CN115057903B - Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof - Google Patents

Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof Download PDF

Info

Publication number
CN115057903B
CN115057903B CN202210716120.6A CN202210716120A CN115057903B CN 115057903 B CN115057903 B CN 115057903B CN 202210716120 A CN202210716120 A CN 202210716120A CN 115057903 B CN115057903 B CN 115057903B
Authority
CN
China
Prior art keywords
reaction
oligonucleotide primer
ring structure
morpholine ring
mrna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210716120.6A
Other languages
Chinese (zh)
Other versions
CN115057903A (en
Inventor
缪佳颖
黄磊
赵万年
沈奇
肖潇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Shenji Biotechnology Co ltd
Original Assignee
Jiangsu Shenji Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Shenji Biotechnology Co ltd filed Critical Jiangsu Shenji Biotechnology Co ltd
Priority to CN202210716120.6A priority Critical patent/CN115057903B/en
Publication of CN115057903A publication Critical patent/CN115057903A/en
Priority to PCT/CN2023/101657 priority patent/WO2023246860A1/en
Application granted granted Critical
Publication of CN115057903B publication Critical patent/CN115057903B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Saccharide Compounds (AREA)

Abstract

The invention provides an initial capping oligonucleotide primer containing a morpholine ring structure, a preparation method and application thereof, wherein the molecular formula of the initial capping oligonucleotide primer containing the morpholine ring structure is m7 GpppA 2'OMe pG of (1) 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 capping oligonucleotide primer containing the morpholine ring structure provided by the invention has lower immunogenicity, higher protein translation efficiency and higher stability.

