CN115109110A - Initial capped oligonucleotide primer containing hexa-membered sugar ring structure and preparation method and application thereof - Google Patents
Initial capped oligonucleotide primer containing hexa-membered sugar ring structure and preparation method and application thereof Download PDFInfo
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- CN115109110A CN115109110A CN202210716077.3A CN202210716077A CN115109110A CN 115109110 A CN115109110 A CN 115109110A CN 202210716077 A CN202210716077 A CN 202210716077A CN 115109110 A CN115109110 A CN 115109110A
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- unsubstituted
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- sugar ring
- alkyl
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- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- BCNZYOJHNLTNEZ-UHFFFAOYSA-N tert-butyldimethylsilyl chloride Chemical compound CC(C)(C)[Si](C)(C)Cl BCNZYOJHNLTNEZ-UHFFFAOYSA-N 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides an initial capped oligonucleotide primer containing a hexatomic sugar ring structure, and a preparation method and application thereof, wherein the molecular formula of the initial capped oligonucleotide primer containing the hexatomic sugar ring structure is m7 GpppA 2’OMe pG, wherein m7 G is N 7 -methylated guanosine or any guanosine analogue, a being any natural, modified or non-natural adenosine or any adenosine analogue. The mRNA synthesized by the initial capped oligonucleotide primer containing the six-membered sugar ring structure has lower immunogenicity and higher proteinTranslation efficiency and higher stability.
Description
Technical Field
The invention relates to the technical field of chemistry and biological engineering, in particular to an initial capped oligonucleotide primer containing a hexatomic sugar ring structure and a preparation method and application thereof.
Background
In eukaryotic cells, the 5' end of most messenger rnas (mrnas) is blocked, or "capped," which contains a 5' -5' triphosphate linkage between two nucleoside moieties and a 7-methyl group on the distal guanine ring, capping of the mRNA facilitates its normal function in the cell. The synthesis of mRNA by in vitro transcription has become an important tool for the introduction of foreign genes and the expression of proteins, and is widely used in the treatment and prevention of diseases, enabling workers to prepare RNA molecules that exhibit the right expression in various biological applications. Such applications include research applications and commercial production of polypeptides, for example, production of polypeptides comprising "unnatural" amino acids at specific sites in cell-free translation systems, or production of polypeptides in culture that require post-translational modification for their activity or stability. In the latter system, synthesis takes significantly longer and therefore more protein is produced. The in vitro transcription yield of mRNA and 5' capping of the analog is a key process in the mRNA preparation process. The prior art is applied to a system for capping by an mRNA chemical method, and higher efficiency cannot be obtained.
Patent CN201680067458.6 reports compositions and methods for synthesizing 5' -capped RNA. Wherein the initial capped oligonucleotide primer has the general form m 7 Gppp[N 2’OMe ] n [N] m Wherein m is 7 G is N 7 -methylated guanosine or any guanosine analogue, N being any natural, modified or non-natural nucleoside, "N" can be any integer from 0 to 4 and "m" can be an integer from 1 to 9.Cleancap belongs to Cap1, and uses dimer (m) with ARCA 7 GpppG) initiates transcription of T7 differently, CleanCap uses trimer (m) 7 GpppAmG) initiates transcription of T7. The method has high yield, 4mg of capped RNA is prepared in a transcription reaction system per ml, the capping efficiency can reach 90%, and the immunogenicity of a transcription product is lower than that of ARCA.
Disclosure of Invention
In order to further improve the expression efficiency of target mRNA in cells, prolong the half-life of a medicament and obviously improve the stability of the mRNA, the application provides an initial capping oligonucleotide primer containing a six-membered sugar ring structure, a preparation method and an application thereof, and compared with a five-membered sugar ring, a cap analogue of a Hexitol Nucleic Acid (HNA) structure contained in the initial capping oligonucleotide primer containing the six-membered sugar ring structure has a spatial structure advantage and can be better combined with a transcription factor protein (elF4E) related to translation of the mRNA; meanwhile, the cap analogue with a non-natural structure resists the degradation capability of endogenous incision enzyme and exonuclease, improves the stability of RNA, prolongs the half-life period of the medicament and obviously improves the stability of mRNA; and the cap analogue of the hexitol nucleic acid structure can improve the expression efficiency of the target mRNA in the cell.
