WO2021162567A1 - Novel mrna 5'-end cap analogs, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide - Google Patents

Novel mrna 5'-end cap analogs, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide Download PDF

Info

Publication number
WO2021162567A1
WO2021162567A1 PCT/PL2021/050007 PL2021050007W WO2021162567A1 WO 2021162567 A1 WO2021162567 A1 WO 2021162567A1 PL 2021050007 W PL2021050007 W PL 2021050007W WO 2021162567 A1 WO2021162567 A1 WO 2021162567A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
gppp
rna molecule
group
rna
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.)
Ceased
Application number
PCT/PL2021/050007
Other languages
French (fr)
Inventor
Macin WARMIŃSKI
Paweł SIKORSKI
Joanna Kowalska
Jacek Jemielity
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.)
Uniwersytet Warszawski
Explorna Therapeutics Sp zoo
Original Assignee
Uniwersytet Warszawski
Explorna Therapeutics Sp zoo
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 Uniwersytet Warszawski, Explorna Therapeutics Sp zoo filed Critical Uniwersytet Warszawski
Priority to AU2021219563A priority Critical patent/AU2021219563A1/en
Priority to CA3167570A priority patent/CA3167570A1/en
Priority to EP21754142.4A priority patent/EP4103577A4/en
Priority to US17/798,099 priority patent/US12606590B2/en
Priority to JP2022548990A priority patent/JP7805938B2/en
Priority to KR1020227030072A priority patent/KR20220163359A/en
Publication of WO2021162567A1 publication Critical patent/WO2021162567A1/en
Anticipated expiration legal-status Critical
Priority to JP2025162162A priority patent/JP2026012182A/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure

Definitions

  • Tri- and tetranucleotide cap analogs were synthesized as described in Examples 1-6 combining solid phase synthesis and solution synthesis methods, followed by isolation using a two-step purification procedure.
  • the starting point was the synthesis of appropriately modified oligonucleotides: dinucleotides (pA * pG, where A * stands for adenosine substituted at the N6 position) or trinucleotides (pA * pGpG) on a highly loaded support using the phosphoramidite method, as described in Example 2.[11]
  • the corresponding phosphoramidites modified at the N6-position of adenosine were obtained by N-alkylation of commercially available adenosine phosphoramidite under phase transfer catalysis conditions, as described in Example 1.
  • the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-0.9 M TEAB) to afford after evaporation a triethylammonium salt of pA*pG dinucleotide.
  • Step 1 Activation of pNpG: Dinucleotide 5'-phosphate was dissolved in DMF (to obtain a 0.05 M solution) followed by addition of imidazole (16 equivalents), 2,2'-dithiodipiridine (6 equivalents), triethylamine (6 equivalents) and triphenylphosphine (6 equivalents). The mixture was stirred at room temperature for 2 h. The product was precipitated by addition of a solution of sodium perchlorate (10 equivalents) in acetonitrile (10 times the volume of DMF). The precipitate was centrifuged at 4°C, washed with cold acetonitrile 3 times and dried under reduced pressure to give a sodium salt of dinucleotide P-imidazolide (Im-pNpG).
  • Step 2 Formation of triphosphate bridge: 7-Methylguanosine b-thiodiphosphate m 7 GDP- ⁇ -S (378 mOD 2 60 nm , 33.2 ⁇ moI; obtained as described earlier and stored in TEAB at -20°C) [12] was evaporated to dryness and suspended in DMF (890 ⁇ L). Then ZnCI 2 (24.1 mg, 177 ⁇ moI) and lm-p Bn6 A m pG (19 mg) were added and the mixture was stirred at room temperature for 2 h.
  • Step 1 Synthesis of p Bn6, 5'S A m pG: Synthesis of dinucleotide was performed manually using a 10 mL syringe equipped with a frit filter.
  • the solid support (5'-O-DMT-2'-O-TBDMS-rG iBu 3'- lcaa PrimerSupport 5G, GE Healthcare, 308 ⁇ mol/g, 650 mg) was placed in a syringe and washed with dry acetonitrile.
  • Step 2 Activation of p Bn6,5'S A m pG: Dinucleotide 5'-phosphorothiolate p Bn6,5'S A m pG (2400 mOD 2 60 nm , 88.5 ⁇ mol) was dissolved in DMF (1.80 mL) followed by addition of imidazole (96.3 mg, 1.42 mmol), 2,2'-dithiodipiridine (117 mg, 531 ⁇ mol), triethylamine (74.2 ⁇ L, 531 ⁇ mol) and triphenylphosphine (139 mg, 531 ⁇ mol). The mixture was stirred at room temperature for 2 h.
  • Step 3 Formation of triphosphate bridge: 7-Methylguanosine 5'-diphosphate (m 7 GDP; 1210 mOD260 nm , 106 ⁇ mol) and lm-p 5'S,Bn6 A m pG (92.2 mg) were suspended in DMF (1 .77 mL). Then ZnCI 2 (144 mg, 1.06 mmol) were added and the mixture was stirred at room temperature for 2 h.
  • the reaction was quenched by addition of a solution (3 mL) of Na 2 EDTA (20 mg/mL) and NaHCO 3 (10 mg/mL) in water and the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-1.2 M TEAB) to afford after evaporation triethylammonium salt of m 7 Gppp 5'S Bn6 A m pG, contaminated with a 2'-O-TBDMS protected cap m 7 Gppp 5'S,Bn6 A m pGi BDM s ⁇
  • the solid was dissolved in DMSO (150 ⁇ L) and TEA (133 ⁇ L) followed by addition of TEA.3HF (78 ⁇ L).
  • the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-1.2 M TEAB) and purified by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous CH 3 COONH 4 buffer pH 5.9 to yield (after evaporation and repeated freeze-drying from water) an ammonium salt of m 7 Gppp Bn6 A m pGpG (2151mOD 260nm , 53,2 ⁇ mol, 27%).
  • Example 7 Preparation of capped mRNA by in vitro transcription method mRNAs encoding Gaussia luciferase were generated on template of pJET_T7_Gluc_128A plasmid digested with restriction enzyme Aarl (ThermoFisher Scientifics).
  • RNA Pol buffer 40 mM Tris-HCI pH 7.9, 10 mM MgCI 2 , 1 mM DTT, 2 mM spermidine
  • RNA Pol buffer 40 mM Tris-HCI pH 7.9, 10 mM MgCI 2 , 1 mM DTT, 2 mM spermidine
  • 10 U/mI T7 RNA polymerase 10 U/mI RiboLock RNase Inhibitor
  • 2 mM ATP/CTP/UTP 0.5 mM GTP
  • 3 mM cap analog of interest 50 ng/mI digested plasmid as a template.
  • transcripts were purified with NucleoSpin RNA Clean-up XS (Macherey-Nagel). Quality of transcripts was checked on native 1.2% 1xTBE agarose gel, whereas concentration was determined spectrophotometrically. To remove uncapped RNA, transcripts were treated with 5’-polyphosphatase (Epicentre) and Xrn1 (New England Biolabs).
  • 3T3-L1 murine embryo fibroblast-like cells, ATCC CL-173 were grown in DMEM (Gibco) supplemented with 10% FBS (Sigma), GlutaMAX (Gibco) and 1 % penicillin/streptomycin (Gibco) at 5% CO 2 and 37 °C.
  • Murine immature dendritic cell line JAWS II ATCC CRL-11904 was grown in RPMI 1640 (Gibco) supplemented with 10% FBS, sodium pyruvate (Gibco), 1% penicillin/streptomycin and 5 ng/ml GM-CSF (PeproTech) at 5% CO 2 and 37 °C.
  • Fig. 1 depicts representative chromatograms demonstrating such separation in comparison with corresponding results obtained for mRNAs obtained with an unmodified reference trinucleotide, which as demonstrated, does not enable separation of capped and uncapped mRNAs under the same conditions.
  • Table 1 compares the chromatographic properties of mRNAs modified with different cap analogs. mRNAs modified with cap analogs according to the invention were eluted from the HPLC column at retention times longer than mRNAs not capped or terminated with reference cap analogs known from the state of the art. This indicates that the introduction of a suitably hydrophobic substituent at the N6-adenosine position facilitates purification of mRNA from uncapped contaminants. Moreover, analysis of the data shown in Fig.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Saccharide Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to new mRNA 5' end cap analogs, RNA molecules containing them, their uses and methods for their in vitro synthesis, as well as a method for protein or peptide synthesis in vitro or in cell cultures, which method encompases translation of the RNA molecule.

Description

Novel mRNA 5’-end cap analogs, RNA molecule incorporating the same, uses thereof and method of synthesizing RNA molecule or peptide
Technical field
This invention relates to novel mRNA 5’-end cap analogs, an RNA molecule incorporating the same, uses thereof and a method of synthesizing the RNA molecule in vitro, as well as a method of synthesizing a protein or peptide in vitro or in cells, said method comprising translating the RNA molecule.