Description

Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof
Technical Field
The invention relates to the technical field of chemistry and bioengineering, in particular to an initial capping oligonucleotide primer containing a morpholine 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, and capping of the mRNA promotes its normal function in the cell. Synthesis of mRNA by in vitro transcription has become an important tool for introducing foreign genes and expressing proteins, and is widely used in the treatment and prevention of diseases, enabling workers to prepare RNA molecules that perform properly in a variety of biological applications. Such applications include research applications and commercial production of polypeptides, for example, in cell-free translation systems to produce polypeptides comprising "unnatural" amino acids at specific sites, or in cultured cells to produce polypeptides that require post-translational modification for their activity or stability. In the latter system, synthesis proceeds for a significantly longer time and thus more protein is produced.
Patent CN201680067458.6 reports compositions and methods for synthesizing 5' -capped RNAs. Wherein the initial capping 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 unnatural nucleoside, "N" may be any integer from 0 to 4 and "m" may be an integer from 1 to 9. Cleanneap belongs to Cap1 and uses dimer with ARCA (m 7 GpppG) initiates T7 transcription differently, cleanCap uses trimer (m 7 Gppppamg) initiates T7 transcription. The method has higher yield, 4mg of capped RNA is prepared per milliliter of transcription reaction system, the capping efficiency can reach 90 percent, and the immunogenicity of the transcription product is lower than that of ARCA.
The 5' -position capping analog is a key structure for reducing the immunogenicity of mRNA, and the immunogenicity of mRNA can be reduced by optimizing the structure of the cap analog. Accordingly, there is a continuing need to optimize the structure of cap analogs, and to find novel cap analogs that are less immunogenic than currently available clearcaps. According to the invention, the five-membered sugar ring is replaced by the morpholine ring structure in the cap analogue structure, so that the binding capacity of mRNA and RIG-I is reduced, and the immunogenicity of mRNA is obviously reduced.
Disclosure of Invention
In order to further improve the stability of RNA, prolong the half life of a drug, obviously improve the stability of mRNA, reduce the binding capacity of mRNA and RIG-I, the application provides an initial capping oligonucleotide primer containing a morpholine ring structure, a preparation method and application thereof. Meanwhile, mRNA synthesized by the cap analogue of the morpholine ring structure has lower binding capacity with RIG-I and lower immunogenicity.
An initial capped oligonucleotide primer comprising a morpholino loop structure comprising the structure:
wherein X is 1 、X 2 And X 3 O, CH each independently of the other 2 Or NH;
Y 1 、Y 2 and Y 3 O, S, se or BH, respectively and independently 3 ；
R a Is thatRb is->And when Ra is->Rb is +.>
R 1 Is hydrogen, hydroxy, substituted or unsubstituted O-alkyl, substituted or unsubstituted S-alkyl, substituted or unsubstituted NH-alkyl, substituted or unsubstituted N-dialkyl, substituted or unsubstitutedAn O-aryl group, a substituted or unsubstituted S-aryl group, a substituted or unsubstituted NH-aryl group, a substituted or unsubstituted O-aralkyl group, a substituted or unsubstituted S-aralkyl group, a substituted or unsubstituted NH-aralkyl group;
R 2 and R is 3 Independently H, OH, alkyl, O-alkyl, halogen;
B 1 and B 2 Independent are natural, or modified, or unnatural nucleobases.
A method for preparing an initial capped oligonucleotide primer containing a morpholino loop structure, comprising the steps of: (1) Synthesis of intermediate F: synthesizing a compound A from guanosine, sequentially carrying out diphosphorylation, N7 methylation and polyphosphoric imidization on the basis of the compound A, and synthesizing an intermediate F; (2) preparation of phosphoester-linked dinucleotides: coupling a phosphoramidite monomer and a disubstituted nucleoside monomer under the action of tetrazole to form a first phosphoester bond, removing a protecting group under the action of acid, introducing a second phosphoric acid, and finally hydrolyzing to obtain a dinucleotide connected with the phosphoester bond; (3) Synthesis of initial capped oligonucleotide primers containing morpholino loop structures: preparing an initial capping oligonucleotide primer containing a morpholine ring structure by reacting the intermediate F with a dinucleotide connected with a phosphate bond;
the structural formula of the (phosphorous) phosphoramide monomer is as follows:
wherein R4 is H, OH, alkyl, O-alkyl, halogen; b (B) 3 And B 4 Independent are natural, or modified, or unnatural nucleobases.
