Initial capping oligonucleotide primer containing HNA structure as well as preparation method and application of initial capping oligonucleotide primer

Abstract

The invention discloses an initial capping oligonucleotide primer containing an HNA structure and a preparation method and application of the initial capping oligonucleotide primer, the structure of the initial capping oligonucleotide primer containing the HNA structure is shown in the formula I. In the formula I, R1 and R2 are independently selected from H, Me, MOE 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. According to the invention, the cap structure is optimized, the found initial capping oligonucleotide primer containing the HNA structure belongs to a new substance, the discovery of the substance provides platform significance for the research of mRNA series and the development of new products, and the range of mRNA research is widened. The initial capping oligonucleotide primer containing the HNA structure can be used for mRNA capping under a T7 RNA polymerase system.

Description

Technical Field

[0001] The present invention relates to an initial capping oligonucleotide primer, in particular to an initial capping oligonucleotide primer containing an HNA structure and a preparation method and application thereof. Background Art

[0002] In eukaryotic cells, in addition to identifying the start of protein synthesis, the 5'-end cap structure also acts as a protective group for exonuclease cleavage from 5' to 3', that is, it has a degradation-resistant effect against 5'-exonuclease. At the same time, during protein synthesis, the cap structure is also 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.

[0003] Currently, the immunogenicity of mRNA is still the focus of this research field. The 5' cap structure is the key structure to reduce immunogenicity, so more cap structures need to be designed to better reduce immunogenicity.

[0004] At present, there are related cap patents to optimize the structure, which can play a certain role. However, it is still the trend of scientific research and application to continuously optimize the structure of cap analogs and find new cap analogs with lower immunogenicity than the existing caps. Summary of the invention

[0005] The purpose of the present invention is to provide an initial capping oligonucleotide primer containing an HNA structure, and a preparation method and application thereof. The discovered initial capping oligonucleotide primer containing an HNA structure is a new substance, and the initial capping oligonucleotide primer containing an HNA structure can be used for mRNA capping under a T7 RNA polymerase system.

[0006] Compared with the existing cap structure analog Clean cap, the mRNA produced by the initial capping oligonucleotide primer containing the HNA structure of the present invention has lower immunogenicity.

[0007] In order to achieve the above object, the present invention provides an initial capping oligonucleotide primer containing an HNA structure, and the structure of the initial capping oligonucleotide primer containing an HNA structure is shown in Formula I:

[0008] In formula I, R1 and R2 are each independently selected from H, Me, methoxyethyl (MOE) 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.

[0009] Another object of the present invention is to provide a method for synthesizing the initial capping oligonucleotide primer containing the HNA structure, the method comprising:

[0010] 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 capping oligonucleotide primer containing an HNA structure as shown in Formula I.

[0011] Preferably, the terminating reaction is performed by adding EDTA-Na solution to terminate the reaction.

[0012] Preferably, the purification uses a DEAE column.

[0013] Preferably, the organic solvent A is selected from DMF.

[0014] Preferably, the method for synthesizing the dinucleotide comprises:

[0015] HNA 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, an iodine-containing dichloromethane solution is added to the reaction solution, after the monitoring reaction is completed, trifluoroacetic acid is added to remove DMTr, after the monitoring reaction is completed, the solution is spin-dried, and the intermediate 1 is passed through a silica gel column; the intermediate 1 is dissolved in an organic solvent C, a phosphine reagent and tetrazole are added, and the reaction is stirred thoroughly, after the monitoring reaction is completed, an iodine-containing dichloromethane solution is added, after the monitoring reaction is completed, the solution is spin-dried, methanol and concentrated ammonia water are added to a spin flask, the reaction is carried out at 40°C, the reaction is monitored, after the reaction is completed, the solution is spin-dried, ultrapure water is added, the solution is purified by ion resin, and the solution is concentrated and freeze-dried to obtain a dinucleotide connected by a phosphate bond.

[0016] Preferably, the organic solvent B is selected from dichloromethane; and the organic solvent C is selected from acetonitrile.

[0017] Preferably, the synthesis method of the intermediate m7GMP-Im comprises:

[0018] 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.

[0019] Preferably, the organic solvent D and the organic solvent E are both selected from DMF.

[0020] Another object of the present invention is to provide the use of the initial capping oligonucleotide primer containing the HNA structure in mRNA synthesis under the T7 RNA polymerase system.

[0021] The initial capping oligonucleotide primer containing HNA structure of the present invention and its preparation method and application have the following advantages: The present invention optimizes the cap structure and provides an initial capping oligonucleotide primer containing an HNA structure and a preparation method thereof. The initial capping oligonucleotide primer containing an HNA structure is a new substance, and there is no literature or patent report at present. The discovery of this substance provides a platform significance for the research of mRNA series and the development of new products, and broadens the scope of mRNA research. The initial capping oligonucleotide primer containing an HNA structure can be used for mRNA capping under the T7 RNA polymerase system.