An initial capped oligonucleotide primer comprising a six-membered sugar ring structure, comprising the structure:
wherein, X 1 、X 2 And X 3 Are each independently O, CH 2 Or NH;
Y 1 、Y 2 and Y 3 Each independently is O, S, Se or BH 3 ；
R a Is composed of
Rb is
And when Ra is
When Rb is
R 1 、R 2 And R 3 、R 6 、R 7 、R 8 Independently hydrogen, hydroxy, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, substituted or unsubstituted NH-aralkyl;
R 4 and R 5 Independently H, OH, alkyl, O-alkyl, halogen;
B 1 and B 2 Independently, a natural, or modified, or non-natural nucleobase.
The preparation method of the initial capped oligonucleotide primer containing the six-membered sugar ring structure comprises the following steps: (1) synthesis of intermediate K: synthesizing a compound A from sorbitol, and sequentially carrying out reactions such as glycosylation, phosphorylation, monophosphorylation, diphosphorylation, methylation of N7, and imidazolation of polyphosphoric acid on the basis of the compound A to synthesize an intermediate K; (2) preparation of a phosphate-linked dinucleotide: coupling a phosphoramidite monomer and a disubstituted nucleoside monomer under the action of tetrazole to form a first phosphate ester bond, removing a protecting group through acid action, introducing a second phosphoric acid, and finally hydrolyzing to obtain a dinucleotide connected with the phosphate ester bond; (3) synthesis of initial capped oligonucleotide primers containing six-membered sugar ring structures: reacting the intermediate K with dinucleotide connected with a phosphate bond to prepare an initial capped oligonucleotide primer containing a six-membered sugar ring structure;
the structural formula of the phosphoramidite monomer is as follows:
B 3 and B 4 Independently, a natural, or modified, or non-natural nucleobase.
The above disubstituted nucleoside monomer is selected from
Any one of the above.
The preparation method of the initial capped oligonucleotide primer containing the hexahydric sugar ring structure specifically comprises the following steps:
(1) synthesis of intermediate F:
s1, weighing 1, 5-anhydride-D-sorbitol, dissolving in DMF, adding benzaldehyde dimethyl acetal and p-toluenesulfonic acid into a reaction solution at room temperature, heating the reaction solution to 60 +/-5 ℃, and stirring for 10 hours. TLC monitors the reaction to completion, and saturated NaHCO is added after the reaction is finished 3 Quenching the aqueous solution for reaction, extracting the aqueous solution for three times by using a proper amount of ethyl acetate, combining organic phases, washing the organic phases by using saturated saline solution, drying the organic phases by using anhydrous sodium sulfate, and crystallizing the organic phases by using normal hexane to obtain a target compound A;
s2, weighing the compound A, dissolving the compound A in anhydrous pyridine, dropwise adding p-toluenesulfonyl chloride into the reaction solution under ice bath, and reacting for 7 days at room temperature. TLC monitors the reaction to be complete, and the product is recrystallized by acetone to obtain the target compound B.
S3, weighing the compound B, dissolving the compound B in dichloromethane, dropwise adding 3M sodium methoxide methanol solution into the reaction solution in ice bath, and heating to room temperature for reaction for 20 hours after dropwise adding. TLC monitoring reaction is carried out until complete, water is added after reaction is finished, reaction liquid is extracted for 2 times by dichloromethane, organic phases are combined, the organic phases are washed by saturated saline water and dried by anhydrous sodium sulfate, and n-heptane is used for crystallization to obtain a target compound C;
s4, weighing the compound C, 2-amino-6-chloropurine, aliquat 336 and potassium carbonate, uniformly dispersing in hexamethylphosphoric triamide, heating the reaction solution to 90 +/-5 ℃ in a nitrogen atmosphere, and stirring for 3 hours. The reaction was monitored by TLC to completion, and the reaction was cooled and poured into ice water, followed by stirring at room temperature for 1 hour and suction filtration. And purifying the filter cake by using a silica gel column to obtain a target compound D.
S5, weighing the compound D and the DABCO, suspending in 1M NaOH, heating to 90 +/-5 ℃, and stirring for 2 hours. And monitoring the reaction to be complete by TLC, cooling the reaction solution, adjusting the pH to be neutral by using 1M HCl aqueous solution, performing suction filtration, washing with water, and drying to obtain a target compound E.