The above-mentioned disubstituted nucleoside monomer is selected fromAny one of them.
The preparation method of the initial capping oligonucleotide primer containing the morpholine ring structure specifically comprises the following steps:
(1) Synthesis of intermediate F:
(1-1) suspending guanosine in ethanolNaIO is processed 4 Dissolving in 40+ -5deg.C water, and adding dropwise to adenosine suspension under vigorous stirring. After 15min (NH) 4 ) 2 B 4 O 7 .4H 2 O. And (3) dropwise adding triethylamine to keep the pH=8.5-9.0 of the reaction system, reacting for 1.5 hours at room temperature, and monitoring the reaction until the adenosine is less than or equal to 1 percent by HPLC. Suction filtration and washing of the filter cake with ethanol. Adding NaBH to the filtrate in portions under ice bath 3 CN, after stirring for 1h at room temperature, TFA was used to adjust ph=3-4, the reaction was continued for 2h, and hpcl monitored to be complete. Residual TFA was removed by distillation under reduced pressure. Preparing and purifying the crude product in reverse phase, and freeze-drying to obtain a compound A;
(1-2) dissolving the compound A in trimethyl phosphate, cooling the reaction solution to 0+/-5 ℃, slowly dropwise adding phosphorus oxychloride, reacting for 4 hours at room temperature, adding 2M ammonium acetate solution to quench the reaction, preparing and purifying in reverse phase, and freeze-drying to obtain a target compound B;
(1-3) taking a compound B, and suspending the compound B with triphenylphosphine, dithiodipyridine, imidazole and TEA in DMF (dimethyl formamide), reacting for 8 hours at room temperature, monitoring the reaction until the raw material is less than or equal to 1% by HPLC (high Performance liquid chromatography), adding 4M sodium perchlorate acetone solution into the reaction solution, carrying out suction filtration, and fully washing a filter cake with acetone to obtain a target compound C;
(1-4) weighing a target compound C, suspending in DMF, adding triethylamine phosphate and manganese chloride, stirring at room temperature to obtain 8h, monitoring the reaction until the raw material is less than or equal to 1% by HPLC, and pouring the reaction solution into water to obtain a crude product aqueous solution of the compound D. Slowly dropwise adding dimethyl sulfate, regulating pH to not more than 5 with 2M sodium hydroxide, monitoring reaction by HPLC, purifying by ion chromatography after reaction, and lyophilizing to obtain target compound E;
(1-5) suspending the compound E, triphenylphosphine, dithiodipyridine, imidazole and TEA in DMF (dimethyl formamide), reacting for 8 hours at room temperature, monitoring the reaction until the raw material is less than or equal to 1% by HPLC (high Performance liquid chromatography), adding 4M sodium perchlorate acetone solution into the reaction solution, carrying out suction filtration, and fully washing a filter cake with acetone to obtain a target compound F;
(2) Preparation of phosphoester-linked dinucleotides:
weighing 2' OMe-rA phosphoramidite monomer, dissolving in dichloromethane, adding 2',3' acetyl guanosine, cooling to 25+ -2deg.C, adding tetrazole under nitrogen blowing, and heating to 25+ -2deg.C for reaction. After the monitoring reaction is finished, adding an iodopyridine solution into the reaction solution, spin-drying after the monitoring reaction is finished, dissolving the concentrated ointment into dichloromethane, adding trifluoroacetic acid, 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, adding an iodopyridine solution into a reaction solution after the reaction is monitored, spin-drying after the reaction is monitored, adding methanol and concentrated ammonia water into a spin bottle, reacting for 4 hours at room temperature, monitoring the reaction, spin-drying after the reaction is finished, adding ultrapure water, entering reverse ion permeation equipment, washing, concentrating, and freeze-drying to obtain dinucleotide connected with a phosphate ester bond;
(3) Synthesis of initial capped oligonucleotide primers containing morpholino loop structures:
dissolving m7UrGDP-Im in a solution containing MnCl 2 And adding to the obtained DMF solution of the phosphoester-linked dinucleotide, dissolving the intermediate F obtained in the step (1) in a solution containing MnCl 2 And added to the DMF solution of A-G-P obtained in step (2). The reaction was stirred at room temperature. After 24h, the reaction was quenched with 0.25M EDTA solution. The mixture was loaded onto a DEAESEPHADEX column (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting the eluted product with HPLC purity of > 97%, concentrating the separated liquid, loading the concentrated solution into strong anion resin, linearly gradient eluting with 0-1.0M sodium acetate eluent, collecting the eluted product with HPLC purity of > 98.5%, combining the high-purity eluted products, removing residual sodium acetate solution by nanofiltration equipment, and concentrating to obtain the target product of the initial capping oligonucleotide primer containing morpholine ring structure.