THE STATE OF ART
The 7-methylguanosine cap (m7G) present at the 5' end of eukaryotic mRNA plays an important role in numerous fundamental cellular processes, mainly by protecting mRNA from premature degradation and by serving as a molecular marker for proteins involved in mRNA transport and translation. [1] Therefore, chemical 5' cap modifications pave the way for the design of molecular tools for the selective modulation of cap-dependent processes and, consequently, mRNA metabolism. [2] The presence of 5' cap is needed to control mRNA and its effective translation under normal conditions. Chemically synthesized m7GpppG cap mRNA analogs are used as in vitro transcription reagents for capped mRNA. [3]
In vitro transcribed (IVT) 5'-capped mRNAs are useful tools for studying translation, transport and processing of mRNAs and are an emerging class of promising therapeutic molecules. IVT mRNA finds application in the expression of proteins in eukaryotic cells, extracts, cell cultures and even in whole organisms. Finally, IVT RNA has recently attracted considerable attention as a tool for safe exogenous protein delivery for anticancer vaccination, antiviral vaccination, and gene replacement therapy [4].
The synthesis of 5'-capped mRNA using cap analogues can be achieved by in vitro transcription. [3] In this method, called co-transcriptional capping, RNA synthesis is performed by RNA polymerase on a DNA matrix in the presence of all 4 ribonucleoside 5'-triphosphates (NTPs: ATP, GTP, CTP, UTP) and a dinucleotide cap such as m7GpppG. The DNA template is designed so that the first transcribed nucleotide is G. Polymerase initiates transcription using GTP or m7GpppG, thereby incorporating one of the nucleotides at the 5' end of the resulting RNA. To increase the percentage of cap analog incorporation (capping efficiency), GTP concentration is reduced relative to other NTPs, and dinucleotide cap concentration is increased (4-10 fold over GTP). Unfortunately, even when using high dinucleotide cap excess over GTP, the capping efficiency is less than 100% and rarely exceeds 90%. Uncapped mRNAs are much less stable and translationally active than capped mRNAs and, moreover, can lead to induction of an unwanted immune response in cells leading to a reduction in translation efficiency also for capped mRNAs [11]. A way to remove uncapped mRNAs is to treat the post-transcriptional mixture with appropriate enzymes (e.g., a mixture of 5'- polyphosphatase and 5'-exonuclease) that degrade uncapped RNA and leave the capped mRNA intact. Another limitation of the mRNA capping method using dinucleotides is the fact that the dinucleotide cap can be reversely incorporated, leading to "Gpppm7G-capped" RNAs that are translationally inactive. This problem was solved by the discovery of 'anti-reverse cap analogs' (ARCAs), which are modified at the 2'- or 3'- positions of 7-methylguanosine (usually by replacing one of the OH groups with an OCH3 group) to block reverse incorporation. [5,6]
It has already been shown that the co-transcriptional capping method enables the incorporation of various modified cap structures at the 5' mRNA end. These modified cap structures can be molecular label carriers or give mRNA molecules new properties such as increased translation efficiency and stability. Especially preferred cap analogues are among those modified in the triphosphate bridge. [7] It has been shown that substitution of even one atom in a 5', 5'-triphosphate bridge can significantly affect mRNA properties. For example, substitution of a single atom in the b position of the cap oligophosphate bridge, depicted as b- S-ARCA, resulted in a significant increase in mRNA translation efficiency in vitro and in vivo, [8,9] while substitution of a single O atom with a CH2 group at the a-b position resulted in a reduction in translation efficiency. [10] These dramatically different biological effects of different single atom substitutions within the cap indicate for the high sensitivity of the translational machinery to oligophosphate chain modifications and suggest that this is an important area for further exploration.
The objective of the invention is to provide new mRNA 5' end cap analogs that will enable higher transcription efficiency of the mRNA capped with them and a higher level of expression of proteins encoded by such mRNA than those obtained with prior art mRNA 5' end cap analogs. A particular objective of the invention is to provide new mRNA 5' end cap analogs that will enable easy separation of capped and uncapped mRNA present in the in vitro reaction mixture without the need for enzymatic treatment. Unexpectedly, the problem defined above was solved by this invention. THE DESCRIPTION OF THE INVENTION
An object of the invention is a compound of the formula:
Figure imgf000004_0001
wherein:
R1, R2, R3 , R4 are selected from the group consisting of: H, CH3, alkyl, wherein the substituents R with different numbers may be the same or different, n is 0 or 1 ,
R5 is selected from the group consisting of: benzyl, substituted benzyl, especially mono- substituted or di-substituted, preferably with a substituent selected from the group consisting of: chlorine, fluorine, bromine, iodine, methyl group, alkyl, nitro group, carboxyl group, azido group, amino group, hydroxyl group, or any combination thereof, wherein the benzyl may be ortho-, meta- or para-substituted, (1-naphthylmethyl), (2-naphthylmethyl), substituted naphthylmethyl, alkylaryl, aryl,
Basei and Base2are independently selected from the group including:
Figure imgf000004_0002
X1, X3 , are selected from a group including: O, S, Se, whereby substituents X with different numbers may be the same or different,
X2, X4, X5 are selected from the group including: O, S, Se, BH3, wherein the X substituents with different numbers may be the same or different, X6 is selected from the group including: O, CH2, CF2, CCI2,
Preferably R5 is benzyl, mono-substituted benzyl, di-substituted benzyl, 1-methylnaphthyl or 2- methylnaphthyl; X1, X4, X5 X6 are O, X2 and X3are O or S, and R3, R4 are H.
In a preferred embodiment, R5 is selected from the group consisting of: benzyl, chlorobenzyl, fluorobenzyl, bromobenzyl, iodobenzyl, methylbenzyl, alkylbenzyl, nitrobenzyl, carboxybenzyl, azidobenzyl, aminobenzyl, hydroxybenzyl, difluorobenzyl.
Preferrably, this compound was selected from a group including: compound m7GpppBn6AmpG of formula:
Figure imgf000005_0001
compound m7Gppp2MeBn6AmpG of formula:
Figure imgf000005_0002
compound rn7Gppp3MeBn6AmpG of formula:
Figure imgf000006_0001
compound m7Gppp4MeBn6AmpG of formula:
Figure imgf000006_0002
compound m7Gppp4FBn6AmpG of formula:
Figure imgf000006_0003
compound m7Gppp3,4diFBn6AmpG of formula:
Figure imgf000007_0001
compound rn7Gppp1Naphm6AmpG of formula:
Figure imgf000007_0002
compound rn7Gppp2Naphm6AmpG of formula:
Figure imgf000007_0003
compound m7GppspBn6AmpG of formula:
Figure imgf000008_0002
compound m7Gppp 5'S,Bn6AmpG of formula:
Figure imgf000008_0003
compound m7GpppBn6AmpGpG of formula:
Figure imgf000008_0001
Preferably, the compound according to the invention consists essentially of a single stereoisomer or comprises a mixture of at least two stereoisomers, a first diastereoisomer and a second diastereoisomer, the diastereoisomers being identical except that they have different stereochemical configurations around a stereogenic phosphorus atom, said stereogenic phosphorus atom being bonded to a sulfur atom, a selenium atom, or a borane group.
Another embodiment of the invention is an RNA molecule which at the 5' end contains a compound according to the invention as defined above.
A further embodiment of the invention is a method for in vitro synthesis of an RNA molecule according to the invention as defined above, said method comprising reacting ATP, CTP, UTP and GTP, a compound according to the invention as defined above, and a polynucleotide template in the presence of an RNA polymerase, under conditions that allow the RNA polymerase to synthesize RNA copies on the polynucleotide template; whereby some of the RNA copies will contain a compound according to the invention, resulting in the production of an RNA molecule according to the invention.
Another embodiment of the invention is a method for synthesizing a protein or peptide in vitro, said method comprising translating an RNA molecule according to the invention, in a cell-free protein synthesis system, the RNA molecule comprising an open reading frame, under conditions that allow translation from the open reading frame of the RNA molecule to form a protein or peptide encoded by the open reading frame.
Another embodiment of the invention is a method for synthesizing a protein or peptide in a living cell, characterized in that it comprises incorporating an RNA molecule according to the invention into a cell, said RNA molecule comprising an open reading frame, under conditions that allow translation from the open reading frame of the RNA molecule with formation of a protein or peptide encoded by said open reading frame, said cell not being contained in a patient's body.
Another embodiment of the invention is a method for purifying an RNA molecule according to the invention characterized in that it comprises using a chromatographic method, preferably a reversed-phase HPLC method, whereby column is equilibrated, sample containing an RNA molecule according to the invention is introduced onto the chromatographic column, separation of the components of the sample in a buffered aqueous solution/organic solvent system takes place, and fractions containing the RNA molecule according to the invention are collected, whereby the RNA molecules according to the invention are separated from RNA molecules that do not contain at the 5' end compounds with structures according to the invention defined above.
Another embodiment of the invention is to use a compound according to the invention in the in-vitro synthesis of an RNA molecule. Another embodiment of the invention is to use an RNA molecule according to the invention in the in-vitro synthesis of a protein or peptide.
Another embodiment of the invention is a compound according to the invention or an RNA molecule according to the invention for use in medicine, pharmacy or diagnostics.
It has surprisingly been found that the tri- or tetra-nucleotide analogues of the mRNA 5' end cap according to the invention enable easy purification of in vitro transcribed mRNA by facilitating the separation of mRNAs capped with them from uncapped mRNAs by available chromatographic methods, especially reversed-phase HPLC.
Equally surprisingly, the invention enables the production of mRNAs that have significantly higher protein expression efficiency than mRNA obtained using known mRNA 5'- end cap analogs.