S6, weighing the compound, uniformly dispersing the compound in acetic acid (80% aqueous solution), heating the reaction solution to 80 +/-5 ℃, and stirring for 2 hours. TLC monitors the reaction to be complete, and after concentrating and removing acetic acid, the crude product is purified by silica gel column to obtain the target compound F.
S7, dissolving the compound F in trimethyl phosphate, cooling the reaction liquid to 0 +/-5 ℃, slowly dropwise adding phosphorus oxychloride, reacting at a low temperature for 4 hours, adding 2M ammonium acetate solution for quenching reaction, and purifying by reversed phase chromatography to obtain a target compound G.
S8, suspending the compound G, triphenylphosphine, dipyridyl disulfide, imidazole and TEA in DMF, reacting at room temperature for 8 hours, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing a filter cake by acetone to obtain a target compound H;
s9, weighing a target compound H, suspending the target compound H in DMF (dimethyl formamide), adding tributylamine phosphate and manganese chloride, stirring at room temperature for 6 hours, monitoring the reaction by HPLC (high performance liquid chromatography) until the raw material is less than or equal to 1%, and pouring the reaction liquid into water to obtain a crude product water solution of the compound I. Slowly dropwise adding dimethyl sulfate, adjusting the pH value to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by using HPLC, and purifying by using ion chromatography after the reaction is finished to obtain a target compound J;
s10, suspending the compound J, triphenylphosphine, dipyridyl disulfide, imidazole and TEA in DMF, reacting at room temperature for 8 hours, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing a filter cake by acetone to obtain a target compound K;
(2) preparation of a phosphate-linked dinucleotide:
weighing 2 ' OMe-rA phosphoramidite monomer, dissolving in dichloromethane, adding 2 ', 3 ' acetyl guanosine, cooling to 25 +/-2 ℃, adding tetrazole under nitrogen blowing, and heating to 25 +/-2 ℃ for reaction. After the monitoring reaction is finished, adding an iodopyridine solution into the reaction solution, performing spin drying after the monitoring reaction is finished, dissolving the concentrated ointment into dichloromethane, adding trifluoroacetic acid, performing spin drying after the monitoring reaction is finished, pulping petroleum ether/dichloromethane according to a certain proportion, and filtering to obtain an intermediate G1; dissolving G1 in acetonitrile, adding a phosphine reagent and tetrazole, fully stirring for reaction, after monitoring reaction is finished, adding an iodopyridine solution into a reaction solution, after monitoring reaction is finished, carrying out spin-drying, adding methanol and concentrated ammonia water into a spinner bottle, reacting at room temperature for 4 hours, monitoring reaction, after reaction is finished, carrying out spin-drying, adding ultrapure water, entering a reverse ion permeation device, washing, concentrating, and freeze-drying to obtain the dinucleotide connected with the phosphate bond;
(3) synthesis of initial capped oligonucleotide primers containing six-membered sugar ring structures:
dissolving the intermediate K in MnCl 2 And added to a DMF solution of A-G-P. The reaction was stirred at room temperature. After 24 hours, the reaction was stopped with 0.25M EDTA solution. The mixture was loaded onto a DEAE Sephadex column (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting the elution product with HPLC purity more than 97%, concentrating the separation liquid, loading the separation liquid to strong anion resin, performing linear gradient elution by using 0-1.0M sodium acetate eluent, collecting the elution product with HPLC purity more than 98.5%, combining the high-purity eluents, removing residual sodium acetate solution through nanofiltration equipment, and concentrating to obtain the initial capped oligonucleotide primer containing the hexahydric sugar ring structure of the target product.
The initial capping oligonucleotide primer containing the six-membered sugar ring structure is used for mRNA capping under a T7 RNA polymerase system. T7 RNA polymerase is a DNA-dependent RNA polymerase with high specificity for the bacteriophage T7 promoter sequence. This enzyme synthesizes large amounts of RNA from the insertion of the T7 promoter into DNA downstream of the transcription vector. The IVT reaction system catalyzed by T7 RNA polymerase is the most mature mRNA preparation system at present.
Typically 20ul of IVT (in vitro transcription) reaction system contains 50U of T7 RNA polymerase, and 1ul of cap analogue (100mM) is used to obtain optimal transcription yield and capping efficiency.