The initial capping oligonucleotide primer containing the morpholine ring structure is used for mRNA capping under a T7 RNase system. T7 RNA polymerase is a DNA dependent RNA polymerase with high specificity for phage T7 promoter sequences. The enzyme synthesizes a large amount of RNA from DNA inserted downstream of the T7 promoter into the transcription vector. The T7 RNA polymerase catalyzed IVT (in vitro transcription) reaction system is the most mature mRNA preparation system at present.
The invention provides an initial capping oligonucleotide primer containing a morpholine ring structure, which is suitable for mRNA (messenger ribonucleic acid) produced by taking a DNA sequence as a template and adopting an in vitro co-transcription method, wherein the DNA sequence can be derived or modified from virus, animal, plant and other species, and the produced mRNA has lower immunogenicity, higher protein translation efficiency and better stability.
Compared with the prior art, the invention has the following advantages:
compared with the existing cap structure analogue Cleencap, the initial capping oligonucleotide primer containing the morpholine ring structure has lower immunogenicity, higher protein translation efficiency and better stability.
Drawings
FIG. 1 is a diagram of the cell phenotype of examples 1-3 and comparative examples 1-2;
FIG. 2 is a graph showing the fluorescence statistics of examples 1-3 and comparative examples 1-2;
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The raw material names and sources used in each example are shown in table 1 below:
TABLE 1
Intermediate F used in each of the following examples was prepared by the following steps:
(1-1) taking 5.0g of guanosineSuspended in 100mL of ethanol, 1.2eq. NaIO 4 Dissolved in 150mL of warm water at 40℃and added dropwise to the adenosine suspension with vigorous stirring. After 15min (NH) 4 ) 2 B 4 O 7 .4H 2 O (1.2 eq.). The reaction system ph=8.5 to 9.0 was maintained by dropwise addition of triethylamine (about 5.0 mL), and after 1.5 hours of reaction at room temperature, the reaction was monitored by HPLC until the adenosine was 1% or less. Suction filtration and washing of the filter cake with ethanol. Adding NaBH to the filtrate in portions under ice bath 3 CN (1.3 eq.) after stirring for 1h at room temperature, ph=3-4 with TFA, the reaction was continued for 2h, and hpcl monitored to be complete. Residual TFA was removed by distillation under reduced pressure. Preparing and purifying the crude product in reverse phase, and freeze-drying to obtain a compound A;
(1-2) dissolving 2g of the compound A in 20ml of trimethyl phosphate, cooling the reaction solution to 0 ℃, slowly dropwise adding 2.0eq of phosphorus oxychloride, reacting for 4 hours at room temperature, adding 2M ammonium acetate solution to quench the reaction, preparing and purifying in reverse phase, and freeze-drying to obtain the target compound B;
(1-3) taking 2g of compound B, 2eq of triphenylphosphine, 2eq of dithiodipyridine, 8eq of imidazole and 1eq of TEA to suspend in 20mL of DMF, reacting for 8 hours at room temperature, monitoring the reaction until the raw material is less than or equal to 1 percent by HPLC, adding 4M sodium perchlorate acetone solution into the reaction solution, carrying out suction filtration, and fully washing a filter cake with acetone to obtain a target compound C;
(1-4) 2g of the target compound C was weighed and suspended in 20mL of DMF, 3.5eq of triethylamine phosphate and 8eq of manganese chloride were added, stirring was carried out at room temperature for 8 hours, the reaction was monitored by HPLC until the starting material was 1% or less, and the reaction solution was poured into 200mL of water to obtain a crude aqueous solution of the compound D. Slowly dropwise adding 8eq of dimethyl sulfate, regulating pH to be not more than 5 by using 2M sodium hydroxide in the process, monitoring the reaction by using HPLC, purifying by ion chromatography after the reaction is finished, and freeze-drying to obtain a target compound E;
(1-5) taking 1g of compound E, 2eq of triphenylphosphine, 2eq of dithiodipyridine, 8eq of imidazole and 1eq of TEA to suspend in 10mL of DMF, reacting for 8 hours at room temperature, monitoring the reaction until the raw material is less than or equal to 1 percent by HPLC, adding 4M sodium perchlorate acetone solution into the reaction solution, carrying out suction filtration, and fully washing a filter cake with acetone to obtain a target compound F;
specific schemes for intermediate F are shown in equation (1):