Unexpectedly, these properties were obtained by introducing a hydrophobic substituent at the N6-adenosine position of the trinucleotide or tetranucleotide cap analogue. This modification results in a significant change in the migration of cap analogs and capped mRNAs on reversed-phase packed chromatography columns, allowing the separation of capped and uncapped mRNAs. Independently, this modification results in increased efficiency of mRNA expression in eukaryotic cells. Furthermore, this modification does not preclude incorporation of other previously identified cap modifications that improve mRNA properties, such as triphosphate bridge modifications or natural epigenetic modifications in the form of methylation of the 2'-O position of the first or second transcribed nucleotide, and can therefore be used together with them.
The publications cited in the description and the references given therein are hereby incorporated as references.
BRIEF DESCRIPTION OF THE FIGURES
For a better understanding of the invention, it has been illustrated in the working examples and the attached drawings, in which:
Figure 1 depicts the reversed-phase HPLC purification of mRNA obtained using various cap analogs.
Figure 2 shows protein expression in 3T3-L1 cells from HPLC-purified mRNA.
Figure 3 shows protein expression in JAWSII cells obtained from mRNAs capped with selected analogs (m7GpppApG, m2 7,2 '-OGpppSG, rn7Gpppm6AmpG, m7GpppG, m2 7,2 '-OGpppG, m2 7,2'- OGppspG D1 , m7GpppBn6AmpG) purified by HPLC .
Figure 4 shows protein expression in JAWSII cells obtained from mRNAs capped with selected analogs (rn7GpppAmpG, m7GpppBn6AmpG, m7Gppp3MeBn6AmpG, m7Gppp2MeBn6AmpG, m7Gppp4MeBn6AmpG, m7Gppp4FBn6AmpG, m7Gppp3,4diFBn6AmpG, rn7Gppp1Naphm6AmpG, rn7Gppp2Naphm6AmpG, m2 7,2 '-OGpppG) purified by HPLC. The term "alkyl" refers to a saturated, linear or branched hydrocarbon substituent with the indicated number of carbon atoms, preferably from 1 to 10 carbon atoms.
Examples of the alkyl substituent are -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n- hexyl, -n-heptyl, -n-octyl, -n-nonyl, and -n-decyl . Representative branched - (C1-C10) alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, -1-methylbutyl, -2- methylbutyl, -3-methylbutyl, -1 , 1-dimethylpropyl, -1 ,2-dimethylpropyl, -1-methylpentyl, -2- methylpentyl, -3-methylpentyl, -4-methylpentyl, -1-ethylbutyl, -2-ethylbutyl, -3-ethylbutyl, -1 , 1- dimethylbutyl, -1 ,2-dimethylbutyl, 1 ,3-dimethylbutyl, -2,2-dimethylbutyl, -2,3-dimethylbutyl, - 3,3-dimethylbutyl, -1-methylhexyl, 2-methylhexyl, - 3-methylhexyl, -4-methylhexyl, -5- methylhexyl, -1 ,2-dimethylpentyl, -1 ,3-dimethylpentyl, -1 ,2-dimethylhexyl, -1 ,3-dimethylhexyl, -3,3-dimethylhexyl , 1 ,2-dimethylheptyl, -1 ,3-dimethylheptyl, and -3,3-dimethylheptyl and the like.
The term "aryl" refers to an unsaturated, ring, aromatic or heteroaromatic (i.e. containing a heteroatom instead of carbon) hydrocarbon substituent having the indicated number of carbon atoms, preferably from 6 to 10 carbon atoms. Examples of aryl are: phenyl, naphthyl, anthracyl, phenanthryl, pyridyl.
The term "alkylaryl" refers to an unsaturated hydrocarbon substituent constructed from an alkyl and aryl portion linked together (as defined above). Examples of alkylaryl are benzyl, phenylethyl, phenylpropyl, naphthylmethyl, naphthylethyl, etc.
The term "heteroatom" means an atom selected from the group oxygen, sulfur, nitrogen, phosphorus and others.
The term "HPLC" means high performance liquid chromatography, and the solvents designated as solvents for "HPLC" mean solvents of adequate purity for HPLC (High Performance Liquid Chromatography) analysis.
The term "NMR" means Nuclear Magnetic Resonance.
WAYS OF IMPLEMENTING THE INVENTION
The following examples are provided solely to illustrate the invention and to clarify its particular aspects, and not to limit it and should not be equated with its entire scope as defined in the appended claims. In the examples below, unless otherwise indicated, standard materials and methods used in the field were used or manufacturer's recommendations for specific materials and methods were followed.
EXAMPLES
Tri- and tetranucleotide cap analogs were synthesized as described in Examples 1-6 combining solid phase synthesis and solution synthesis methods, followed by isolation using a two-step purification procedure. The starting point was the synthesis of appropriately modified oligonucleotides: dinucleotides (pA*pG, where A* stands for adenosine substituted at the N6 position) or trinucleotides (pA*pGpG) on a highly loaded support using the phosphoramidite method, as described in Example 2.[11] The corresponding phosphoramidites modified at the N6-position of adenosine were obtained by N-alkylation of commercially available adenosine phosphoramidite under phase transfer catalysis conditions, as described in Example 1. Oligonucleotides were cleaved from the support, deprotected and isolated by ion exchange chromatography as triethylammonium salts suitable for activation into P-imidazolides and subsequent coupling reaction in the presence of ZnCI2. Activated oligonucleotides were subjected to a coupling reaction with m7GDP [12] to obtain m7GpppA*(pG)k analogues, wherein A* denotes the N6-modified adenosine, and k equals 1 or 2, as described in Examples 3 and 6.
Example 3 shows examples of preparation of trinucleotide cap analogs modified at N6 position of adenosine with groups such as benzyl, substituted benzyl, 1-naphthylmethyl, 2- naphthylmethyl. Other trinucleotides, unmodified within the triphosphate bridge according to claim 1 , can be synthesized using a procedure analogous to that described in Example 3, using pA*pG dinucleotides (appropriately modified at N6 position of adenosine), obtained as described in Example 2. Examples 4 and 5 show examples of preparation of trinucleotide cap analogs modified at N6 position of adenosine with benzyl moiety and additionally containing modification within 5',5'-triphosphate bridge. The synthesis of b-thiophosphate (b- phosphorothioate) analogues (Example 4) required the transformation of pBn6AmpG into the corresponding P-imidazolide, which was then coupled with m7GDP^-S [12]. The compounds were isolated by ion exchange chromatography and further purified by RP HPLC to give ammonium salts suitable for biological studies. In the case of b-phosphorothioates, the product was obtained as a mixture of two stereoisomers that could not be separated at the RP HPLC purification stage, so they were isolated as a mixture the synthesis of 5'-phosphorothiolate cap analog (Example 5), required a 2-step modification of 5'-deprotected oligonucleotide on solid support, in order to incorporate sulfur atom at the 5' position of adenosine. Other tri- or tetranucleotides modified within triphosphate bridge can be synthesized using strategies described in Examples 1-6, using appropriate N6-modified adenosine phosphoramidites and appropriate N6-modfiied oligonucleotides (pA*pG or pA*pGpG), combined with methods of triphosphate bridge modifications described in the literature for dinucleotide cap analogs. 17, 18,
19 20 21
Example 6 shows an example of preparation of tetranucleotide cap analog, modified at N6 of adenosine position with benzyl group. Other tetranucleotides, unmodified within the triphosphate bridge according to claim 1 , can be synthesized using a procedure analogous to that described in Example 6, using pA*pGpG trinucleotides appropriately modified at N6 position of adenosine.
T ranscripts incorporating at the 5' end the compounds according to the invention or benchmark (reference) compounds were obtained by in vitro transcription in the presence of T7 RNA polymerase and DNA template containing the F6.5 promoter sequence for this polymerase. To analyze the protein expression efficiency in mammalian cells, mRNA transcripts containing compounds of the invention or reference compounds and encoding Gaussia luciferase as a reporter gene were obtained. The in vitro transcription reaction was carried out under the conditions described in Example 7. The obtained mRNAs were pre-purified and analyzed by reversed-phase HPLC under the conditions described in Example 8. The results of this analysis juxtaposed with the results of analogous analysis for mRNA obtained using the most structurally similar unmodified trinucleotide cap analog [11] (rn7Gpppm6AmpG) are shown in Fig. 1. Prior to testing for protein expression efficiency, mRNAs capped with trinucleotide cap analogs of the invention were subjected to RP HPLC purification to remove double-stranded RNA impurities as described in Example 8. In each case, an increase in the retention time of the mRNA was observed compared to the non-capped mRNA or reference mRNAs (capped with m7GpppApG, rn7GpppAmpG or rn7Gpppm6AmpG), with the retention time of a given mRNA increasing with increasing hydrophobicity of the substituent at the N6-adenosine position (Table 1). Reference mRNAs were also subjected to HPLC purification as described in Example 8, but they were beforehand subjected to a procedure for the enzymatic removal of uncapped (5'-triphosphate) mRNA as described in Example 7. The resulting mRNA capped with compounds of the invention or reference compounds were introduced into mammalian cell lines (fibroblasts - 3T3-L1 and denitric cells - JAWS II) by transfection using lipofectamine, followed by measuring the level of Gaussia luciferase expression in the extracellular medium at appropriate intervals as described in Example 9. The results of these experiments are shown in Fig. 2, Fig. 3 and Fig. 4, which show the overall (total) expression level of Gaussia luciferase obtained throughout the experiment (88 h), being the sum of Gaussia luciferase expression levels obtained at individual time points.