The invention provides an initial capped oligonucleotide primer containing a hexabasic sugar ring structure, which is suitable for mRNA produced by using a DNA sequence as a template by an in vitro co-transcription method, wherein the DNA sequence can be derived or modified from virus, animals, plants and other species, and the produced mRNA has higher protein translation efficiency and better stability.
Compared with the prior art, the invention has the following advantages:
compared with the prior cap structure analog Cleancap, the mRNA produced by the initial capping oligonucleotide primer containing the six-membered sugar ring structure has higher protein translation efficiency and better stability.
Drawings
FIG. 1 is a cell phenotype diagram of examples 1 to 3 and comparative examples 1 to 2;
FIG. 2 is a graph showing the fluorescence statistics of examples 1 to 3 and comparative examples 1 to 2;
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The raw material names and sources used in each example are given in table 1 below:
TABLE 1
The intermediate K used in each of the following examples was prepared by the following steps:
s1, weighing 5g of 1, 5-anhydride-D-sorbitol, dissolving in 50mL of DMF, adding benzaldehyde dimethyl acetal (2.0eq.) and p-toluenesulfonic acid (0.2eq.) into the reaction solution at room temperature, heating the reaction solution to 60 ℃, and stirring for 10 hours. TLC monitored the reaction to completion, after the reaction was complete 200mL of saturated NaHCO was added 3 Quenching the aqueous solution for reaction, extracting the aqueous solution for three times by using a proper amount of ethyl acetate, combining organic phases, washing the organic phases by using saturated saline solution, drying the organic phases by using anhydrous sodium sulfate, and crystallizing the organic phases by using normal hexane to obtain a target compound A;
s2, weighing 5g of the compound A, dissolving the compound A in 60mL of anhydrous pyridine, dropwise adding p-toluenesulfonyl chloride (5.5eq.) into the reaction solution in ice bath, and reacting at room temperature for 7 days. TLC monitors the reaction to be complete, and the product is recrystallized by acetone to obtain the target compound B.
S3, weighing 2.8g of the compound B, dissolving the compound B in 50mL of dichloromethane, dropwise adding 20mL of 3M sodium methoxide methanol solution into the reaction solution under ice bath, and heating to room temperature for reaction for 20 hours after dropwise addition. TLC monitoring reaction is carried out until the reaction is complete, 50mL of water is added after the reaction is finished, the reaction solution is extracted for 2 times by using dichloromethane, then organic phases are combined, the organic phases are washed by saturated saline solution and dried by using anhydrous sodium sulfate, and the target compound C is obtained by using n-heptane crystallization;
s4, weighing 2.1g of the compound C, 2-amino-6-chloropurine (2.1eq.), aliquat 336(1.0eq.) and potassium carbonate (2.0eq.) and uniformly dispersing in 35mL of hexamethylphosphoric triamide, and heating the reaction solution to 90 ℃ under a nitrogen atmosphere and stirring for 3 hours. The reaction was monitored by TLC to completion, and the reaction solution was cooled and poured into 350mL of ice water, followed by stirring at room temperature for 1 hour and suction filtration. And purifying the filter cake by using a silica gel column to obtain a target compound D.
S5, 1.2g of compound D and DABCO (2.9eq.) are weighed and suspended in 1M NaOH (50mL), and the mixture is heated to 90 ℃ and stirred for 2 hours. And monitoring the reaction to be complete by TLC, cooling the reaction solution, adjusting the pH to be neutral by using 1M HCl aqueous solution, performing suction filtration, washing with water, and drying to obtain a target compound E.
S6, weighing 1.2g of the compound, uniformly dispersing the compound in 20mL of acetic acid (80% aqueous solution), heating the reaction solution to 80 ℃, and stirring for 2 hours. TLC monitors the reaction to be complete, and after concentrating and removing acetic acid, the crude product is purified by silica gel column to obtain the target compound F.
S7, dissolving 2G of the compound F in 20ml of trimethyl phosphate, cooling the reaction liquid to 0 ℃, slowly dropwise adding 1.5eq of phosphorus oxychloride, reacting at a low temperature for 4 hours, adding 2M ammonium acetate solution, quenching, and purifying by reversed phase chromatography to obtain the target compound G.