the synthetic route for A-G-P used in Synthesis example 1 was as follows: 5kg of 2' OMe-rA phosphoramidite monomer is weighed into a single-mouth bottle, dissolved by 50L of dichloromethane, added with 2.73kg of 2',3' acetyl guanosine, cooled to 25+/-2 ℃, added with 880g of tetrazole under nitrogen blowing, and heated to 25+/-2 ℃ for reaction. After the monitoring reaction is finished, adding 1.2eq of iodopyridine solution into the reaction solution, spin-drying after the monitoring reaction is finished, dissolving the concentrated ointment into 4L of dichloromethane, adding 1.1eq of trifluoroacetic acid, 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 4L acetonitrile, adding 1.2eq of phosphine reagent and 1.2eq of tetrazole, fully stirring for reaction, adding 1.2eq of iodopyridine solution into the reaction liquid after monitoring the reaction, spin-drying after monitoring the reaction, adding 3L of methanol and 3L of concentrated ammonia water into a spin bottle, reacting for 4 hours at room temperature, monitoring the reaction, spin-drying after the reaction, adding 20L of ultrapure water, entering reverse ion permeation equipment, washing, concentrating, and freeze-drying to obtain a target compound A-G-P, wherein the reaction route flow is as shown in the following equation (2):
synthesis example 2 the synthesis of morpholine-substituted A-G-P was carried out according to the method of synthesis of A-G-P in example 1, followed by the scheme of the morpholine-substituted A-G-P, as shown in the following equation (3):
wherein the preparation of C1 comprises the following steps: 1) 10g of A1 and DMAP (0.1 eq.) were suspended in 10 volumes of pyridine, TBDPSCl (1.1 eq.) was added dropwise to the reaction solution at room temperature, and the reaction was completed at room temperature. Concentrating the reaction solution to remove pyridine, and recrystallizing the crude product with methanol to obtain A1; 2) A synthesis step wherein the synthesis of B1 refers to intermediate a; 3) 5g of B1 was dissolved in a mixed solvent of dichloromethane and acetonitrile (1: 1, 40 mL), a solution of lithium bromide (3.2 eq.) in acetonitrile (10 mL) was added dropwise to the reaction solution at 0 ℃, and after stirring for 2 minutes DBU (3.2 eq.) was added dropwise and stirring was continued for 2 minutes. N, N-dimethylphosphino-amino dichloride (1.6 eq.) was added dropwise to the reaction solution, followed by reaction at room temperature for 1 hour, and TLC monitored the reaction was complete. The reaction solution was concentrated and purified by column chromatography to obtain C1.
Example 1: synthesis method of initial capping oligonucleotide primer containing morpholine ring structure by using intermediates F and A-G-P as raw materials
Dissolving intermediate F (2 mol) in a solution containing MnCl 2 (20 mol) in DMF and added to a solution of A-G-P (1.8 mol) in DMF. The reaction was stirred at room temperature. After 24 hours, the reaction was stopped with 10L of 0.25MEDTA solution. The mixture was loaded onto a DEAESEPHADEX column (30X 500 cm). The product was eluted using a linear gradient of 0-1.0M TEAB eluent. Collecting the eluted product with HPLC purity of more than 97%, concentrating the separated liquid, loading the concentrated product into strong anion resin, linearly gradient eluting with 0-1.0M sodium acetate eluent, collecting the eluted product with HPLC purity of more than 98.5%, combining the high-purity eluted product, removing residual sodium acetate solution by nanofiltration equipment, and concentrating to obtain the target product, wherein the reaction route flow is as shown in the following equation (4):
example 2: synthesis method of initial capping oligonucleotide primer containing morpholine ring structure by using intermediate F and morpholine substituted A-G-P as raw materials
The starting capped oligonucleotide primers of example 2 were obtained by the synthesis of the target product of reference example 1 starting from intermediate F and morpholine-substituted A-G-P. Scheme of reaction scheme, equation (5) below:
example 3: synthesis method of initial capping oligonucleotide primer containing morpholine ring structure by using intermediate N and morpholine substituted A-G-P as raw materials
The starting capped oligonucleotide primers of example 2 were obtained by the synthesis of the target product of reference example 1 starting from intermediate N and morpholine-substituted A-G-P. Scheme of reaction scheme, equation (6) below:
wherein, intermediate N is obtained by the following steps: 1) Weighing 5g of guanosine, dispersing in 50mL of DMF, carrying out ice bath to enable the internal temperature of the reaction liquid to be lower than 10 ℃, adding 1.2eq of TBSCl into the reaction liquid in two batches, monitoring the reaction until the raw material is less than or equal to 5% by HPLC, 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 solution to 0 ℃, slowly dropwise adding 1.2eq phosphorus oxychloride, reacting for 4 hours at low temperature, adding 2M ammonium acetate solution to quench the reaction, purifying by reverse phase chromatography to obtain a target compound f, fully reacting the obtained compound f with 1eq triphenylphosphine, 2eq dithiodipyridine and 4eq imidazole, adding the reaction solution into 4M sodium perchlorate acetone solution to precipitate, and fully washing the filter cake with acetone to obtain the target compound g;