[0022] Compared with the existing cap structure analog Clean cap, the mRNA produced by the initial capping oligonucleotide primer containing the HNA structure of the present invention has lower immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a purity chart of the HNA-containing initial capping oligonucleotide primer GUG prepared in Example 1 of the present invention.

[0024] Figure 2 This is a purity chart of the HNA-containing initial capping oligonucleotide primer GAG prepared in Example 2 of the present invention.

[0025] Figure 3 This is a purity chart of the HNA-containing initial capping oligonucleotide primer GCG prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0026] 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.

[0027] The following example is a specific description of the method for synthesizing an initial capping oligonucleotide primer containing an HNA structure provided by the present invention.

[0028] Example 1 Synthesis of the initial capping oligonucleotide primer GUG containing HNA structure The structural formula of the initial capping oligonucleotide primer GUG containing the HNA structure is as follows:

[0029] In the formula, X=O, S or Se; R1 and R2 are each independently selected from H, Me, MOE (methoxyethyl) or F; Uridine is a uracil base.

[0030] The synthesis method of the initial capping oligonucleotide primer GUG containing the HNA structure is as follows: (1) Synthesis of intermediate m7GMP-Im

[0031] 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 152g 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 180g of the target compound guanosine monophosphate imidazole salt GMP.Im; C) Weigh 180g 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 220g of the target compound m7GMP-Im; (2) Synthesis of intermediate dinucleotide

[0032] Weigh 10g of HNA 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, pass through a silica gel column to obtain 12g of intermediate 1, whose chemical structure is as follows:

[0033] 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 8g of dinucleotide linked by phosphate bond; (3) Synthesis of initial capping oligonucleotide primer containing HNA structure:

[0034] 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 initial capping oligonucleotide primer GUG containing HNA structure was obtained by DEAE column purification. The purity results are shown in Figure 1 .

[0035] When R1 and R2 are H and X is O, the purity of the synthesized initial capping oligonucleotide primer GUG containing HNA structure is 99.8%, and the NMR characterization results are: 1H NMR (400 MHz, Chloroform ) δ 12.08 (s, 1H), 11.32 (s, 1H), 10.24 (s, 1H), 8.98-8.95 (d, J=9.9 Hz, 1H), 8.90 (s, 1H) 7.96 (s, 1H),7.45-7.44 (d, J = 5.6 Hz, 1H), 6.64 (s, 4H), 6.19-6.13 (m, 2H), 5.53-5.51 (d,J = 8.9 Hz, 1H), 4.79 -4.72 (m, 2H), 4.54 (m, 4H), 4.50-4.37 (m, 5H), 4.26(m, 4H), 4.01 (m, 2H), 3.92 (s, 3H), 3.88-3.72 (m, 3H), 3.52-3.49 (m, 2H).

[0036] Example 2 Synthesis of the initial capping oligonucleotide primer GAG containing HNA structure The structural formula of the initial capping oligonucleotide primer GAG containing the HNA structure is as follows:

[0037] In the formula, X=O, S or Se; R1 and R2 are each independently selected from H, Me, MOE or F; adenosine is adenine base.

[0038] The synthetic route of the initial capping oligonucleotide primer GAG containing the HNA structure is as follows:

[0039] The intermediate dinucleotide AG and the intermediate m7GMP-Im were used as raw materials, and the target product, i.e., the starting capping oligonucleotide primer GAG containing the HNA structure, was synthesized according to the method of Example 1. The purity results are shown in Figure 2 .

[0040] When R1 and R2 are H and X is O, the purity of the synthesized initial capping oligonucleotide primer GAG containing HNA structure is 98.0%, and the NMR characterization results are: 1H NMR (500 MHz, Chloroform ) δ 12.08 (s, 1H), 10.23 (s,1H), 8.97-8.94 (d, J = 9.9 Hz, 1H), 8.16 (s, 1H), 8.05 (s, 1H), 7.97 (s, 1H),7.09 (s, 2H), 6.63 (s, 4H), 6.19-6.13 (m, 2H), 4.79-4.72 (m, 2H), 4.54 (m,4H), 4.50-4.37 (m, 8H), 4.28-4.03 (m, 4H), 3.92 (s, 3H), 3.80-3.60(m, 3H),3.51-3.47 (m, 2H).

[0041] Example 3 Synthesis of the initial capping oligonucleotide primer GCG containing HNA structure The structural formula of the initial capping oligonucleotide primer GCG containing the HNA structure is as follows:

[0042] In the formula, X=O, S or Se; R1 and R2 are each independently selected from H, Me, MOE or F; cytosine is a cytosine base.