S8, suspending 2G of the compound G and 2eq of triphenylphosphine, 2eq of dithiodipyridine, 8eq of imidazole and 1eq of TEA in 20mL of DMF, reacting at room temperature for 8 hours, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M of sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing a filter cake by acetone to obtain a target compound H;
s9, weighing 2g of target compound H, suspending the target compound H in 20mL of DMF (dimethyl formamide), adding 3eq of tributylamine phosphate and 8eq of manganese chloride, stirring at room temperature for 6 hours, monitoring the reaction by HPLC (high performance liquid chromatography) until the raw material is less than or equal to 1%, and pouring the reaction liquid into 200mL of water to obtain a crude water solution of the compound I. Slowly dropwise adding 8eq of dimethyl sulfate, adjusting the pH value to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by using HPLC, and purifying by using ion chromatography after the reaction is finished to obtain a target compound J;
s10, suspending 1g of the compound J and 2eq of triphenylphosphine, 2eq of dithiodipyridine, 8eq of imidazole and 1eq of TEA in 10mL of DMF, reacting at room temperature for 8 hours, monitoring the reaction by HPLC until the raw materials are less than or equal to 1%, adding 4M of sodium perchlorate acetone solution into the reaction solution, performing suction filtration, and fully washing a filter cake by acetone to obtain a target compound K;
the specific reaction scheme of intermediate K is as follows equation (1):
the synthetic route of A-G-P used in Synthesis example 1 was: weighing 5kg of 2 ' OMe-rA phosphoramidite monomer in a single-mouth bottle, dissolving with 50L of dichloromethane, adding 2.73kg of 2 ', 3 ' acetyl guanosine, cooling to 25 +/-2 ℃, adding 880g of tetrazole under the action of nitrogen blowing, and heating to 25 +/-2 ℃ for reaction. Monitoring the reaction, adding 1.2eq of iodopyridine solution into the reaction solution, monitoring the reaction, performing spin-drying, dissolving the concentrated ointment into 4L of dichloromethane, adding 1.1eq of trifluoroacetic acid, monitoring the reaction, performing spin-drying, pulping petroleum ether/dichloromethane according to a certain ratio, and filtering to obtain an intermediate G1; dissolving G1 in 4L acetonitrile, adding 1.2eq phosphine reagent and 1.2eq tetrazole, fully stirring for reaction, monitoring after the reaction is finished, adding 1.2eq iodopyridine solution into the reaction solution, monitoring after the reaction is finished, spin-drying, adding 3L methanol and 3L strong ammonia water into a spinner bottle, reacting for 4 hours at room temperature, monitoring for reaction, spin-drying after the reaction is finished, adding 20L ultrapure water, entering a reverse ion permeation device, washing, concentrating, and freeze-drying to obtain a target compound A-G-P, wherein the reaction route flow is as follows equation (2):
synthesis of A-G-P substituted with six-membered sugar Ring used in Synthesis example 2 referring to the A-G-P synthesis method of example 1, the scheme of the reaction scheme of A-G-P substituted with six-membered sugar Ring, the following equation (3):
example 1: synthesis method of initial capped oligonucleotide primer containing hexa-membered sugar ring structure by taking intermediate K and A-G-P as raw materials
Dissolving the intermediate K (2mol) in MnCl 2 (20mol) in DMF and added to A-G-P (1.8mol)In DMF solution. The reaction was stirred at room temperature. After 24 hours, the reaction was terminated with 10L of 0.25MEDTA solution. The mixture was loaded onto a DEAE Sephadex column (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting an elution product with HPLC purity more than 97%, concentrating the separation solution, loading the separation solution to strong anion resin, performing linear gradient elution by using 0-1.0M sodium acetate eluent, collecting an elution product with HPLC purity more than 98.5%, combining high-purity eluates, removing residual sodium acetate solution through nanofiltration equipment, and concentrating to obtain a target product, wherein the reaction route flow is as follows:
example 2: synthesis method of initial capped oligonucleotide primer containing hexahydric sugar ring structure by taking intermediate K and hexahydric sugar ring substituted A-G-P as raw materials