2) Weighing 2g of target compound g, dissolving DMF, adding 3eq of tributylamine phosphate, fully stirring to obtain target compound h, adding 20eq of aqueous solution into the reaction solution, cooling the reaction solution to 4 ℃, slowly dropwise adding dimethyl sulfate, regulating Ph with 2M sodium hydroxide to be not more than 5 in the process, monitoring the reaction by HPLC, and purifying by ion chromatography after the reaction is finished to obtain 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 the reaction solution into 4M sodium perchlorate acetone solution to precipitate, and fully washing a filter cake with acetone to obtain a target compound N; scheme of reaction scheme, equation (7) below:
comparative example 1: m7 GpppA 2’Ome pG
m7 GpppA 2’Ome the synthesis of pG is described in the above examples by reference to the scheme of the reaction scheme, equation (8) below:
the structures of the starting capped oligonucleotide primers containing a morpholine ring structure obtained in each example and the capped analogues obtained in the 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 initial capped oligonucleotide primers containing morpholino loop structures: linearizing the plasmid with NotI, and enzyme cutting at 4 ℃ overnight; extracting a DNA template; mRNA was synthesized by in vitro transcription using the initial capping oligonucleotide primers containing the morpholino ring structure of examples 1-3 and comparative example 1, respectively, as the cap structure.
The reaction system is shown in Table 3:
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 analogue 1μl
Inorganic pyrophosphatase 0.05U
Nuclease inhibitors 20U
Sterile water Make up to 20. Mu.l
Template 1μg
Remarks: in the experimental process, the volume of materials required by the system is calculated first, and then the sample is added. Firstly, adding sterile and sterile water into a system, then sequentially adding 10 Xbuffer, NTPs and cap analogues, mixing uniformly, lightly centrifuging, then adding nuclease inhibitor, inorganic pyrophosphatase, T7 RNA polymerase and linearized DNA template, fully mixing uniformly, lightly centrifuging, and incubating at 37 ℃. DNase I1U was added after 2 hours and incubation was continued for 30 minutes at 37℃to remove the DNA template, followed by RNA purification, typically using a magnetic bead purification method. Purified mRNA was solubilized with sterile, aqueous water and subsequently quantitatively detected using a Nanodrop One.
Liquid chromatography mass spectrometry (LC-MS) was used to detect IVT capping rates of mRNA of different starting cap analogs; firstly, a section of DNA probe with a label matched with the initial base of mRNA of a transcription product needs to be designed, a general label is a biotin label, a streptavidin-labeled magnetic bead is washed and then incubated with the synthesized DNA probe, mRNA and 10X RNase H reaction buffer for 30 minutes at room temperature, the DNA probe, mRNA and 10X RNase H reaction buffer are slowly mixed while being incubated, and then 20ul RNase H (5U/ul) is added for incubation for 3 hours at 37 degrees, and the mixture is mixed every half hour. After the incubation, the beads were washed, 100. Mu.L of 75% methanol heated to 80℃was added to the washed beads, the mixture was heated to 80℃on a hot plate, kept for 3 minutes, and the supernatant was then sucked up on a magnetic rack and dried at room temperature for 45 minutes to 10. Mu.L 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 RNA capping during transcription. Since the capped and uncapped bases are significantly different in molecular weight, the difference in molecular mass can be used to determine the capping rate of mRNA transcription initiated by different cap analogs. The specific results are shown in Table 4.
TABLE 4 Table 4
From the experimental results, the initial capping oligonucleotide primers containing the morpholino loop structure of the present application have the same level of mRNA in vitro transcription yield and capping efficiency as compared to the comparative examples.
Test example 2: determination of mRNA binding Capacity to RIG-I
RIG-I consists essentially of two repetitive caspase activation and recruitment domains at the N-terminus (caspase activation and recruitment domain, CARD), an intermediate helicase structure and a C-terminal RNA domain. The N-terminal CARD domain of RIG-I promotes secretion of type I Interferon (IFN) by cells even in the absence of viral infection, and is therefore primarily responsible for signaling downstream.
