four essential basic monomers in RNA synthesis. Due to its structural characteristics, the selection of its amino protecting group must have a certain degree of stability and be relatively easy to remove (such as Figure 1 Currently, isobutyryl (i-Bu) is used as the protecting group for guanine amino group. However, its disadvantage is that the final removal of this protecting group requires a long aminolysis time (more than 12 hours) and a high temperature (more than 55°C), which can lead to different degrees of RNA degradation (such as Figure 1 ), especially in RNA sequences that contain additional alkali- or temperature-sensitive modification groups, such as fluorescent groups and drug molecules. Therefore, selecting appropriate protecting groups and shortening the aminolysis time are the keys to improving the purity and synthesis efficiency of crude RNA. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a guanosine RNA monomer containing a protecting group and a synthesis method thereof, wherein an acetyl group is used as a protecting group at position 1, and an O-methyl, fluoro, O-polyethylene glycol chain, O-tert-butyldimethylsilyl, O-alkyl chain or O-acetamide is used as a protecting structure at position 2, and the two have good compatibility, and the protecting groups on the synthesized guanosine RNA monomer are stable, which greatly reduces the aminolysis temperature and time during the RNA synthesis process, and reduces the generation of impurities.

[0005] The first object of the present invention is to provide an N-acetyl protected guanosine RNA monomer, the structure of which conforms to the following general formula:

[0006]

[0007] The R is selected from one of the following structures:

[0008]

[0009] m is an integer between 1 and 10, and n is an integer between 2 and 20. The protected structure at position 1 of the present invention is N-acetyl; the protected structure at position 2 is O-methyl, O-alkyl chain (carbon chain length 2-20 carbons) and O-polyethylene glycol chain (ethylene glycol unit number 1-10), O-tert-butyldimethylsilyl, O-acetic acid amide and fluoro.

[0010] The second object of the present invention is to provide a method for preparing the guanosine RNA monomer, comprising the following steps:

[0011] S1, reacting the compound represented by formula I with acetic anhydride in an organic solvent to obtain a compound represented by formula II;

[0012] S2, reacting the compound represented by formula II with 4,4'-bismethoxytrityl chloride in an organic solvent to obtain a compound represented by formula III;

[0013] S3, reacting the compound represented by formula III with bis(diisopropylamino)(2-cyanoethoxy)phosphine in the presence of diisopropylammonium tetrazolium in an inert gas atmosphere to obtain the guanosine RNA monomer;

[0014] in,

[0015]

[0016]

[0017] Furthermore, in step S1, the reaction temperature is 80-120°C.

[0018] Further, in step S1, the reaction is carried out under microwave conditions.

[0019] Furthermore, in step S1, the organic solvent includes N,N-dimethylformamide.

[0020] Furthermore, in step S2, the reaction temperature is -10 to 5°C.

[0021] Further, in step S2, the organic solvent includes pyridine.

[0022] Furthermore, in step S3, the reaction temperature is 25-30°C.

[0023] Furthermore, in step S3, the reaction is carried out in the presence of an organic solvent, and the organic solvent includes dichloromethane or acetone.

[0024] Furthermore, the compounds represented by formula I are all purchased.

[0025] The third object of the present invention is to provide the use of the guanosine RNA monomer in nucleic acid synthesis.

[0026] Furthermore, the nucleic acid includes RNA.

[0027] Furthermore, the method of nucleic acid synthesis includes solid phase synthesis.

[0028] Furthermore, the solid phase synthesis method comprises the following steps:

[0029] S1, connecting the nucleotide with the protecting group to the solid phase carrier, and gradually adding the next nucleotide with the protecting group until a sequence with a solid phase carrier and a protecting group of the target length is obtained;

[0030] S2, performing aminolysis on the sequence with the solid phase carrier and the protecting group in S1, and removing the solid phase carrier after the aminolysis is completed to obtain the target nucleic acid.

[0031] Further, a purification step is included after removing the solid support.