The initial capped oligonucleotide primer of example 2 was obtained by the method of synthesizing the target product of reference example 1 using the intermediate K and a-G-P substituted with a six-membered sugar ring as raw materials. Scheme, equation (5) below:
example 3: synthesis method of initial capped oligonucleotide primer containing hexahydric sugar ring structure by taking intermediate N and hexahydric sugar ring substituted A-G-P as raw materials
The initial capped oligonucleotide primer of example 3 was obtained by the method of synthesizing the target product of reference example 1 using the intermediate N and A-G-P substituted with a six-membered sugar ring as raw materials. Scheme, equation (6) below:
wherein the intermediate N is obtained by the following steps: 1) weighing 5g of guanosine, dispersing the guanosine in 50mL of DMF, carrying out ice bath to ensure that the internal temperature of the reaction solution is lower than 10 ℃, adding 1.2eq of TBSCl in two batches, monitoring the reaction by HPLC (high performance liquid chromatography) until the raw material is less than or equal to 5%, adding 100mL of water after the reaction is finished to precipitate a product, filtering and washing a filter cake; dissolving 2g of filter cake in 10ml of trimethyl phosphate, cooling the reaction liquid to 0 ℃, slowly adding 1.2eq of phosphorus oxychloride dropwise, reacting at a low temperature for 4 hours, adding 2M of ammonium acetate solution, quenching, purifying by reverse phase chromatography to obtain a target compound f, fully reacting the obtained compound f with 1eq of triphenylphosphine, 2eq of dipyridyl disulfide and 4eq of imidazole, adding the reaction liquid into 4M of sodium perchlorate acetone solution, precipitating, and fully washing the filter cake with acetone to obtain a target compound g;
2) weighing 2g of target compound g, dissolving the target compound g in DMF, adding 3eq of tributylamine phosphate, fully stirring to obtain a target compound h, adding 20eq of aqueous solution into reaction liquid, cooling the reaction liquid to 4 ℃, slowly dropwise adding dimethyl sulfate, adjusting Ph to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by HPLC, and purifying by ion chromatography after the reaction is finished to obtain a target compound i;
3) dissolving 4g of a compound i in 50mL of DMF, fully reacting with 1eq of triphenylphosphine, 2eq of dithiodipyridine and 4eq of imidazole, adding a reaction solution into 4M of a sodium perchlorate acetone solution, precipitating, and fully washing a filter cake with acetone to obtain a target compound N; scheme, equation (7) below:
comparative example 1: m7 GpppA 2’OMe pG
m7 GpppA 2’OMe pG Synthesis method referring to the synthesis method of the above example, the reaction scheme, the following equation (8):
comparative example 2: only Rb is a capped analog with a ring-opened structure, the synthesis method refers to the synthesis method of the above example, the reaction scheme, and the following equation (9):
the synthetic route of intermediate L is shown in the following equation, wherein 5g of intermediate F is dissolved in 50.0mL of pyridine, and acetic anhydride (3.5eq) is added dropwise to the reaction solution. Stir at rt for 5 h and monitor the reaction to completion by TLC. And concentrating to remove pyridine to obtain a crude intermediate M. The intermediate M is suspended in 70% acetic acid water solution and heated to 60 ℃ for reaction for 6 hours. TLC monitored the reaction to completion. Concentrating to remove the solvent, and performing column chromatography to obtain an intermediate n. Synthesis of intermediate L from intermediate n referring to the A-G-P synthesis in example 1, scheme, equation (10) below
The initial capped oligonucleotide primers containing six-membered sugar ring structures obtained in each example and the capping analog structures obtained in comparative examples are shown in Table 2 below,
TABLE 2
Test example 1: determination of mRNA in vitro transcription yield and capping efficiency
In vitro synthesis of mRNA using an initial capped oligonucleotide primer containing a six-membered sugar ring substitution structure: firstly, carrying out NotI linearization on plasmids, and carrying out enzyme digestion at 4 ℃ overnight; extracting a DNA template; mRNA was synthesized by in vitro transcription using the initial capped oligonucleotide primers containing the six-membered sugar ring substitution structure of examples 1-3 and comparative examples 1-2, respectively, as the cap structure.
The reaction system is shown in Table 3:
TABLE 3
| System of | Dosage of |
| T7 RNA polymerase | 50U |
| 10X buffer | 2μl |
| 100mM ATP | 1μl |
| 100mM GTP | 1μl |
| 100mM CTP | 1μl |
| 100mM N1-Me-pUTP | 1μl |
| 100mM cap analogs | 1μl |
| Inorganic pyrophosphatase | 0.05U |
| Nuclease inhibitors | 20U |
| Sterile enzyme-free water | Make up to 20. mu.l |
| Form panel | 1μg |
Remarking: in the experimental process, the volume of the materials needed by the system is calculated firstly, and then the sample is added. Firstly, adding sterile enzyme-free water into a system, then sequentially adding 10X buffer, NTPs and a cap analogue, mixing uniformly, then gently centrifuging, then adding a nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase and a linearized DNA template, fully mixing uniformly, gently centrifuging, and incubating at 37 ℃. After 2 hours DNase I1U was added and incubation continued at 37 ℃ for 30 minutes to remove DNA template and then RNA purification was performed, typically using magnetic bead purification. The purified mRNA was dissolved in sterile, enzyme-free water, followed by quantitative detection using Nanodrop One.
Liquid chromatography mass spectrometry (LC-MS) was used to detect IVT capping rates of mrnas of different starting cap analogs; firstly, a section of labeled DNA probe matched with mRNA starting base of a transcription product is required to be designed, the common label is labeled with biotin, streptavidin-labeled magnetic beads are washed and incubated with the synthesized DNA probe, mRNA and 10 XRNase H reaction buffer for 30 minutes at room temperature, the incubation and the slow mixing are carried out simultaneously, then 20ul RNase H (5U/ul) is added for incubation for 37 ℃ for 3 hours, and the mixing is carried out once every half hour. And (3) cleaning the magnetic beads after the incubation is finished, adding 100 mu L of 75% methanol heated to 80 ℃ into the cleaned magnetic beads, heating the mixture on a heating plate to 80 ℃, keeping the mixture for 3 minutes, then placing the mixture on a magnetic frame to absorb supernatant, and drying the supernatant at room temperature for 45 minutes to 10 mu L by using an evaporation centrifuge. The sample was then resuspended in 50. mu.l of 100. mu.M EDTA/1% MeOH and used for LC-MS analysis to determine the capping of the RNA in the transcription reaction. Since the capped and uncapped bases are clearly distinguished in molecular weight, the capping rate of mRNA transcription initiated by different cap analogs can be determined by using the difference in molecular mass. The specific results are shown in Table 4.
TABLE 4
| Numbering | Yield (. mu.g) | Capping ratio (%) |
| Example 1 | 52 | 94.0 |
| Example 2 | 45 | 92.0 |
| Example 3 | 46 | 93.0 |
| Comparative example 1 | 50 | 93.5 |
| Comparative example 2 | 20 | 25.0 |
As can be seen from the experimental results, the initial capping oligonucleotide primers containing the six-membered sugar ring substitution structure of the present application have the same level of mRNA in vitro transcription yield and capping efficiency as compared to the comparative examples. Meanwhile, the five-membered sugar ring of the third nucleotide in the comparative example 2 is replaced by the six-membered sugar ring, so that the in vitro transcription yield and the capping efficiency of mRNA are obviously reduced.
Test example 2: cellular protein expression assay
In vitro transcription was performed using the cap analogs of the examples as the start, using the eGFP coding sequence as the DNA template. The reaction system is shown in Table 5.
TABLE 5
| System of | Amount of the composition |
| T7 RNA polymerase | 50U |
| 10X buffer | 2μl |
| 100mM ATP | 1μl |
| 100mM GTP | 1μl |
| 100mM CTP | 1μl |
| 100mM N1-Me-pUTP | 1μl |
| 100mM cap analogs | 1μl |
| Inorganic pyrophosphatase | 0.05U |
| Nuclease inhibitors | 20U |
| Sterile enzyme-free water | Make up to 20. mu.l |
| Form panel | 1μg |
In the experimental process, the volume of the materials needed by the system is calculated firstly, and then the sample is added. Firstly, adding sterile enzyme-free water into a system, then sequentially adding 10X buffer, NTPs and a cap analogue, mixing uniformly, then gently centrifuging, then adding a nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase and a linearized DNA template, fully mixing uniformly, gently centrifuging, and incubating at 37 ℃. After 2 hours DNase I1U was added and incubation continued at 37 ℃ for 30 minutes to remove DNA template and then RNA purification was performed, typically using magnetic bead purification.
The different mRNA products obtained were subsequently transfected into 293T cells. 293T cells were plated (24-well plates) at (0.5-1). times.105 cells, and transfection experiments with cells within 50 passages were recommended. Cells were required to be passaged again 24 hours before transfection and addition of antibiotics to the medium had no effect on the transfection effect. The cell density is generally 60-80% and 2. mu.g of mRNA per well is transfected, and the Transfection Reagent is Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and is used according to the method of use. Transfected cells were placed at 37 ℃ CO 2 In the incubator, the medium was replaced with fresh complete medium 4-6 hours after transfection. CO at 37 deg.C 2 After incubation in the incubator for 24 hours, fluorescence microscope was used to observe the fluorescence intensity of GFP therein.
The results are shown in FIGS. 1 and 2, and it is evident from the results that the expression efficiency of mRNA of the present invention is significantly higher than that of the comparative example, while neither of them causes significant cell death, indicating that the capped analog of the present application has higher expression efficiency; namely, the effective protein translation efficiency of the capped analog containing the six-membered sugar ring structure applied to mRNA synthesis is obviously higher than that of the cap structure of Cleancap (comparative example 1). We believe that the cap analogue of the hexitol nucleic acid structure contained in the starting capped oligonucleotide primer structure containing a six-membered sugar ring structure has a steric structural advantage over the five-membered sugar ring, and can bind better to the transcription factor protein (elF4E) involved in translation of mRNA; meanwhile, the cap analogue with a non-natural structure resists the degradation capability of endogenous incision enzyme and exonuclease, improves the stability of RNA, prolongs the half-life period of the medicament and obviously improves the stability of mRNA. Finally, the hexabasic sugar ring structure cap analogue has better protein translation effect.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. An initial capped oligonucleotide primer containing a six-membered sugar ring structure, comprising the structure:
wherein, X 1 、X 2 And X 3 Each independently is O, CH 2 Or NH;
Y 1 、Y 2 and Y 3 Each independently is O, S, Se or BH 3 ；
R a Is composed of
Rb is
And when Ra is
When Rb is
R 1 、R 2 And R 3 、R 6 、R 7 、R 8 Independently hydrogen, hydroxy, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dihydrocarbyl, substituted or unsubstituted O-aryl, substituted or unsubstituted S-aryl, substituted or unsubstituted NH-aryl, substituted or unsubstituted O-aralkyl, substituted or unsubstituted S-aralkyl, substituted or unsubstituted NH-aralkyl;
R 4 and R 5 Independently H, OH, alkyl, O-alkyl, halogen;
B 1 and B 2 Independently, a natural, or modified, or non-natural nucleobase.
2. The method for preparing the initial capped oligonucleotide primer containing a six-membered sugar ring structure of claim 1, comprising the steps of: (1) synthesis of intermediate K: synthesizing a compound A from sorbitol, and sequentially carrying out reactions such as glycosylation, phosphorylation, monophosphorylation, diphosphorylation, methylation of N7, and imidazolation of polyphosphoric acid on the basis of the compound A to synthesize an intermediate K; (2) preparation of a phosphate-linked dinucleotide: coupling a phosphoramidite monomer and a disubstituted nucleoside monomer under the action of tetrazole to form a first phosphate ester bond, removing a protecting group through acid action, introducing a second phosphoric acid, and finally hydrolyzing to obtain a dinucleotide connected with the phosphate ester bond; (3) synthesis of initial capped oligonucleotide primers containing six-membered sugar ring structures: reacting the intermediate K with dinucleotide connected with a phosphate bond to prepare an initial capped oligonucleotide primer containing a six-membered sugar ring structure;
the phosphoramidite monomer has a structural formula:
B 3 and B 4 Independently, a natural, or modified, or non-natural nucleobase.
3. The use of the initial capped oligonucleotide primer containing a six-membered sugar ring structure according to claim 1, wherein: the mRNA of the initial capped oligonucleotide primer containing the six-membered sugar ring structure was capped using T7 RNA polymerase.
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| CN116768950B (en) * | 2023-08-16 | 2023-11-03 | 江苏申基生物科技有限公司 | Initial capping oligonucleotide primer and application thereof |
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