Example 1: Synthesis of N6-modified 2'-O-methyladenosine 3’-O-phosphoramidites (A*- CEP)
3'-0-phosphoramidite of 5'-0-dimethoxytrityl-N6-phenoxyacetyl-2'-O-methyladenosine (5 '-O- DMT-2'-O-Me-rAPac) (1 equivalent) and benzyl bromide (4 equivalents) were dissolved in CH2CI2 (to get 0.1 M solution of phosphoramidite) and mixed with an solution of tetrabutylammonium bromide (1 equivalent) in 1 M NaOH (1 volume of the reaction mixture). The mixture was stirred vigorously for 30 min and then diluted with 50 mL of water and 50 mL of diethyl ether . The layers were separated and the aqueous phase was extracted twice with diethyl ether (50 mL). Organic layers were combined, dried over anhydrous Na2SO4 and evaporated. The residue was dissolved in CH2CI2 with triethylamine (0.5% v/v) and evaporated with silica-gel. The product was isolated by flash chromatography on 20 g silica-gel column using gradient elution (0→100% ethyl acetate in n-hexane) to afford after evaporation a mixture of diastereomers of A*-CEP phosphoramidite as a white foam.
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Bn6Am-CEP: 1H NMR (500 MHz, CDCI3, 25°C): <δ = 8.60 (s, 1H, H8), 8.58 (s, 1H, H8), 8.26 (s, 1H, H2),
8.19 (s, 1H, H2), 7.46-6.60 (m, 46H, ArH), 6.15 (d, 3JH,H = 5.4 Hz, 1H, H1'), 6.13 (d, 3JH,H = 5.0 Hz, 1H, H1'), 5.65 (s, 4H, CH2 (bn6)) 5.13 (s, 4H, CH2 (Pac)), 4.67 (m, 2H, H3'), 4.54 (m, 2H, H2'), 4.41 (m, 1H, H4'), 4.35 (m, 1H, H4'), 3.90 (m, 2H, OCH2CH2CN), 3.77 (s, 12H, OCH3 DMT), 3.72-3.52 (m, 8H, OCH2CH2CN, H5', CHiPr), 3.48 (s, 6H, CH32'-O), 3.38 (dd, 2JH,H = 10.6 Hz, 3JH,H = 3.8 Hz, 2H, H5"), 2.63 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 1.19 (m, 24 H, CH3 iPr) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): <5 = 151 .0 (s, 1 P, P), 150.3 (s, 1 P, P) ppm;
2MeBn6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): δ = 8.62 (s, 1H, H8), 8.60 (s, 1H, H8), 8.24 (s, 1H, H2), 8.17 (s, 1H, H2), 7.45-6.62 (m, 44H, ArH), 6.13 (d, 3JH,H = 4.8 Hz, 1H, H1'), 6.11 (d, 3JH,H = 5.0 Hz, 1H, H1'), 5.63 (s, 4H, CH2 (2MeBn)) 5.15 (s, 4H, CH2 (Pac)), 4.65 (m, 1H, H3'), 4.57 (m, 2H, H3', H2'), 4.50 (m, 1H, H2'), 4.40 (m, 1H, H4'), 4.34 (m, 1H, H4'), 3.88 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH3 (DMTr)), 3.71-3.50 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.46 (6H, CH3 (2'-O)), 3.36 (m, 2H, H5"), 2.62 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3JH,H = 6.4 Hz, 2H, OCH2CH2CN), 2.33 (s, 6H, CH3 (2MeBn))
1.19 (m, 18 H, CH3 (iPr)), 1.07 (d, 3JH,H = 6.8 Hz, 6H, CH3 (iPr)) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): δ = 151.0 (s, 1 P, P), 150.4 (s, 1 P, P) ppm;
3MeBn6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): δ = 8.60 (s, 1H, H8), 8.59 (s, 1H, H8), 8.26 (s, 1H, H2), 8.20 (s, 1H, H2), 7.46-6.61 (m, 44H, ArH), 6.15 (d, 3JH,H = 5.2 Hz, 1H, H1'), 6.14 (d, 3JH,H = 5.1 Hz, 1H, H1'), 5.62 (s, 4H, CH2 (3MeBn)) 5.14 (s, 4H, CH2 (Pac)), 4.66 (m, 1H, H3'), 4.60 (m, 1H, H3'), 4.58 (m, 1H, H2'), 4.54 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.89 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH3 (DMTr)), 3.73-3.50 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.48 (2x s, 6H, CH3 ( 2'-O)), 3.37 (m, 2H, H5"), 2.63 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.23 (s, 6H, CH3 (3MeBn)) 1.20 (m, 18 H, CH3 0Rg)), 1.08 (d, 3JH,H = 6.8 Hz,' qH, CH3 (iPr)) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): δ = 151.1 (s, 1 P, P), 150.4 (s, 1 P, P) ppm;
4MeBn6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): <5 = 8.59 (s, 1H, H8), 8.57 (s, 1H, H8), 8.26 (s, 1H, H2), 8.19 (s, 1H, H2), 7.45-6.61 (m, 44H, AM), 6.15 (d, 3JH,H = 5.1 Hz, 1H, H1'), 6.13 (d, 3JH,H = 5.0 Hz, 1H, H1'), 5.61 (s, 4H, CH2 (4MeBn)) 5.12 (s, 4H, CH2 (Pac)), 4.67 (m, 1H, H3'), 4.60 (m, 1H, H3'), 4.58 (m, 1H, H2'), 4.54 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.89 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH3 (DMTr)), 3.73-3.50 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.48 (6H, CH3 (2'-O)), 3.36 (m, 2H, H5"), 2.63 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.37 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.22 (s, 6H, CH3 (4MeBn)) 1.20 (m, 18 H, CH3 (iPr)), 1.08 (d, 3JH,H = 6.8 Hz, 6H, CH3 (iPr)) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): δ = 151 .0 (s, 1 P, P), 150.4 (s, 1 P, P) ppm;
1Naph6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): δ = 8.59 (s, 1H, H8), 8.58 (s, 1H, H8), 8.24 (s, 1H, H2), 8.16 (s, 1H, H2), 8.14 (d, 3JH,H = 8.4 Hz, 2H, ArtHNaph), 7.78 (d, 3JH,H = 8.1 Hz, 2H, ArHNaph), 7.63 (d, 3JH,H = 8.2 Hz, 2H, ArHNaph), 7.51 (m, 2H, ArHNaph), 7.45 (m, 2H, ArHNaph), 7.43-6.63 (m, 40H, AM), 6.13 (s, 4H, CH2 (1NaphCH2)), 6.11 (d, 3JH,H = 4.9 Hz, 1H, H1'), 6.10 (d, 3JH,H = 5.0 Hz, 1H, H1'), 5.17 (s, 4H, CH2 (Pac)), 4.63 (m, 1H, H3'), 4.56 (m, 2H, H3', H2'), 4.48 (m, 1H, H2'), 4.38 (m, 1H, H4'), 4.33 (m, 1H, H4'), 3.87 (m, 2H, OCH2CH2CN), 3.75 (4x s, 12H, OCH3 (DMTr)), 3.70-3.53 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.44 (6H, CH3 (2'-O)), 3.34 (m, 2H, H5"), 2.60 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.34 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 1.17 (d, 3JH,H = 6.7 Hz, 18 H, CH3 (iPr)), 1.06 (d, 3JH,H = 6.7 Hz, 6H, CH3 (iPr)) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): δ = 151.0 (s, 1 P, P), 150.4 (s, 1 P, P) ppm;
2Naph6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): <δ = 8.52 (s, 1H, H8), 8.51 (s, 1H, H8), 8.21 (s, 1H, H2), 8.14 (s, 1H, H2), 7.66 (m, 4H, ArHNaph), 7.60 (m, 4H, ArHNaph), 7.39-6.55 (m, 42H, AM), 6.07 (d, 3JH,H = 4.8 Hz, 1H, H1'), 6.05 (d, 3JH,H = 4.8 Hz, 1H, H1'), 5.74 (s, 4H, CH2 (1NaphCH2)), 5.09 (s, 4H, CH2 (Pac)), 4.58 (m, 1H, H3'), 4.50 (m, 2H, H3', H2'), 4.44 (m, 1H, H2'), 4.32 (m, 1H, H4'), 4.27 (m, 1H, H4'), 3.79 (m, 2H, OCH2CH2CN), 3.69 (4x s, 12H, OCH3 (DMTr)), 3.64-3.44 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.38 (6H, CH3 (2'-O)), 3.28 (m, 2H, H5"), 2.53 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.28 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 1.10 (m, 18 H, CH3 (iPr)), 0.99 (d, 3JH,H = 6.8 Hz, 6H, CH3 (iPr)) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 151 .0 (s, 1 P, P), 150.4 (s, 1 P, P) ppm;