The study transfected 293T cells with the initially capped oligonucleotide primers containing the morpholino ring structure of examples 1-3 and the cap analogues of comparative examples 1-2 as mRNA for initiating in vitro transcription, collected cells after 24 hours, co-immunoprecipitated intracellular protein RIG-I with its associated RNA by RNA co-immunoprecipitation, and finally reverse transcribed and real-time quantitative PCR of these mRNA.
Specific cell culture conditions are the same as above, cells are collected after transfection for 24 hours, firstly, a fixing solution is added for incubation, and after 10 minutes, a glycine solution with proper concentration is added for stopping reaction, and the cells are collected. The collected cells were lysed by means of lysate, centrifuged at 12000rpm at 4℃for 10min, and the supernatants were incubated with RIG-I or IgG antibodies, respectively, on a 4℃shaker overnight. Then adding 20 μl Protein A/G magnetic beads into the mixture, incubating the mixture at 4 ℃ for 4 hours, washing the mixture on a magnetic rack, and extracting RNA after washing is finished, thus the RNA can be used for subsequent RT-qPCR verification of the expression result. Binding capacity of different cap analogue nucleotide mRNAs to RIG-1 results are shown in Table 5 below:
TABLE 5
Different cap analogues Binding ability to RIG-I
Example 1 1.5±0.11
Example 2 2.1±0.32
Example 3 2.5±0.33
Comparative example 1 3.2±0.52
As can be seen from the experimental data in Table 4 above, the initial capping oligonucleotide primers containing a morpholino ring structure of the present invention are significantly less immunogenic than the comparative Cleancap when applied to mRNA synthesis cells.
Test example 3: intracellular translation effect detection of mRNA
Transfection of 293T cells was performed with different mRNA products obtained from IVT. 293T cells were plated (24-well plate) in (0.5-1). Times.105 cells, and transfection experiments using cells within 50 passages were recommended. Cells were required to be passaged again 24 hours prior to transfection, and the addition of antibiotics to the medium had no effect on the transfection effect. The cell density at transfection is generally 60-80% and 2. Mu.g mRNA per well is transfected, and the transfection reagent is selected from Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and is manipulated by reference to its method of use. The transfected cells were placed at 37℃and CO 2 In the incubator, after 4-6 hours of transfection, the medium was replaced with fresh complete medium. CO at 37 DEG C 2 After 24 hours incubation in the incubator, fluorescence intensity of GFP was observed by fluorescence microscope. The results are shown in FIGS. 1 and 2, in which it is evident that the mRNA of the present invention is expressed more efficiently than the comparative example, while neither causes significant cell death.
This result indicates that the initial capping oligonucleotide primer containing the morpholine ring structure of example 1 of the present application has higher expression efficiency; that is, the initial capping oligonucleotide primer containing the morpholino ring nucleoside structure in the invention has obviously higher protein translation efficiency when applied to mRNA synthesis effective protein translation efficiency than the clearcap (comparative example 1) cap structure.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein 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 initially capped oligonucleotide primer comprising a morpholino loop structure, characterized by the following structure:
wherein X is 1 、X 2 And X 3 Each independently is O;
Y 1 、Y 2 and Y 3 Each independently is O;
R a is thatRb is->And when Ra is->Rb is +.>
R 1 Is hydrogen, hydroxy, methoxy;
R 2 and R is 3 H, OH, halogen independently;
B 1 and B 2 Independently a natural nucleobase.
2. The morpholine ring structure-containing starting capping oligonucleotide primer of claim 1, which is any one of the following structures:
3. use of a starting capped oligonucleotide primer comprising a morpholino ring structure according to claim 1, characterized in that:
the mRNA of the initial capping oligonucleotide primer containing the morpholino loop structure was capped using the IVT reaction system of T7 RNA polymerase.
CN202210716120.6A 2022-06-22 2022-06-22 Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof Active CN115057903B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210716120.6A CN115057903B (en) 2022-06-22 2022-06-22 Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof
PCT/CN2023/101657 WO2023246860A1 (en) 2022-06-22 2023-06-21 Initially capped oligonucleotide primer, method for preparing same, and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210716120.6A CN115057903B (en) 2022-06-22 2022-06-22 Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115057903A CN115057903A (en) 2022-09-16
CN115057903B true CN115057903B (en) 2024-03-29