[0043] The synthetic route of the initial capping oligonucleotide primer GCG containing the HNA structure is as follows:

[0044] The intermediate dinucleotide CG and the intermediate m7GMP-Im were used as raw materials, and the target product, i.e., the starting capping oligonucleotide primer GCG containing the HNA structure, was synthesized by referring to the experimental method of Example 1. The purity results are shown in Figure 3 .

[0045] When R1 and R2 are H and X is O, the purity of the synthesized initial capping oligonucleotide primer GCG containing HNA structure is 98.3%, and the NMR characterization results are: 1H NMR (500 MHz, Chloroform ) δ 12.07 (s, 1H), 10.24 (s,1H), 8.95-8.92 (d, J = 9.9 Hz, 1H), 8.90 (s, 1H), 7.97 (s, 1H), 6.67(s, 2H),6.63 (s, 4H), 6.19-6.13 (m, 2H), 5.55-5.52 (d, J=9.6 Hz, 1H), 4.79-4.72 (m,2H), 4.40-4.51 (m, 6H), 4.36-4.32 (m, 4H), 4.28-4.03 (m, 6H), 3.91 (s, 3H),3.80-3.60 (m, 3H), 3.43-3.42 (m, 2H).

[0046] 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 HNA 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 HNA structure (R1 and R2 are H, X is O). The results are shown in Table 1.

[0047] 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% 91% 81% 79% 75% Example 5 Effect of Chromatographic Columns with Different Fillers on Reaction Referring to the method of Example 1, keeping other conditions unchanged, only changing the type of chromatography column, the initial capping oligonucleotide primer GUG containing HNA structure (R1, R2 are H, X is O) was prepared. The results are shown in Table 2.

[0048] Table 2 Effect of different chromatography columns on reaction yield serial number 1 2 3 Chromatography column type DEAE ICE QA CM Yield 91% 79% 70% Note: The chromatography columns in the table were purchased from Nanovita Technologies.

[0049] 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 HNA structure (R1 and R2 are H, and X is O). The results are shown in Table 3.

[0050] Table 3 Effect of different solvents on reaction yield serial number 1 2 3 4 5 Solvent type DMSO DMF THF Acetone ACN Yield 85% 91% 83% 87% 81% Example 7 Testing mRNA in vitro transcription yield and capping efficiency In vitro synthesis of mRNA using an initial capping oligonucleotide primer containing an HNA structure: in vitro transcription was used to synthesize mRNA, and Examples 1 to 3 (R1 and R2 are both H, and X is both O) and the comparative example Clean CAP GAG (Cap1-GAG m7G(5')ppp(5')(2'OMeA)pG, purchased from Shanghai Zhaowei Technology Development Co., Ltd.) were used for determination.

[0051] First, add 20 μL of sterile enzyme-free water, then add 2 μL of 10X buffer, 1 μL of each 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. The results are shown in Table 4.

[0052] Table 4 mRNA in vitro transcription yield and capping efficiency of Examples 1 to 3 and Comparative Example 1 serial number Yield (μg) Cap rate (%) Example 1 55 94.5 Example 2 50 97.3 Example 3 48 95.2 Comparative Example 1 50 92.0 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 measure the mRNA in vitro transcription yield and capping efficiency. The results are shown in Table 5.

[0053] 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 94.5 2 S H H 53 93.5 3 Se H H 51 92.6 4 O H Me 52 92.9 5 O H MOE 54 93.1 6 O H F 50 91.9 7 O Me H 51 92.8 8 O MOE H 53 93.2 9 O F H 50 91.8 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 HNA structure discovered by the present invention is a new substance, and there is no literature or patent report at present. The discovery of this substance provides a platform significance 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 HNA structure is used for mRNA capping under the T7RNA polymerase system. Compared with the existing cap structure analog Clean cap, the starting capping oligonucleotide primer containing HNA structure of the present invention produces mRNA with lower immunogenicity.

[0054] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0055] 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 HNA structure, characterized in that: The structure of the initial capping oligonucleotide primer containing the HNA structure 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. A method for synthesizing an initial capping oligonucleotide primer containing an HNA structure 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 capping oligonucleotide primer containing an HNA structure 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: HNA 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; The intermediate 1 is dissolved in an organic solvent C, and a phosphine reagent and tetrazole are added and stirred to react. After monitoring the reaction, an iodine-containing dichloromethane solution is added, and the reaction is dried by spin drying. Methanol and concentrated ammonia water are added to a spin flask, and the reaction is carried out at 40° C. The reaction is monitored, and the reaction is dried by spin drying after the reaction is completed. Ultrapure water is added, and the reaction is purified by ion resin, and concentrated and freeze-dried to obtain a dinucleotide connected by a phosphate bond.

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 initial capping oligonucleotide primer containing an HNA structure as claimed in claim 1 in mRNA synthesis under a T7 RNA polymerase system.

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