[0032] By means of the above scheme, the present invention has at least the following advantages:

[0033] 1. The introduction of acetyl protecting groups in guanosine RNA monomers is mainly through the efficient amidation reaction of cheap and readily available acetic anhydride and amino groups. The reaction has a high yield and is easy to purify, which reduces the cost of monomer synthesis. On the other hand, the deacetylation of amino groups produces acetic acid, which has the characteristics of low toxicity (acetic acid rat oral LD50: 3530 mg / kg, isobutyric acid rat oral LD50: 280 μL / kg produced by removal of i-Bu) and environmental friendliness. It solves the problem of RNA degradation caused by high aminolysis temperature and long time, and also improves the environmental unfriendly problem of the removal product.

[0034] 2. The acetyl group has good compatibility with the 2'-substituents of guanosine, including O-methyl, O-alkyl chain and O-polyethylene glycol chain, O-tert-butyldimethylsilyl, O-acetamide and O-fluoro, which improves the stability of RNA synthesis process.

[0035] 3. The introduction of the acetyl protecting group in the guanosine RNA monomer and the appropriate compatible protecting group at position 2 increases the rate of aminolysis and significantly reduces the temperature. Under the reaction conditions of 25°C, dissociation from the carrier and removal of the acetyl group can be completed in 2-3 hours. Thus, on the one hand, the risk of RNA degradation is reduced, the purity of the crude product is improved, the purification pressure is reduced, and the separation yield of RNA is increased. On the other hand, it is better compatible with alkaline sensitive groups. Thus, the problem of poor group tolerance is solved.

[0036] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0038] Figure 1 Schematic diagram of the aminolysis of RNA with N-isobutyrylguanine structure.

[0039] Figure 2 It is the structural formula of the N-acetyl protected guanosine RNA monomer of the present invention.

[0040] Figure 3 This is a synthetic route for the N-acetyl-protected guanosine RNA monomer of the present invention.

[0041] Figure 4 These are the NMR and HPLC results of the 2'-O-methoxy-N-acetylguanosine RNA monomer in Example 1.

[0042] Figure 5These are the NMR and HPLC results of the 2'-fluoro-N-acetylguanosine RNA monomer in Example 2.

[0043] Figure 6 These are the NMR and HPLC results of the N-acetyl O-2'-MOE guanosine RNA monomer in Example 3.

[0044] Figure 7 This is the result of RNA structure detection synthesized by the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0046] Example 1

[0047] Taking the synthesis of 2'-O-methoxy-N-acetylguanosine RNA monomer as an example, the synthesis route is as follows:

[0048]

[0049] The specific steps are as follows:

[0050] (1) Synthesis of N-acetyl O-2'-methoxyguanosine: 2'-Methoxyguanosine (3.0 g, 10.0 mmol) and acetic anhydride (2.82 mL, 30.0 mmol) were dissolved in 30 mL of anhydrous N,N-dimethylformamide and placed in a Biotage Initiator+ microwave reactor. The mixture was reacted at 80-120°C for 10-20 minutes. TLC showed that the reaction was complete. The solvent and acetic anhydride were evaporated under reduced pressure and the mixture was recrystallized from ethyl acetate to obtain pure N-acetyl O-2'-methoxyguanosine as a white solid (3.2 g, yield 94.6%).

[0051] (2) Synthesis of DMT-N-acetyl O-2'-methoxyguanosine: N-acetyl O-2'-methoxyguanosine (3.2 g, 9.4 mmol) was dissolved in 60 mL of anhydrous pyridine. The reaction system was cooled to 0°C, and then a pyridine solution of 4,4'-dimethoxytrityl chloride (3.81 g, 9.5 mol) was added dropwise. 4 mL of methanol was added to quench the reaction. Pyridine was evaporated under reduced pressure, and the residue was extracted with ethyl acetate and water. The ethyl acetate layers were combined and dried over anhydrous sodium sulfate. The mixture was evaporated to dryness and column chromatography (methanol: dichloromethane = 1:100 to 1:10) was performed to obtain DMT-N-acetyl O-2'-methoxyguanosine as a white foaming solid (4.5 g, yield 74.1%).

[0052] (3) Synthesis of N-acetyl O-2'-methoxyguanosine RNA monomer: 500 mL of dichloromethane was added to the reaction vessel, nitrogen was continuously introduced into the reaction vessel, and DMT-N-acetyl O-2'-methoxyguanosine (4.5 g, 7.0 mmol) and diisopropylammonium tetrazolium salt (2.4 g, 14.0 mol) were added to the reaction vessel under the protection of nitrogen atmosphere, stirred to fully dissolve, and then bis(diisopropylamino)(2-cyanoethoxy)phosphine (4.2 g, 14.0 mmol) was added dropwise to the reaction vessel, and the reaction system was reacted at 25-30°C for 4-5 hours, and the progress of the reaction was controlled by HPLC. After the reaction was complete, 500 mL of water was added to the reaction system, and the organic layer was extracted and separated, and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure at 25-30°C, and purified by column chromatography, with the eluent being dichloromethane / ethyl acetate = 3 / 1-2 / 1. The pure component was concentrated under reduced pressure at 20-30°C to obtain N-acetyl O-2'-methoxyguanosine RNA monomer, a white foamy solid (5.0 g, yield 84.2%). 1 H NMR (400MHz, DMSO-d6) δ12.06(s,1H),11.68(s,1H),8.17(s,1H),7.30(ddd,J=24.9,15.8,8.1H z,9H),6.85(dt,J=9.1,5.5Hz,4H),5.92(dd,J=16.5,6.0Hz,1H),5.76(s,1H),4.60–4.32(m,2H ),4.30–4.11(m,1H),3.83–3.74(m,7H),3.62–3.49(m,3H),3.44–3.24(m,6H),2.80(t,J=5.9Hz ,1H),2.60(t,J=5.9Hz,1H),2.19(s,3H),2.08(s,1H),1.27–1.09(m,9H),1.01(d,J=6.7Hz,3H). 31 PNMR (162MHz, DMSO-d6): δ149.76, 149.74. HPLC purity>95%. H NMR spectrum, P NMR spectrum and HPLC were as follows: Figure 4 A-4C.

[0053] Example 2

[0054]

[0055] The 2'-methoxyguanosine in step (1) was replaced by 2'-fluoroguanosine, and the rest was the same as in Example 1. N-acetyl O-2'-fluoroguanosine RNA monomer was obtained as a white foamy solid (3.7 g, yield 63.1%). 1H NMR(300MHz,DMSO-d6)δ12.06(s,1H),11.67(s,1H),8.14(s,1H),7.48–7.13(m,9H),6.95–6.68(m, 4H),5.91(dd,J=12.4,6.4Hz,1H),4.70(dd,J=8.0,3.5Hz,1H),4.43(dd,J=10.5,4.0Hz,1H),4.20(d ,J=26.0Hz,1H),3.85–3.68(m,7H),3.68–3.43(m,5H),3.47–3.36(m,2H),3.19(dd,J=23.9,4.0Hz,4 H),2.80(d,J=5.9Hz,1H),2.64–2.51(m,3H),2.19(s,3H),1.30–1.07(m,9H),1.01(d,J=6.7Hz,3H). 31 P NMR (162MHz, DMSO-d6): δ149.43. HPLC purity>95%. H NMR spectrum, P NMR spectrum and HPLC were as follows: Figure 5 A-5C.

[0056] Example 3

[0057]

[0058] The 2'-methoxyguanosine in step (1) was replaced by 2'-MOE guanosine, and the rest was the same as in Example 1. N-acetyl O-2'-MOE guanosine RNA monomer was obtained as a white solid (4.4 g, yield 71.1%). 1 HNMR (300MHz, DMSO) δ12.00(s,1H),11.74(s,1H),8.11(d,J=1.0Hz,1H),7.50–7.10(m,9H),6.85(ddd,J=8.8,6.6,2 .1Hz,4H),6.21(ddd,J=9.1,7.8,1.7Hz,1H),5.70–5.54(m,1H),5.63(d,J=3.8Hz,1H),5.48(dd,J=11.6,3.3Hz,1H), 4.67(dd,J=9.3,5.6Hz,1H),4.22(s,1H),3.72(t,J=4.5Hz,8H),3.54(ddd,J=12.4,9.0,2.7Hz,3H),3.38(d,J=8.3H z,1H),2.76(t,J=5.9Hz,1H),2.60(t,J=5.9Hz,1H),2.19(d,J=0.8Hz,3H),1.18–1.03(m,9H),0.95(d,J=6.8Hz,3H).31 P NMR (162MHz, DMSO) δ150.27 (d, J = 8.9 Hz), 149.59 (d, J = 8.3 Hz). HPLC purity> 95%. H NMR spectrum, P NMR spectrum and HPLC are as follows: Figure 6 A-6C.