Family

ID=83201900

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210716120.6A Active CN115057903B (en) 2022-06-22 2022-06-22 Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN115057903B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023246860A1 (en) * 2022-06-22 2023-12-28 江苏申基生物科技有限公司 Initially capped oligonucleotide primer, method for preparing same, and use thereof
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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107223128A (en) * 2014-12-16 2017-09-29 诺华股份有限公司 The nucleic acid molecules that end is capped
CN108366604A (en) * 2015-09-21 2018-08-03 垂林克生物技术公司 For synthesizing the 5 '-compositions and method for capping RNA

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009544754A (en) * 2006-07-28 2009-12-17 アプライド バイオシステムズ， エルエルシー Dinucleotide MRNA cap analog
US11866754B2 (en) * 2015-10-16 2024-01-09 Modernatx, Inc. Trinucleotide mRNA cap analogs
CA3001014A1 (en) * 2015-10-16 2017-04-20 Modernatx, Inc. Mrna cap analogs and methods of mrna capping
WO2018075827A1 (en) * 2016-10-19 2018-04-26 Arcturus Therapeutics, Inc. Trinucleotide mrna cap analogs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107223128A (en) * 2014-12-16 2017-09-29 诺华股份有限公司 The nucleic acid molecules that end is capped
CN108366604A (en) * 2015-09-21 2018-08-03 垂林克生物技术公司 For synthesizing the 5 '-compositions and method for capping RNA

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Deoxynucleoside phosphoramidites-A new class of key intermediates for deoxypolynucleotide synthesis;S. L. Beaucage and M. H. Caruthers;《Tetrahedron Letters》;第22卷(第20期);第1859-1862页 *
吗啉核苷类似物及其磺胺衍生物的合成及初步抗牛病毒性腹泻病毒（BVDV）活性研究;华迎春 等;《有机化学》;第38卷;第1147-1154页 *

Also Published As

Publication number Publication date
CN115057903A (en) 2022-09-16

Similar Documents

Publication Publication Date Title
CN115057903B (en) Initial capping oligonucleotide primer containing morpholine ring structure and preparation method and application thereof
CN114853836B (en) Initial capping oligonucleotide primer containing GNA structure and preparation method and application thereof
CN114685588B (en) Initial capping oligonucleotide primer containing open-loop nucleoside structure
EP3521456B1 (en) Methods and means for enhancing rna production
EP3394280A1 (en) Method of rna in vitro transcription using a buffer containing a dicarboxylic acid or tricarboxylic acid or a salt thereof
CN114540444B (en) Capping composition, preparation method thereof and in-vitro transcription reaction system
US20050191689A1 (en) Novel nucleic acid base pair
CN111971396A (en) Method for producing single-stranded RNA
CN116143854A (en) Ribose ring modified mRNA cap analogue and preparation method and application thereof
CN116143855B (en) Vinyl phosphonic acid modified mRNA cap analogue and preparation method and application thereof
CN116478226A (en) Lock nucleoside cap analogue and application
CN115109110B (en) Initial capping oligonucleotide primer containing six-membered sugar ring structure, and preparation method and application thereof
Matuszewski et al. Efficient conversion of N 6-threonylcarbamoyladenosine (t 6 A) into a tRNA native hydantoin cyclic form (ct 6 A) performed at nucleoside and oligoribonucleotide levels
WO2023246860A1 (en) Initially capped oligonucleotide primer, method for preparing same, and use thereof
Hobartner et al. Engineering a selective small-molecule substrate binding site into a deoxyribozyme
CN116375781B (en) TNA modified cap analogue and preparation method and application thereof
EP3309250A1 (en) Amino acid-modified nucleic acid and utilization thereof
Grosjean et al. RNA‐modifying and RNA‐editing enzymes: Methods for their identification
WO2021024467A1 (en) Production method for single-strand rna
CN116554250A (en) PACE structure modified cap analogue and application thereof
TWI837122B (en) Method of producing single-strand rna
US20240218412A1 (en) Methods and means for enhancing rna production
Koyama et al. Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest
TWI541249B (en) 5-position modified pyrimidines and their use
CN117820388A (en) Deuterated capping compound for mRNA synthesis and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant