R-configuration-containing initial capping oligonucleotide primer and synthesis method thereof

Abstract

The invention discloses an initial capping oligonucleotide primer containing an R configuration and a synthesis method thereof, the structure of the initial capping oligonucleotide primer containing the R configuration is shown as a formula I. In the formula I, R1 and R2 are independently selected from H, Me, methoxyethyl or F; x is selected from O, S or Se; the Base is selected from an adenine base, a guanine base, a cytosine base, a 5-methylcytosine base or uracil. The initial capping oligonucleotide primer containing the R configuration belongs to a new substance, and all existing cap compounds are R + S type mixtures, which may be one of reasons causing mRNA low immunogenicity. The R-type cap compound with a single configuration is prepared, so that the cap compound is single in structure and relatively high in purity. The discovery of the substance provides platform significance for the research of mRNA series and the development of new products, and widens the range of mRNA research. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a method for synthesizing an initial capping oligonucleotide primer, and in particular to an initial capping oligonucleotide primer containing an R configuration and a method for synthesizing the same. Background Art

[0002] During protein synthesis, the cap structure is a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation, and nuclear export. It also serves as an anchor for recruiting initiation factors, which helps the ribosome recognize and bind to mRNA, allowing translation to start correctly. At present, there are related cap patents to optimize the structure, which can have a certain effect. However, the trend of scientific research applications is still to continuously optimize the structure of cap analogs and discover new cap analogs with lower immunogenicity than the existing caps.

[0003] The 5' cap structure is the key structure to reduce the immunogenicity of mRNA, so more cap structures need to be designed to better reduce immunogenicity. Currently, all cap structures are R / S mixtures. We applied to synthesize a single-configuration R-type cap to reduce the immunogenicity of the cap and reduce the harmfulness of the vaccine. Developing a cap structure with a single configuration and high purity to reduce immunogenicity is the trend of current scientific research applications. Summary of the invention

[0004] The purpose of the present invention is to provide an initial capping oligonucleotide primer containing an R configuration and a synthesis method thereof. The initial capping oligonucleotide primer containing an R configuration is a new substance and can be used for mRNA capping under a T7 RNA polymerase system, thereby broadening the scope of mRNA research.

[0005] In order to achieve the above object, the present invention provides an initial capping oligonucleotide primer containing an R configuration, and the structure of the initial capping oligonucleotide primer containing an R configuration is shown in Formula I: In formula I, R1 and R2 are each independently selected from H, Me, methoxyethyl or F; X is selected from O, S or Se; Base is selected from adenine base, guanine base, cytosine base, 5-methylcytosine base or uracil.

[0006] The R-configuration-containing starting capped oligonucleotide primer of the present invention is a new substance, which has not been reported in any literature or patent so far. It broadens the scope of oligonucleotides, and compared with mixed configurations, single configurations are less toxic.

[0007] Another object of the present invention is to provide a method for synthesizing the initial capping oligonucleotide primer containing an R configuration, the method comprising: The dinucleotide and the intermediate m7GMP-Im are dissolved in an organic solvent A containing MgCl2, and the reaction is stirred at room temperature; after the reaction is completed, the reaction is terminated and purified to obtain an initial capped oligonucleotide primer containing an R configuration as shown in Formula I.

[0008] Preferably, the terminating reaction is performed by adding EDTA-Na solution to terminate the reaction.

[0009] Preferably, the purification uses a DEAE column.

[0010] Preferably, the organic solvent A is selected from DMF.

[0011] Preferably, the method for synthesizing the dinucleotide comprises:

[0012] S-type phosphoramidite monomer and 2',3'-diacetylguanosine are placed in organic solvent B, tetrazole is added under an inert gas atmosphere, and the temperature is raised to 25±2℃ for reaction; after the reaction is completed, iodine-containing dichloromethane solution is added to the reaction solution, after monitoring the reaction, trifluoroacetic acid is added to remove DMTr, after monitoring the reaction, the solution is spin-dried and passed through a silica gel column to obtain intermediate 1; intermediate 1 is dissolved in organic solvent C, phosphine reagent and tetrazole are added, stirred and reacted, iodine-containing dichloromethane solution is added after monitoring the reaction, the solution is spin-dried after monitoring the reaction, methanol and concentrated ammonia water are added to the spin flask, react at 40 degrees, monitor the reaction, spin-dry after the reaction, add ultrapure water, purify with ion resin, and concentrate and freeze-dry to obtain phosphate-linked dinucleotides.

[0013] Preferably, the organic solvent B is selected from dichloromethane; and the organic solvent C is selected from acetonitrile.

[0014] Preferably, the synthesis method of the intermediate m7GMP-Im comprises:

[0015] A) m7G was heated and dissolved in trimethyl phosphate, the reaction solution was cooled to 0±5°C, phosphorus oxychloride was slowly added dropwise, reacted at room temperature, triethylamine-glacial acetic acid solution was added to quench the reaction, and the target compound guanosine monophosphate triethylamine salt GMP.TEA was obtained by freeze-drying; B) guanosine monophosphate triethylamine salt GMP.TEA, triphenylphosphine, dithiodipyridine, imidazole and triethylamine are suspended in an organic solvent D, reacted at room temperature, and after the reaction is complete, sodium perchlorate acetone solution is added, filtered, and the filter cake is fully washed with acetone to obtain the target compound guanosine monophosphate imidazole salt GMP.Im; C) Guanosine monophosphate imidazolate GMP.Im was suspended in organic solvent E, triethylamine phosphate was added, and the mixture was stirred at room temperature. After the reaction was complete, the mixture was used in the next step in one pot, triphenylphosphine, dithiodipyridine, imidazole, and TEA were added, and the mixture was reacted at room temperature. The reaction was monitored by HPLC until the starting material was ≤1%. Sodium perchlorate acetone solution was added to the reaction solution, and the mixture was filtered with suction. The filter cake was washed thoroughly with acetone to obtain the target compound m7GMP-Im.

[0016] Preferably, the organic solvent D and the organic solvent E are both selected from DMF.

[0017] Another object of the present invention is to provide the use of the initial capping oligonucleotide primer containing the R configuration in mRNA synthesis under the T7 RNA polymerase system.

[0018] The R-configuration-containing initial capping oligonucleotide primer and the synthesis method thereof of the present invention have the following advantages: The R-configuration-containing starting capping oligonucleotide primer of the present invention is a new substance, and there is no literature or patent report on it. All existing cap compounds are R+S type mixtures, which may be one of the reasons for the low immunogenicity of mRNA. The present invention prepares a single-configuration R-type cap compound, so that the cap compound has a single structure and high purity. The discovery of this substance provides a platform for the research of mRNA series and the development of new products, and broadens the scope of mRNA research. The R-configuration starting capping oligonucleotide is used for mRNA capping under the T7 RNA polymerase system. Compared with the existing cap structure analog Cleancap, the R-type starting capping oligonucleotide primer of the present invention produces mRNA with lower immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a purity diagram of the initial capping oligonucleotide primer GUG containing an R-type structure in Example 1 of the present invention.

[0020] Figure 2 This is a purity diagram of the initial capping oligonucleotide primer GAG containing an R-type structure in Example 2 of the present invention.

[0021] Figure 3 This is a purity diagram of the initial capping oligonucleotide primer GCG containing an R-type structure in Example 3 of the present invention. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The following example is a specific description of the synthesis method of an initial capping oligonucleotide primer GUG containing an R configuration provided by the present invention.

[0024] Example 1 Synthesis of the initial capping oligonucleotide primer GUG containing the R configuration The structural formula of the initial capping oligonucleotide primer GUG containing the R configuration is as follows:

[0025] In the formula, X=O, S or Se; R1 and R2 are each independently selected from H, Me, MOE (methoxyethyl) or F.

[0026] The method for synthesizing the initial capping oligonucleotide primer GUG with a controllable R configuration is as follows: (1) Synthesis of intermediate m7GMP-Im

[0027] A) 100g of raw material m7G (7-methylguanosine) was heated and dissolved in 1000mL of trimethyl phosphate. The reaction solution was cooled to 0±5°C, phosphorus oxychloride was slowly added dropwise, and reacted at room temperature for 4h. Then, 5000mL of 2M (concentration of triethylamine in glacial acetic acid) triethylamine-glacial acetic acid solution was added to quench the reaction, and lyophilized to obtain 154g of the target compound guanosine monophosphate triethylamine salt GMP.TEA; B) 100g of guanosine monophosphate triethylamine salt GMP.TEA, 62g of triphenylphosphine, 56g of dithiodipyridine, 41g of imidazole, and 200g of triethylamine were suspended in 700mL of DMF and reacted at room temperature for 8h. After the reaction was complete, 4M sodium perchlorate acetone solution was added, filtered, and the filter cake was thoroughly washed with acetone to obtain 183g of the target compound guanosine monophosphate imidazole salt GMP.Im; C) Weigh 183g of guanosine monophosphate imidazole salt GMP.Im and suspend it in 800mL DMF, add 100g of triethylamine phosphate, stir at room temperature for 8h, and after the reaction is complete, put it into the next step in one pot, add 92g of triphenylphosphine, 85g of dithiodipyridine, 70g of imidazole, and 200g of TEA, react at room temperature for 8h, monitor the reaction by HPLC until the raw material is ≤1%, add 6L of 4M sodium perchlorate acetone solution to the reaction solution, filter it, and wash the filter cake with acetone to obtain 225g of the target compound m7GMP-Im; (2) Synthesis of intermediate dinucleotide

[0028] Weigh 10g of S-uridine phosphoramidite monomer into a single-mouth bottle, dissolve it with dichloromethane, add 8g of 2',3'-diacetylguanosine, add 3.8g of tetrazole under nitrogen blowing, and heat to 25±2℃ for reaction. After the reaction is completed, add 20mL of dichloromethane solution containing 0.1g of iodine to the reaction solution, monitor the reaction and add 2mL of trifluoroacetic acid to remove DMTr. After the reaction is completed, spin dry, and pass through a silica gel column to obtain 12g of intermediate 1; Dissolve 12g of intermediate 1 in 70mL of acetonitrile, add 6g of phosphine reagent and 3.8g of tetrazole, stir well to react, add 20mL of dichloromethane solution containing 0.1g of iodine after monitoring the reaction, spin dry after monitoring the reaction, add 100mL of methanol and 30mL of concentrated ammonia water in a spin flask, react at 40 degrees for 4h, monitor the reaction, spin dry after the reaction, add ultrapure water, purify with ion resin, concentrate and freeze-dry to obtain 8.3g of phosphate-linked dinucleotide; (3) Synthesis of initial capping oligonucleotide primers containing R-type structures:

[0029] 8 g of dinucleotide and 7.6 g of intermediate m7GMP-Im were dissolved in 100 mL of DMF solution containing 12 g of MgCl2 and stirred at room temperature for reaction. After 24 hours, the reaction was terminated with 2 L of 0.25 M EDTA-Na solution. The starting capped oligonucleotide primer GUG containing an R-type structure was obtained by DEAE column purification.

[0030] When R1 and R2 are H and X is O, the purity of the initial capping oligonucleotide primer GUG containing an R-type structure is 99.6%, and the NMR characterization results are: 1 H NMR (400 MHz, Chloroform ) δ 12.09 (s, 1H), 11.32 (s, 1H),10.25 (s, 1H), 8.92 (s, 1H), 7.97 (s, 1H), 7.41-7.39 (d, J = 7.6 Hz, 1H),6.63 (s, 4H), 6.16-6.15 (d, J = 2.6 Hz, 1H), 5.85-5.84 (d, J = 2.9 Hz, 1H), 5.47-5.45 (d, J = 7.4 Hz, 1H), 4.75-4.73 (m, 2H), 4.55-4.47 (m, 6H), 4.44 -4.35 (m, 6H), 4.28-4.26 (m, 4H), 4.03-4.01 (m, 2H), 3.92 (s, 3H), 3.88 – 3.72 (m, 1H).

[0031] Example 2 Synthesis of the initial capping oligonucleotide primer GAG containing an R-type structure The structural formula of the initial capping oligonucleotide primer GAG containing an R-type structure is as follows:

[0032] In the formula, X=O, S or Se; R1 and R2 are each independently selected from H, Me, MOE (methoxyethyl) or F.

[0033] The synthetic route of the initial capping oligonucleotide primer GAG containing an R-type structure is as follows:

[0034] Using the intermediate dinucleotide AG and the intermediate m7GMP-Im as raw materials, the target product, the starting capping oligonucleotide primer GAG containing an R-type structure, was synthesized according to the method of Example 1.

[0035] When R1 and R2 are H and X is O, the purity of the initial capping oligonucleotide primer GAG containing the R-type structure is 99.7%, and the NMR characterization results are: 1 H NMR (400 MHz, Chloroform ) δ 12.09 (s, 1H), 10.25 (s, 1H), 8.92 (s, 1H), 8.68 (s, 1H), 7.97 (s, 1H), 7.91 (s, 1H), 6.63 (s, 4H), 6.39(s, 2H), 6.16-6.14 (m, 2H), 4.88 (m, 1H), 4.76-4.73 (m, 2H), 4.51 (m, 4H), 4.40 - 4.35 (m, 6H), 4.28 - 4.03 (m, 6H), 3.92 (s, 3H), 3.90 - 3.85 (m, 2H),, 2.65 (s, 1H), 1.43 (s, 1H).

[0036] Example 3 Synthesis of the initial capping oligonucleotide primer GCG containing an R-type structure The structural formula of the initial capping oligonucleotide primer GCG containing an R-type structure is as follows:

[0037] The synthetic route of the initial capping oligonucleotide primer GCG containing an R-type structure is as follows:

[0038] Using the intermediate dinucleotide CG and the intermediate m7GMP-Im as raw materials, the target product, the starting capping oligonucleotide primer GCG containing an R-type structure, was synthesized according to the method of Example 1.

[0039] When R1 and R2 are H and X is O, the purity of the initial capping oligonucleotide primer GCG containing the R-type structure is 98.4%, and the NMR characterization results are: 1 H NMR (400 MHz, Chloroform ) δ 12.09 (s, 1H), 10.25 (s, 1H), 8.95 (s, 1H), 8.92 (s, 1H), 7.97 (s, 1H), 6.67 (s, 2H), 6.63 (s, 4H), 6.16-6.14 (m, 2H), 5.85-5.86 (m, 1H), 5.55-5.53(d, J = 7.8 Hz, 1H), 4.75 (m, 2H), 4.51-4.47 (m, 4H), 4.45- 4.35 (m, 6H), 4.28-4.10 (m, 4H), 3.92 (s, 3H), 3.85-3.83 (m, 1H).

[0040] Example 4 Effects of different metal chelators on the reaction Referring to the method of Example 1, keeping other conditions unchanged, in the step of synthesizing the starting capping oligonucleotide primer containing an R-type structure from the dinucleotide and the intermediate m7GMP-Im, only the type of chelating agent was changed to prepare the starting capping oligonucleotide primer GUG containing an R-type structure (R1 and R2 are H, and X is O).

[0041] Table 1 Effect of different chelating agents on reaction yield serial number 1 2 3 4 5 Chelating agents EDDHA-Na EDTA-Na DTPA-Na HEDP-Na IDHA-Na Yield 83% 90% 82% 76% 73% Example 5 Effect of Chromatographic Columns with Different Fillers on Reaction Referring to the method of Example 1, other conditions were kept unchanged, and only the type of chromatography column was changed to prepare the initial capping oligonucleotide primer GUG containing an R-type structure (R1 and R2 were H, and X was O).

[0042] Table 2 Effect of different chromatography columns on reaction yield serial number 1 2 3 Chromatography column type DEAE ICE QA CM Yield 90% 75% 73% Note: The chromatography columns in the table were purchased from Nanovita Technologies.

[0043] Example 6 Effect of different solvents on the reaction Referring to the method of Example 1, other conditions were kept unchanged, and only the type of solvent was changed to prepare the initial capping oligonucleotide primer GUG containing an R-type structure (R1 and R2 were H, and X was O).

[0044] Table 3 Effect of different solvents on reaction yield serial number 1 2 3 4 5 Solvent type DMSO DMF THF Acetone ACN Yield 83% 90% 81% 85% 80% Example 7 Testing mRNA in vitro transcription yield and capping efficiency In vitro synthesis of mRNA using an initial capping oligonucleotide primer containing an R-type structure: in vitro transcription was used to synthesize mRNA, and the Clean CAP GAG (Cap1-GAG m7G(5')ppp(5')(2'OMeA)pG purchased from Shanghai Zhaowei Technology Development Co., Ltd.) of Examples 1 to 3 (R1 and R2 are H, X is O) and Comparative Example 1 were used for determination.

[0045] First, add 20 μL of sterile enzyme-free water, then add 2 μL of 10X buffer, 1 μL of NTPs, and 1 μL of cap analogs in sequence, mix well and centrifuge gently, then add 20 μL of nuclease inhibitor, 0.05U of inorganic pyrophosphatase, 50U of T7 RNA polymerase, and linearized DNA template, mix thoroughly and centrifuge gently, and incubate at 37 °C. After 2 hours, add 1U of DNase I and continue incubation at 37 °C for 30 minutes to remove the DNA template, and then purify the RNA, usually using magnetic bead purification. The purified mRNA is dissolved in sterile enzyme-free water and then quantitatively detected using Nanodrop One. After washing the streptavidin-labeled magnetic beads, incubate with the synthesized DNA probe, mRNA, and 10×RNase H reaction buffer at room temperature for 30 minutes. At the end of the incubation, place on a magnetic stand to absorb the supernatant, use an evaporating centrifuge to dry at room temperature for 40 minutes to 10 μL, and then use LC-MS analysis to determine the capping of RNA in the transcription reaction.

[0046] Table 4 shows the yield and capping efficiency of mRNA in vitro synthesis using primers from different examples serial number Yield (μg) Cap rate (%) Example 1 55 95.5 Example 2 50 96.3 Example 3 48 94.2 Comparative Example 1 50 92.3 Example 8 Effect of different substituents in the structural formula on the application effect Referring to the method of Example 7, other conditions were kept unchanged, and when the base was uracil, only the type of substituent was changed to determine the mRNA in vitro transcription yield and capping efficiency.

[0047] Table 5 Effects of different substituents on the determination of mRNA in vitro transcription yield and capping efficiency serial number X <![CDATA[R1]]> <![CDATA[R2]]> Yield (μg) Cap rate (%) 1 O H H 55 95.5 2 S H H 52 92.3 3 Se H H 51 93.7 4 O H Me 50 91.8 5 O H MOE 53 92.4 6 O H F 52 90.6 7 O Me H 50 91.3 8 O MOE H 52 91.7 9 O F H 51 92.6 In summary, the raw materials involved in the method of the present invention are all commercially available, easy to obtain, and the synthesis route is short, the operation is simple, and it is suitable for large-scale production. In addition, the starting capping oligonucleotide primer containing an R-type structure discovered by the present invention is a new substance, and there is currently no literature or patent report. The discovery of this substance provides a platform for the research of mRNA series and the development of new products, and broadens the scope of mRNA research. The synthesis of the starting capping oligonucleotide primer containing an R-type structure is used for mRNA capping under the T7 RNA polymerase system. Compared with the existing cap structure analog Cleancap, the mRNA produced by the starting capping oligonucleotide primer containing an R-type structure of the present invention has lower immunogenicity.

[0048] All features defined in the present invention in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0049] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. An initial capping oligonucleotide primer containing an R configuration, characterized in that: The structure of the initial capping oligonucleotide primer containing the R configuration is shown in Formula I: In formula I, R1 and R2 are each independently selected from H, Me, methoxyethyl or F; X is selected from O, S or Se; Base is selected from adenine base, guanine base, cytosine base, 5-methylcytosine base or uracil.

2. The method for synthesizing an initial capping oligonucleotide primer containing an R configuration as claimed in claim 1, characterized in that: The synthesis method comprises: Dissolve the dinucleotide and the intermediate m7GMP-Im in an organic solvent A containing MgCl2 and stir the reaction at room temperature; After the reaction is completed, the reaction is terminated and purified to obtain an initial capped oligonucleotide primer containing an R configuration as shown in Formula I.

3. The synthesis method according to claim 2, characterized in that The termination reaction is terminated by adding EDTA-Na solution.

4. The synthesis method according to claim 2, characterized in that The purification was performed using a DEAE column.

5. The synthesis method according to claim 2, characterized in that The organic solvent A is selected from DMF.

6. The synthesis method according to claim 2, characterized in that The method for synthesizing the dinucleotide comprises: S-type phosphoramidite monomer and 2',3'-diacetylguanosine are placed in an organic solvent B, tetrazole is added under an inert gas atmosphere, and the temperature is raised to 25±2°C for reaction; after the reaction is completed, a dichloromethane solution containing iodine is added to the reaction solution, and after the reaction is monitored, trifluoroacetic acid is added to remove DMTr, and after the reaction is monitored, the solution is spin-dried and passed through a silica gel column to obtain intermediate 1; Dissolve intermediate 1 in organic solvent C, add phosphine reagent and tetrazole, stir well to react, add iodine-containing dichloromethane solution after monitoring the reaction, spin dry after monitoring the reaction, add methanol and concentrated ammonia water in a spin flask, react at 40 degrees, monitor the reaction, spin dry after the reaction, add ultrapure water, purify with ion resin, concentrate and freeze-dry to obtain phosphate-linked dinucleotides.

7. The synthesis method according to claim 6, characterized in that The organic solvent B is selected from dichloromethane; the organic solvent C is selected from acetonitrile.

8. The synthesis method according to any one of claims 2 to 7, characterized in that The synthesis method of the intermediate m7GMP-Im comprises: A) m7G was heated and dissolved in trimethyl phosphate, the reaction solution was cooled to 0±5°C, phosphorus oxychloride was slowly added dropwise, reacted at room temperature, triethylamine-glacial acetic acid solution was added to quench the reaction, and the target compound guanosine monophosphate triethylamine salt GMP.TEA was obtained by freeze-drying; B) guanosine monophosphate triethylamine salt GMP.TEA, triphenylphosphine, dithiodipyridine, imidazole and triethylamine are suspended in an organic solvent D, reacted at room temperature, and after the reaction is complete, sodium perchlorate acetone solution is added, filtered, and the filter cake is fully washed with acetone to obtain the target compound guanosine monophosphate imidazole salt GMP.Im; C) Guanosine monophosphate imidazolate GMP.Im was suspended in organic solvent E, triethylamine phosphate was added, and the mixture was stirred at room temperature. After the reaction was complete, the mixture was used in the next step in one pot, triphenylphosphine, dithiodipyridine, imidazole, and TEA were added, and the mixture was reacted at room temperature. The reaction was monitored by HPLC until the starting material was ≤1%. Sodium perchlorate acetone solution was added to the reaction solution, and the mixture was filtered with suction. The filter cake was washed thoroughly with acetone to obtain the target compound m7GMP-Im.

9. The synthesis method according to claim 8, characterized in that The organic solvent D and the organic solvent E are both selected from DMF.

10. Use of the R-containing initial capping oligonucleotide primer according to claim 1 in mRNA synthesis under a T7 RNA polymerase system.

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