[0059] Of course, the 2' position is other R groups such as The N-acetyl protected guanosine RNA monomers were also prepared in the same way, that is, 2'-methoxyguanosine was replaced by guanosine containing the corresponding R group, and the yield was between 85-90%.

[0060] Example 4 RNA Synthesis

[0061] The N-Ac protected guanosine RNA monomers prepared in Example 1 and Example 2 were used to prepare RNAs with the same sequence (as shown in Table 1) for comparison. The specific general synthesis steps are as follows:

[0062] Place 2'-methoxy phosphoramidite monomers, 2'-fluoro phosphoramidite monomers, acetonitrile, deprotection reagents, oxidants, coupling reagents and capping reagents in the corresponding positions of the ABI394 solid phase synthesizer, enter the sequence information, and start the synthesis program. Using the phosphoramidite solid phase synthesis method, the Uniylinker solid phase carrier is the starting cycle, and the phosphoramidite monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement. Each connection of a phosphoramidite monomer includes four steps of deprotection, coupling, capping, and oxidation to obtain a sequence with a solid phase carrier and a protecting group. The conditions for each step of the reaction are as follows:

[0063] (1) Deprotection: Add decapping agent and the reaction time is 2 minutes.

[0064] (2) Coupling reaction: Add activator and the coupling time is 3 minutes.

[0065] (3) Oxidation reaction: Add 0.05 M iodine solution and the oxidation time is 25 seconds.

[0066] (4) Capping reaction: Add capping agent A / capping agent B (1 / 1, v / v) solution, and the capping time is 20 seconds.

[0067] The sequence with a solid phase carrier and a protecting group was added to a 2 mL centrifuge tube, and 1 mL of 25% to 28% ammonia water was added, and aminolysis was performed at different temperatures and times. After the aminolysis is completed, the solid phase carrier is removed by filtering and then washing three times with 0.5 mL of 50% ethanol aqueous solution. The filtrate was concentrated and dried to obtain the crude products of the sense chain and the antisense chain. The superiority of using N-Ac to protect guanosine RNA monomers over N-iBu to protect guanosine RNA monomers was reflected by comparing the aminolysis temperature, aminolysis time, and crude purity of the two sense chains and the two antisense chains (see Table 2 and Figure 7 ).

[0068] The crude products of two sense chains and two antisense chains were dissolved in 1 mL of RNA-free enzyme water and purified by ion-pair reverse phase chromatography (RP-HPLC, Waters 2545). The analysis was then performed using a Waters X-Bridge C18 column (5 μm, 10 mm × 250 mm), with mobile phase A being 0.1 M TEAA and mobile phase B being ACN. The collected fractions were detected by HPLC and MS, and the combined fractions were quantified by Nanodrop One and freeze-dried to obtain pure products of two sense chains and two antisense chains, and the yields of the two comparison groups were compared (see Table 2).

[0069] Table 1 Nucleic acid sequences used in this example

[0070]

[0071] Comparative Example 1

[0072] The acetic anhydride in step (1) was replaced by acetyl chloride. Experimental steps: Synthesis of N-acetyl O-2'-methoxyguanosine: 2'-methoxyguanosine (3.0 g, 10.0 mmol) and acetyl chloride (2.1 mL, 30.0 mmol) were dissolved in 30 mL of anhydrous N,N-dimethylformamide, placed in a Biotage Initiator+ microwave reactor, and reacted at 80-120°C for 10-20 minutes. TLC showed that the reaction was complete. The solvent and acetic anhydride were evaporated under reduced pressure, and the pure N-acetyl O-2'-methoxyguanosine was recrystallized from ethyl acetate to obtain a white solid (1.5 g, yield 45.3%).

[0073] Comparative Example 2

[0074] The microwave reaction in step (1) was replaced by heating. Experimental steps: Synthesis of N-acetyl O-2'-methoxyguanosine: 2'-methoxyguanosine (3.0 g, 10.0 mmol) and acetic anhydride (2.82 mL, 30.0 mmol) were dissolved in 30 mL of anhydrous N,N-dimethylformamide and heated to 120°C for 10-20 minutes. TLC showed that the reaction was complete. The solvent and acetic anhydride were evaporated under reduced pressure and recrystallized from ethyl acetate to obtain pure N-acetyl O-2'-methoxyguanosine as a white solid (0.85 g, yield 25.8%).