4FBn6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): δ = 8.61 (s, 1H, H8), 8.59 (s, 1H, H8), 8.28 (s, 1H, H2), 8.21 (s, 1H, H2), 7.45-6.59 (m, 43H, AM), 6.16 (d, 3JH,H = 4.7 Hz, 1H, H1'), 6.15 (d, 3JH,H = 4.9 Hz, 1H, H1'), 5.57 (s, 4H, CH2 (4FBn)) 5.12 (s, 4H, CH2 (Pac)), 4.69 (m, 1H, H3'), 4.64-4.60 (m, 2H, H3', H2'), 4.55 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.89 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH3 (DMTr)), 3.73-3.51 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.48 (6H, CH3 (2'-O)), 3.36 (m, 2H, H5"), 2.63 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.38 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 1.20 (m, 18 H, CH3 (iPr)), 1.08 (d, 3JH,H = 6.8 Hz, 6H, CH3 (iPr)) ppm; 19F NMR (470.6 MHz, D2O, NaF, 25°C): δ = -115.4 (m, 1 F, F) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): δ = 151.0 (s, 1 P, P), 150.4 (s, 1 P, P) ppm;
3’4diFBn6Am-CEP: 1H NMR (500 MHz, CDCI3, TMS, 25°C): δ = 8.62 (s, 1H, H8), 8.61 (s, 1H, H8), 8.30 (s, 1H, H2), 8.23 (s, 1H, H2), 7.46-6.58 (m, 42H, AM), 6.17 (d, 3JH,H = 4.9 Hz, 1H, H1'), 6.16 (d, 3JH,H = 4.7 Hz, 1H, H1'), 5.53 (s, 4H, CH2 (3,4diFBn)) 5.13 (s, 4H, CH2 (Pac)), 4.70 (m, 1H, H3'), 4.65-4.58 (m, 2H, H3', H2'), 4.54 (m, 1H, H2'), 4.41 (m, 1H, H4'), 4.36 (m, 1H, H4'), 3.88 (m, 2H, OCH2CH2CN), 3.77 (4x s, 12H, OCH3 (DMTr)), 3.73-3.51 (m, 8H, OCH2CH2CN, H5', CH(iPr)), 3.48 (6H, CH3 (2'-O)), 3.37 (m, 2H, H5"), 2.63 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 2.38 (t, 3JH,H = 6.3 Hz, 2H, OCH2CH2CN), 1.20 (m, 18 H, CH3 (iPr)), 1 .08 (d, 3JH,H = 6.8 Hz, 6H, CH3 (iPr)) ppm; 19F NMR (470.6 MHz, D2O, NaF, 25°C): δ = -137.8 (m, 1 F, F), -140.0 (m, 1 F, F) ppm; 31P NMR (202.5 MHz, CDCI3, H3PO4, 25°C): δ = 150.9 (s, 1 P, P), 150.4 (s, 1 P, P) ppm; Example 2: Synthesis of 5'-phosphorylated di- and trinucleotides (pA*pG or pA*pGpG)
Synthesis of dinucleotide was performed manually using a 10 mL syringe equipped with a frit filter. The solid support (5'-O-DMT-2'-O-TBDMS-rGiBu 3 -lcaa PrimerSupport 5G, GE Healthcare, 308 μmol/g) was placed in a syringe and washed with dry acetonitrile. In the coupling step, 1.2-1 .5 equivalents of phosphoramidite (A* -CEP ,G-CEP or bis-(2-cyanoethyl)- N,N-diisopropylphosphoramidite) dissolved in 1 mL of anhydrous acetonitrile and 1.5 mL of 0.30 M 5-(benzylthio)-1-H-tetrazole in acetonitrile were shaken for 15 min in a syringe capped with a plunger. A solution of 3% (v/v) trichloroacetic acid in dichloromethane was used as a detritylation reagent and 0.05 M iodine in pyridine/water (9:1 v/v) for oxidation. After the last cycle of synthesis, RNAs, still on the solid support, were treated with 20% (v/v) diethylamine in acetonitrile to remove 2-cyanoethyl protecting groups. Finally, the solid support was washed with acetonitrile and dried with argon. The product was cleaved from the solid support and deprotected with AMA (40% methylamine / 33% ammonium hydroxide 1 :1 v/v; 55 °C, 1 h), evaporated to dryness and redissolved in DMSO (200 μL). The TBDMS group was removed using triethylammonium trihydrofluoride (TEA.3HF; 250 μL, 65 °C, 3 h), and then the mixture was cooled down and diluted with 0.25 M NaHCO3 in water (20 mL). The product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-0.9 M TEAB) to afford after evaporation a triethylammonium salt of pA*pG dinucleotide. The yield was estimated by UV absorption at 260 nm, assuming the extinction coefficient e = 27.1 L/mmol/cm for dinucleotides or 39.0 L/mmol/cm for trinucleotides.
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
p2MeBn6AmpG (TEAH+): 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.41 (s, 1H, H8A), 8.14 (s, 1H, H2A), 7.93 (s, 1H, H8G), 7.30-7.15 (m, 4H, ArH2MeBn), 6.11 (d, 3JH,H = 5.1 Hz, 1H, H1'A), 5.83 (d, 3JH,H = 5.2 Hz, 1H, H1'G), 4.92 (m, 1H, H3'A), 4.76 (m,2H, CH overlapped with H DO), 4.72 (m, 1H, H2'G), 4.50-4.47 (m, 2H, H2'A, H3'G), 4.46 (s, 1H, H4'A), 4.34 (s, 1H, H4'G), 4.25-4.17 (m, 2H, H5'G, H5"G), 4.12-4.04 (m, 2H, H5'A, H5"A), 3.50 (s, 3H, CH3, 2-O[A]), 3.36-3.04 (m, CH2, TEAH+), 2.36 (s, 3H, CH3, 2MeBn), 1.27 (m, CH3,TEAH+) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 1.38 (s, 1 P, P5'A), 0.04 (s, 1 P, PA-G) ppm; p4MeBn6AmpG (TEAH+): 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.39 (s, 1H, H8A), 8.14 (s, 1H, H2A), 7.93 (s, 1H, H8G), 7.27 (d, 3JH,H = 8.0 Hz, 2H, ArH4MeBn(3&5)), 7.18 (d, 3JH,H = 7.9 Hz, 2H, ArH4MeBn(2&6)), 6.10 (d, 3JH,H = 5.0 Hz, 1H, H1'A), 5.83 (d, 3JH,H = 5.2 Hz, 1H, H1'G), 4.92 (m, 1H, H3'A), 4.79 (m, 2H, CH2, Bn, overlapped with HDO), 4.72 (m, 1H, H2'G), 4.50-4.45 (m, 3H, H2'A, H3'G, H4'A), 4.34 (s, 1H, H4'G), 4.25-4.17 (m, 2H, H5'G, H5"G), 4.12-4.05 (m, 2H, H5'A, H5"A), 3.50 (s, 3H, CH3, 2-O[A]), 3.20 (q, 3JH,H = 7.3 Hz, CH2, TEAH+), 2.29 (s, 3H, CH3,2MeBn), 1.28 (t, 3JH,H = 7.3 Hz, CH3, TEAH+) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): <δ 1=.06 (s, 1 P, P5'A), 0.05 (s, 1 P, PA-G) ppm; p1Naphm6AmpG (TEAH+): 1H NMR (50o MHz, DIQ TMS, 25°C): δ = 8.30 (s, 1H, H8A), 8.07 (s, 1H, H2A), 8.04 (m, 1H, ArHNaph), 7.91 (s, 1H,H8G), 7.88 (m, 1H, ArHNaph), 7.80 (m, 1H, ArHNaph), 7.57-7.40 (m,4H, ArHNaph), 6.05 (d, 3JH,H = 5.0 Hz, 1H, H1'A), 5.80 (d, 3JH,H = 5.2 Hz, 1H, H1'G), 5.17 (d, 2JH,H = 14.9 Hz, 1H, CH2,Naphm), 5.05 (d, 2JH,H = 14.9 Hz, 1H, CH2, Naphm), 4.92 (m, 1H, H3'A), 4.71 (m, 1H,’ H2'G), 4.50- 4.45 (m, 3H, H2'A, H3'G, H4'A), 4.34 (s, 1H, H4'G), 4.26-4.17 (m, 2H, H5'G, H5"G), 4.09 (m, 2H, H5'A,
H5"A), 3.51 (s, 3H, CH3,2’-O[A]), 3.33-3.03 (m, CH2,TEAH+), 1.29-1.24 (m, CH3, TEAH+) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ= 1.39 (s, 1 P, P5'A), 0.06 (s, 1 P, PA-G) ppm; p2Naphm6AmpG (TEAH+): 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.37 (s, 1H, H8A), 8.02 (s, 1H, H2A), 7.88 (s, 1H, H8G), 7.78 (m, 2H, ArHNaph), 7.71 (m, 2H, ArHNaph), 7.43 (m, 3H, ArHNaph), 5.97 (d, 3JH,H = 4.7 Hz, 1H, H1'A), 5.78 (d, 3JH,H = 5.1 Hz, 1H, H1'G), 4.94-4.86 (m, 3H, H3'A, CH2, Naphm), 4.67 (m, 1H, H2'G), 4.49-4.43 (m, 3H, H2'A, H3'G, H4'A), 4.33 (s, 1H, H4'G), 4.25-4.16 (m, 2H, H5'G, H5"G), 4.11 (m, 2H, H5'A, H5"A), 3.49 (s, 3H, CH3, 2-o[A]), 3.35-3.03 (m, CH2, TEAH+), 1.29-1.24 (m, CH3, TEAH+) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 1 .31 (s, 1 P, P5'A), 0.04 (s, 1P, PA-G) ppm; p4FBn6AmpG (TEAH+): 1H NMR (500 MHz, D2O, TMS, 25°C): <δ = 8.40 (s, 1H, H8A), 8.15 (s, 1H, H2A), 7.93 (s, 1H, H8G), 7.39 (m, 2H, ArH4FBn), 7.09 (m, 2H, ArH4FBn), 6.11 (d, 3JH,H = 5.1 Hz, 1H, H1'A), 5.83 (d, 3JH,H = 5.2 Hz, 1H, H1'G), 4.93 (m, 1H, H3'A), 4.83 (m, 2H, CH2, Bn, overlapped with HDO), 4.73 (m, 1H, H2'G), 4.50-4.44 (m, 3H, H2'A, H3'G, H4'A), 4.35 (s, 1H, H4'G), 4.25-4.17 (m, 2H, H5'G, H5"G), 4.12- 4.04 (m, 2H, H5'A, H5"A), 3.50 (s, 3H, CH3, 2'-O[A]), 3.20 (q, 3JH,H = 7.3 Hz, CH2, TEAH+), 1.28 (t, 3JH,H = 7.3 Hz, CH3, TEAH+) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C ): <δ = 1.08 (s, 1 P, P5'A), 0.06 (s, 1 P, PA-G) ppm; p3'4diFBn6AmpG (TEAH+): 1H NMR (500 MHz, D2O, TMS, 25°C): 5 = 8.42 (s, 1H, H8A), 8.14 (s, 1H, H2A), 7.92 (s, 1H, H8G), 7.30-7.14 (m, 3H, ArH3,4diFBn), 6.12 (d, 3JH,H = 5.1 Hz, 1H, H1'A), 5.83 (d, 3JH,H = 5.3 Hz, 1H, H1'G), 4.93 (m, 1H, H3'A), 4.83 (m, 2H, CH overlapped with HDO), 4.74 (m, 1H, H2'G), 4.51- 4.45 (m, 3H, H2'A, H3'G, H4'A), 4.35 (s, 1H, H4'G), 4.26-4.17 (m, 2H, H5'G, H5"G), 4.12-4.05 (m, 2H, H5'A, H5”A), 3.50 (s, 3H, CH3, 2'-O[A]), 3.20 (q, 3JH,H = 7.3 Hz, CH2 , EAH+), 1.28 (t, 3JH,H = 7.3 Hz, CH3, TEAH+ ) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): 5 = 1.20 (s, 1 P, P5'A), 0.06 (s, 1 P, PA-G) ppm; pBn6AmpGpG (TEAH+): 1H NMR (500 MHz, D2O, TMS, 25°C) d = 8.39 (s, 1H), 8.09 (s, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.47 - 7.29 (m, 5H), 6.07 (d, J = 5.3 Hz, 1H), 5.82 (d, J = 5.5 Hz, 1H), 5.76 (d, J = 5.0 Hz, 1H), 4.95 - 4.89 (m, 1H), 4.79 (1H, overlapped with HDO), 4.79 (1H, overlapped with HDO), 4.71 (t, J = 5.5 Hz, 1H), 4.48 (t, J = 5.3 Hz, 1H), 4.47 - 4.43 (m, 4H), 4.34 - 4.30 (m, 1H), 4.29 - 4.14 (m, 4H), 4.08 - 3.98 (m, 2H), 3.46 (s, 3H), 3.20 (q, J = 7.3 Hz), 1 .28 (t, J = 7.3 Hz) ppm; 31P NMR (202 MHz, D2O, H3PO4, 25°C) δ = 1.73 - 1.43 (m, 1 P), 0.41 - 0.27 (m, 1 P), -0.04 - -0.13 (m, 1 P) ppm.
Example 3: Synthesis of trinucleotide cap analogs m7GpppA*pG
Step 1. Activation of pNpG: Dinucleotide 5'-phosphate was dissolved in DMF (to obtain a 0.05 M solution) followed by addition of imidazole (16 equivalents), 2,2'-dithiodipiridine (6 equivalents), triethylamine (6 equivalents) and triphenylphosphine (6 equivalents). The mixture was stirred at room temperature for 2 h. The product was precipitated by addition of a solution of sodium perchlorate (10 equivalents) in acetonitrile (10 times the volume of DMF). The precipitate was centrifuged at 4°C, washed with cold acetonitrile 3 times and dried under reduced pressure to give a sodium salt of dinucleotide P-imidazolide (Im-pNpG).
Step 2. Formation of triphosphate bridge: 7-Methylguanosine 5'-diphosphate (m7GDP; 1.5 equivalent) and Im-pNpG (1 equivalent) were suspended in DMF (to obtain a 0.05 M solution of P-imidazolide). Then ZnCI2 (8 equivalents) were added and the mixture was stirred at room temperature for 2 h. The reaction was quenched by addition of a solution of Na2EDTA (20 mg/mL ; 8 equivalents) and NaHCO3 (10 mg/mL ) in water and the product was isolated by ion- exchange chromatography on DEAE Sephadex (gradient elution 0-1 .2 M TEAB) to afford after evaporation a triethylammonium salt of m7Gpppbn6AmpG. Additional purification by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous CH3COONH4 buffer pH 5.9 provided (after repeated freeze-drying from water) an ammonium salt of m7GpppA*pG. The yield was estimated by UV absorption at 260 nm, assuming the extinction coefficient e = 35.0 L/mmol/cm).
Figure imgf000022_0001
Figure imgf000023_0001
m7GpppBn6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 9.06 (s, 1H, H8m7G), 8.46 (s, 1H, H8A), 8.19 (s, 1H, H2A), 8.03 (s, 1H, H8G), 7.44-7.31 (m, 5H, ArHBn), 6.03 (d, 3JH,H = 5.6 Hz, 1H, H1'A), 5.88 (d, 3JH,H = 3.7 Hz, 1H, H1'm7G), 5.84 (d, 3JH,H = 5.8 Hz, 1H, H1'G), 4.93 (m, 1H, H3'A), 4.81 (m, 1H, H2'G, overlapped with HDO ), 4.85-4.74 (m, 2H, CH2, Bn, overlapped with HDO), 4.59 (m, 1H, H2'm7G), 4.53-4.49 (m, 2H, H4'A, H3'G), 4.48-4.44 (m, 2H, H2'A, H3'm7G), 4.38-4.16 (m, 8H, H4'm7G, H4'G, H5'm7G, H5"m7G, H5'A, H5"A, H5'G, H5"G), 4.02 (s, 3H, CH3, m7G), 3.43 (s, 3H, CH3, 2'-O) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.03 (s, 1 P, PA-G), - 10.58 (m, 2P, Pα, PY), -21.98 (t, 2JP,P = 18.4 Hz, 1P, Pβ) ppm; m7Gppp2MeBn6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): d = 9.08 (s, 1H, H8m7G), 8.50 (s, 1H, H8A), 8.21 (s, 1H, H2A), 8.06 (s, 1H, H8G), 7.29-7.12 (m, 4H, ArH2MeBn), 6.04 (d, 3JH,H = 5.5 Hz, 1H, H1'A), 5.89 (d, 3JH,H = 3.4 Hz, 1H, H1'm7G), 5.85 (d, 3JH,H = 5.6 Hz, 1H, H1'G), 4.95 (m, 1H, H3'A), 4.81 (m, 1H, H2'G, overlapped with HDO), 4.75 (m, 2H, CH2, Bn, overlapped with HDO), 4.59 (m, 1H, H2'm7G), 4.54-4.44 (m, 4H, H2'A, H3'm7G, H3'G, H4'A), 4.38-4.15 (m, 8H, H4'm7G, H4'G, H5'm7G, H5"m7G, H5'A, H5"A, H5'G, H5”G), 4.02 (s, 3H, CH3, m7G), 3.43 (s, 3H, CH3, 2'-O), 2.34 (s, 3H, CH3, 2MeBn) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.04 (s, 1 P, PA-G), -10.56 (m, 2P, Pα, PY), -21.98 (m, 1P, Pβ) ppm; m7Gppp3MeBn6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): d = 9.07 (s, 1H, H8m7G), 8.48 (s, 1H, H8A), 8.20 (s, 1H, H2A), 8.04 (s, 1H, H8G), 7.29-7.12 (m, 4H, ArH3MeBn), 6.03 (d, 3JH,H = 5.5
Hz, 1H, H1'A), 5.88 (d, 3JH,H = 3.5 Hz, 1H, H1'm7G), 5.84 (d, 3JH,H = 5.7 Hz, 1H, H1'G), 4.93 (m, 1H, H3'A), 4.83 (m, 1H, H2'G, overlapped with HDO), 4.76 (m, 2H, CH2, Bn, overlapped with
HDO), 4.58 (m, 1H, H2'm7G), 4.52-4.44 (m, 4H, H2'A, H3'm7G, H3'G, H4'A), 4.38-4.15 (m, 8H,
H4'm7G, H4'G, H5'm7G, H5"m7G, H5'A, H5"A, H5'G, H5”G), 4.01 (s, 3H, CH3, m7G), 3.42 (s, 3H, CH3, 2'-O), 2.27 (s, 3H, CH3, 2MeBn) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.02 (s, 1 P,
PA-G), -10.60 (m, 2P, Pα, PY), -22.00 (t, 2JP,P = 18.4 Hz, 1P, Pβ) ppm; m7Gppp4MeBn6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): d = 9.07 (s, 1H, H8m7G), 8.49 (s, 1H, H8A), 8.19 (s, 1H, H2A), 8.06 (s, 1H, H8G), 7.22 (m, 2H, ArH4MeBn), 7.11 (m, 2H, ArH4MeBn), 6.02 (d, 3JH,H = 5.4 Hz, 1H, H1'A), 5.88 (d, 3JH,H = 3.6 Hz, 1H, H1'm7G), 5.84 (d, 3JH,H = 5.6 Hz, 1H, H1'G), 4.94 (m, 1H, H3'A), 4.79 (m, 1H, H2'G, overlapped with HDO), 4.71 (m, 2H, CH2, Bn), 4.58 (m, 1H, H2'm7G), 4.54-4.48 (m, 2H, H3'G, H4'A), 4.45 (m, 2H, H2'A, H3'm7G), 4.39-4.16 (m, 8H, H4'm7G, H4'G, H5'm7G, H5"m7G, H5'A, H5"A, H5'G, H5"G), 4.01 (s, 3H, CH3, m7G), 3.44 (s, 3H, CH3, 2'-O), 2.24 (s, 3H, CH3, 2MeBn) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.02 (s, 1P, PA-G), -10.56 (m, 2P, Pα, PY), -21.92 (m, 1P, Pβ) ppm; rn7Gppp1Naphm6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.97 (s, 1H, H8m7G), 8.33 (s, 1H, H8A), 8.03 (s, 2H, H2A, H8G), 7.80-7.25 (m, 7H, ArHNaph), 5.94 (m, 1H, H1'A), 5.79 (d, 3JH,H = 5.3 Hz, 1H, H1'G), 5.73 (m, 1H, Hrm7G), 4.97 (m, 1H, H3'A), 4.83 (m, 2H, CH2, Bn, overlapped with HDO), 4.73 (m, 1H, H2'G), 4.54 (m, 1H, H4'A), 4.51-4.17 (m, 12H, H2'm7G, H2'A, H3'm7G, H3'G, H4'm7G, H4'G, H5'm7G, H5''m7G, H5'A, H5"A, H5'G, H5"G), 3.84 (s, 3H, CH3, m7G), 3.50 (s, 3H, CH3, 2'-O) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.02 (s, 1 P, PA-G), -10.56 (m, 2P, Pα, Pγ), -21.85 (m, 1P, Pβ) ppm; m7Gppp2Naphm6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 8.93 (s, 1H, H8m7G), 8.46 (s, 1H, H8A), 8.04 (s, 2H, H2A, H8G), 7.72-7.19 (m, 7H, ArHNaph), 5.89 (m, 1H, H1'A), 5.79 (d, 3JH,H = 5.3 Hz, 1H, H1'G), 5.71 (m, 1H, Hrm7G), 4.97 (m, 1H, H3'A), 4.83 (m, 2H, CH2, Bn, overlapped with HDO), 4.71 (m, 1H, H2'G), 4.54 (m, 1H, H4'A), 4.51-4.16 (m, 12H, H2'm7G, H2'A, H3'm7G, H3'G, H4'm7G, H4'G, H5'm7G, H5''m7G, H5'A, H5"A, H5'G, H5"G), 3.77 (s, 3H, CH3, m7G), 3.49 (s, 3H, CH3, 2'-O) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.03 (s, 1 P, PA-G), -10.56 (m, 2P, Pc Py), -21.75 (m, 1P, Pβ) ppm; m7Gppp4FBn6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 9.08 (s, 1H, H8m7G), 8.47 (s, 1H, H8A), 8.19 (s, 1H, H2A), 8.05 (s, 1H, H8G), 7.38 (m, 2H, ArH4FBn), 7.06 (m, 2H, ArH4FBn), 6.03 (d, 3JH,H = 5.5 Hz, 1H, H1'A), 5.88 (d, 3JH,H = 3.3 Hz, 1H, H1'm7G), 5.84 (d, 3JH,H = 5.7 Hz, 1H, H1'G), 4.94 (m, 1H, H3'A), 4.81 (m, 1H, H2'G, overlapped with HDO), 4.84-4.74 (m, 2H, CH2, Bn, overlapped with HDO), 4.58 (m, 1H, H2'm7G), 4.54-4.48 (m, 2H, H3'G, H4'A), 4.46 (m, 2H, H2'A, H3'm7G), 4.39-4.15 (m, 8H, H4'm7G, H4'G, H5'm7G, H5”m7G, H5'A, H5"A, H5'G, H5"G), 4.02 (s, 3H, CH3, m7G), 3.43 (s, 3H, CH3, 2'-O) ppm; 19F NMR (470.6 MHz, D2O, NaF, 25°C): δ = -115.35 (s, 1 F, F) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.04 (s, 1 P, PA-G), -10.58 (m, 2P, Pα, Py), -21.99 (m, 1P, Pβ) ppm; m7Gppp3,4diFBn6AmpG: 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 9.09 (s, 1H, H8m7G), 8.48 (s, 1H, H8A), 8.19 (s, 1H, H2A), 8.08 (s, 1H, H8G), 7.30-7.14 (m, 3H, ArH3,4diFBn), 6.03 (d, 3JH,H = 5.6 Hz, 1H, H1'A), 5.88 (d, 3JH,H = 3.6 Hz, 1H, Hrm7G), 5.85 (d, 3JH,H = 5.7 Hz, 1H, H1'G), 4.94 (m, 1H, H3'A), 4.81 (m, 1H, H2'G, overlapped with HDO), 4.80 (m, 2H, CH2 Bn, overlapped with HDO), 4.59 (m, 1H, H2'm7G), 4.52 (m, 1H, H4'A), 4.50 (m, 1H, H3'G), 4.48-4.44 (m, 2H, H2'A, H3'm7G), 4.38-4.16 (m, 8H, H4'm7G, H4'G, H5'm7G, H5"m7G, H5'A, H5"A, H5'G, H5"G), 4.02 (s, 3H, CH3, m7G), 3.43 (s, 3H, CH3, 2'-O) ppm; 19F NMR (470.6 MHz, D2O, NaF, 25°C): δ = -138.22 (s, 1 F, F), -140.39 (s, 1 F, F) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 0.03 (s, 1 P, PA- G), -10.58 (m, 2P, Pα, PY), -21.97 (m, 1 P, Pβ) ppm;
Example 4: Synthesis of b-phosphorothioate trinucleotide cap analog m7GppspBn6AmpG
Step 1. Activation of pBn6AmpG: Dinucleotide 5 -phosphate pBn6AmpG (615 mOD260nm, 22.7 μmoI) was dissolved in DMF (400 μL) followed by addition of imidazole (24.7 mg, 363 μmoI), 2,2'-dithiodipiridine (30 mg, 136 μmoI), triethylamine (9.5 μL, 68 μmoI) and triphenylphosphine (35.7 mg, 136 μmoI). The mixture was stirred at room temperature for 48 h. The product was precipitated by addition of a solution of lithium perchlorate (24.1 mg, 227 μmoI) in acetonitrile (4.0 mL ). The precipitate was centrifuged at 4°C, washed with cold acetonitrile 3 times and dried under reduced pressure to give a lithium salt of dinucleotide P-imidazolide lm-pBn6AmpG (19 mg).
Step 2. Formation of triphosphate bridge: 7-Methylguanosine b-thiodiphosphate m7GDP-β-S (378 mOD260nm, 33.2 μmoI; obtained as described earlier and stored in TEAB at -20°C)[12] was evaporated to dryness and suspended in DMF (890 μL). Then ZnCI2 (24.1 mg, 177 μmoI) and lm-pBn6AmpG (19 mg) were added and the mixture was stirred at room temperature for 2 h. The reaction was quenched by addition of a solution of Na2EDTA (72.5 mg) and NaHCO3 (36 mg) in water (3.6 mL) and the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-1.2 M TEAB) to afford after evaporation triethylammonium salt of m7GppspBn6AmpG. Additional purification by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous CH3COONH4 buffer pH 5.9 provided (after repeated freeze-drying from water) a P-diastereomeric mixture of m7GppSpBn6AmpG (217 mOD260nm, 6.8 μmol, 31%) as an ammonium salt. m7GppsPBn6AmpG: RP HPLC (gradient elution 0-50% MeOH in CH3COONH4 pH 5.9 in 7,5 min, then isocratically): Rt = 8,311 min; HRMS ESI(-): m/z 1250.17041 (calcd. for C39H48N15O23P4S- [M-H]- 1250.17241); 1H NMR (500 MHz, D2O, TMS, 25°C): d = 9.13 (s, 1H, H8m7G), 8.55 (s, 1H, H8A), 8.22 (s, 1H, H2A), 8.07 (s, 1H, H8G), 7.41-7.25 (m, 5H, ArHBn), 6.05 (2x d, 3JH,H = 5.9 Hz, 1H, H1'A), 5.91 (2x d, 3JH,H = 3.7 Hz, 1H, H1'm7G), 5.85 (d, 3JH,H = 5.6 Hz, 1H, H1'G), 5.00 (m, 1H, H3'A), 4.81 (m, 1H, H2'G overlapped with HDO), 4.85-4.73 (m, 2H, CH2, Bn, overlapped with HDO), 4.63 (m, 1H, H2'm7G), 4.58-4.49 (m, 4H, H2'A, H3'm7G, H3'G, H4'A), 4.42-4.16 (m, 8H, H4'm7G, H4'G, H5'm7G, H5''m7G, H5'A, H5"A, H5'G, H5"G), 4.04-4.01 (2x s, 3H, CH3, m7G), 3.45-3.43 (2x s, 3H, CH3 2'.O) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): δ = 30.92 (m, 1 P, Pβ), 0.06- 0.01 (2x s, 1 P, PA-G), -11.45 (m, 2P, Pα, PY) ppm; Example 5: Synthesis of 5'-phosphorothiolate trinucleotide cap analog m7Gppp5 S,Bn6AmpG
Step 1. Synthesis of pBn6, 5'S AmpG: Synthesis of dinucleotide was performed manually using a 10 mL syringe equipped with a frit filter. The solid support (5'-O-DMT-2'-O-TBDMS-rGiBu 3'- lcaa PrimerSupport 5G, GE Healthcare, 308 μmol/g, 650 mg) was placed in a syringe and washed with dry acetonitrile. In the coupling step, 1.2 - 1.5 equivalents of Bn6Am phosphoramidite dissolved in 1 mL of anhydrous acetonitrile and 1.5 mL of 0.30 M 5-(benzylthio)-1-H-tetrazole in acetonitrile were shaken for 30 min in a syringe capped with a plunger. A solution of 3% (v/v) trichloroacetic acid in dichloromethane was used as a detritylation reagent and 0.05 M iodine in pyridine/water (9:1 v/v) for oxidation. After the last cycle of synthesis, the support was treated with 20% (v/v) diethylamine in acetonitrile to remove 2-cyanoethyl protecting groups, washed with acetonitrile and dried with argon. Dinucleotide, still on a solid support, was then converted into 5'-iodo derivative by shaking the support with a solution of triphenoxymethylphosphonium iodide (1 .08 g) in DMF (5 mL) for 15 minutes. The resin was then washed with DMF (15 mL) and acetonitrile (50 ml), dried and transferred to a flask containing a cold solution of triethylammonium thiophosphate (ca. 0.15 M) and triethylamine (0.30 M) in DMF (4 mL). The slurry was shaken at 2-4°C overnight, filtered and washed with DMF (15 mL) and then acetonitrile (50 mL). The product was cleaved and deprotected using AMA (methylamine/ammonium hydroxide 1 :1 v/v; 55 °C, 1 h) and isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-0.9 M TEAB) to afford after evaporation a triethylammonium salt of p5'S,Bn6AmpG (2400 mOD, 88.5 μmol).