[0075] Test Case

[0076] The aminolysis time, aminolysis temperature, crude product purity and purified yield during the RNA synthesis process of Example 4 were recorded. The results are shown in the table below.

[0077] Table 2 Synthesis results

[0078] sample Ammonolysis time (min) Ammonolysis temperature (℃) Purity of crude RNA (%) Yield after RNA purification (%) N-iBu Justice Chain 16 55 67.4 55.2 N-Ac positive chain 4 25 90.4 84.3 N-iBu antisense strand 16 55 65.9 54.7 N-Ac antisense strand 4 25 70.7 65.9

[0079] From the comparative examples and the synthesis of the examples, it can be seen that guanosine with different substitutions at the 2'-position can be reacted with microwaves in the presence of acetic anhydride to obtain N-acetyl protected guanosine with a yield of 85-95%. If acetic anhydride is replaced with acetyl chloride in this method, the yield is reduced to 40-50%. Moreover, if microwaves are replaced with heating, the yield is significantly reduced to 25-30%.

[0080] As can be seen from the above table, compared with the guanosine RNA monomer protected by N-iBu, the RNA prepared with the guanosine RNA monomer protected by N-Ac of the present invention has higher yield and purity, and the aminolysis time and aminolysis temperature are significantly reduced, which shows that the removal of the protecting group is easier and can improve the degradation during RNA synthesis.

[0081] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. An N-acetyl protected guanosine RNA monomer, characterized in that: The structure of the guanosine RNA monomer conforms to the following general formula: The R is selected from one of the following structures: m is an integer between 1 and 10, and n is an integer between 2 and 20.

2. The method for preparing the guanosine RNA monomer according to claim 1, characterized in that: The following steps are involved: S1, reacting the compound represented by formula I with acetic anhydride in an organic solvent to obtain a compound represented by formula II; S2, reacting the compound represented by formula II with 4,4'-bismethoxytrityl chloride in an organic solvent to obtain a compound represented by formula III; S3, reacting the compound represented by formula III with bis(diisopropylamino)(2-cyanoethoxy)phosphine in the presence of diisopropylammonium tetrazolium in an inert gas atmosphere to obtain the guanosine RNA monomer; in, 3. The preparation method according to claim 2, characterized in that: Contain at least one of the following characteristics: (1) In step S1, the reaction temperature is 80 to 120°C; (2) In step S1, the reaction is carried out under microwave conditions; (3) In step S1, the organic solvent includes N,N-dimethylformamide.

4. The preparation method according to claim 2, characterized in that: Contain at least one of the following characteristics: (1) In step S2, the reaction temperature is -10 to 5°C; (2) In step S2, the organic solvent includes pyridine.

5. The preparation method according to claim 2, characterized in that: Contain at least one of the following characteristics: (1) In step S3, the reaction temperature is 25 to 30° C.; (2) In step S3, the reaction is carried out in the presence of an organic solvent, wherein the organic solvent includes dichloromethane or acetone; (3) In step S3, the inert gas includes nitrogen.

6. Use of the guanosine RNA monomer according to claim 1 or the guanosine RNA monomer prepared by the preparation method according to any one of claims 2 to 5 in nucleic acid synthesis.

7. The use according to claim 6, characterized in that: The nucleic acid includes RNA.

8. The use according to claim 7, characterized in that: Methods for nucleic acid synthesis include solid phase synthesis.

9. The use according to claim 8, characterized in that: The solid phase synthesis method comprises the following steps: S1, connecting the nucleotide with the protecting group to the solid phase carrier, and gradually adding the next nucleotide with the protecting group until a sequence with a solid phase carrier and a protecting group of the target length is obtained; S2, performing aminolysis on the sequence with the solid phase carrier and the protecting group in S1, and removing the solid phase carrier after the aminolysis is completed to obtain the target nucleic acid.

10. The use according to claim 9, characterized in that: After removing the solid phase support, a purification step is also included.

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