Step 2. Activation of pBn6,5'SAmpG: Dinucleotide 5'-phosphorothiolate pBn6,5'SAmpG (2400 mOD260nm, 88.5 μmol) was dissolved in DMF (1.80 mL) followed by addition of imidazole (96.3 mg, 1.42 mmol), 2,2'-dithiodipiridine (117 mg, 531 μmol), triethylamine (74.2 μL, 531 μmol) and triphenylphosphine (139 mg, 531 μmol). The mixture was stirred at room temperature for 2 h. The product was precipitated by addition of a solution of sodium perchlorate (108 mg, 885 μmol) in cold acetonitrile (20 mL). The precipitate was centrifuged at 4°C, washed with cold acetonitrile 3 times and dried under reduced pressure to give a sodium salt of dinucleotide P-imidazolide lm-pbn6AmpG (92.2 mg).
Step 3. Formation of triphosphate bridge: 7-Methylguanosine 5'-diphosphate (m7GDP; 1210 mOD260nm, 106 μmol) and lm-p5'S,Bn6AmpG (92.2 mg) were suspended in DMF (1 .77 mL). Then ZnCI2 (144 mg, 1.06 mmol) were added and the mixture was stirred at room temperature for 2 h. The reaction was quenched by addition of a solution (3 mL) of Na2EDTA (20 mg/mL) and NaHCO3 (10 mg/mL) in water and the product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-1.2 M TEAB) to afford after evaporation triethylammonium salt of m7Gppp5'S Bn6AmpG, contaminated with a 2'-O-TBDMS protected cap m7Gppp5'S,Bn6AmpGiBDMs· To remove the TBDMS group, the solid was dissolved in DMSO (150 μL) and TEA (133 μL) followed by addition of TEA.3HF (78 μL). The mixture was shaken at 60°C for 1 h and the reaction was quenched by addition of water (15 mL). The product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-1.2 M TEAB) and additionally purified by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous CH3COONH4 buffer pH 5.9 to give (after repeated freeze-drying from water) an ammonium salt of m7Gppp5'S,Bn6AmpG (1530 mOD, 47.7 μmol). m7Gppp5'S,Bn6AmpG: RP HPLC (gradient elution 0-50% MeOH in CH3COONH4 pH 5.9 in 7,5 min, then isocratically): Rt = 8.496 min; HRMS ESI(-): m/z 1250.17363 (calcd. for C39H48N15O23P4S- [M-H]- 1250.17241); 1H NMR (500 MHz, D2O, TMS, 25°C): δ = 9.08 (s, 1H, H8m7G), 8.37 (s, 1H, H8A), 8.22 (s, 1H, H2A), 8.11 (s, 1H, H8G), 7.43-7.25 (m, 5H, ArHBn), 6.00 (d, 3JH,H = 5.8 Hz, 1H, H1'A), 5.89 (d, 3JH,H = 2.9 Hz, 1H, H1'm7G), 5.86 (d, 3JH,H = 5.5 Hz, 1H, H1'G), 4.86 (m, 1H, H3'A), 4.80 (m, 1H, H2'G, overlapped with HDO), 4.78 (m, 2H, CH2, Bn, overlapped with HDO), 4.58 (m, 1H, H2'm7G), 4.56-4.49 (m, 3H, H2'A, H3'G, H4'A), 4.47 (m, 1H, H3'm7G), 4.42-4.18 (m, 6H, H4'm7G, H4'G, H5'm7G, H5"m7G, H5'G, H5"G), 4.02 (s, 3H, CH3, m7G), 3.40 (s, 3H, CH3,2'-O), 3.33 (m, 2H, H5'A, H5"A) ppm; 31P NMR (202.5 MHz, D2O, H3PO4, 25°C): 5 = 8.19 (m, 1 P, Pα), -0.09 (s, 1P, PA-G), -10.59 (m, 1P, PY), -22.67 (m, 1 P, Pβ) ppm;
Example 6: Synthesis of tetranucleotide cap analog (m7GpppBn6AmpGpG)
Step 1. Synthesis of lm-pBn6AmpGpG: Triethylammonium salt of pBn6AmpGpG (5366 mOD260nm, 138 μmol) was dissolved in DMF (2800 μL) followed by addition of imidazole (150 mg, 2201 μmol), 2,2'-dithiodipiridine (182 mg, 826 μmol), triethylamine (84 μL, 826 μmol) and triphenylphosphine (216 mg, 826 μmol). The mixture was stirred at room temperature for 2 h and then the product was precipitated by addition of a cold solution of sodium perchlorate (169 mg, 1376 μmol) in acetonitrile. The precipitate was centrifuged at 4°C, washed with cold acetonitrile 3 times and dried under reduced pressure over P2O5 to give a sodium salt of lm-pBn6AmpGpG (183 mg). lm-pBn6AmpGpG: RP-HPLC (gradient elution 0-50% MeOH in CH3COONH4 pH 5.9 in 7,5 min, then isocratically): Rt = 8.865 min;
Step 2. Synthesis of m7GpppBn6AmpGpG: The sodium salt of lm-pbn6AmpGpG (5366 mOD260nm, 138 μmol) was dissolved in DMF (2750 μL) followed by addition of m7GDP (1882 mOD260nm, 99 mg, 165 μmol) and anhydrous ZnCI2 (225 mg, 1651 μmol). The mixture was stirred at room temperature for 1 h and then the reaction was quenched by addition of a solution of Na2EDTA (676 mg) and NaHCO3 (338 mg) in water (27.5 mL ). The product was isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-1.2 M TEAB) and purified by RP HPLC (C18) using a linear gradient of acetonitrile in aqueous CH3COONH4 buffer pH 5.9 to yield (after evaporation and repeated freeze-drying from water) an ammonium salt of m7GpppBn6AmpGpG (2151mOD260nm, 53,2 μmol, 27%). The yield was estimated by UV absorption at 260 nm, assuming the extinction coefficient e = 40.5 L/mmol/cm). m7GpppBn6AmpGpG: RP-HPLC (gradient elution 0-50% MeOH in CH3COONH4 pH 5.9 in 7,5 min, then isocratically): Rt = 7.840 min; HRMS ESI(-): m/z 1579.24313 (calcd. for C49H60N20O31P5- [M-H]- 1579.24269); 1H NMR (500 MHz, D2O, TMS, 70°C) d = 9.55 (s, 1H), 8.96 (s, 1H), 8.72 (s, 1H), 8.55 (s, 1H), 8.53 (s, 1H), 7.92 - 7.76 (m, 6H), 6.50 (d, J = 6.2 Hz, 1H), 6.39 (d, J = 4.1 Hz, 1H), 6.33 (d, J = 5.4 Hz, 1H), 6.28 (d, J = 5.9 Hz, 1H), 5.42 - 5.31 (m, 5H), 5.30 - 5.25 (m, 1H), 5.18 (t, J = 5.3 Hz, 1H), 5.10 (t, J = 4.6 Hz, 1H), 4.98 - 4.88 (m, 6H), 4.73 - 4.61 (m, 7H), 4.51 (s, 3H), 3.82 (s, 3H) ppm; 31P NMR {1H} (203 MHz, D2O, H3PO4, 70°C) δ = 0.82 (s, 1 P), 0.40 (s, 1 P), -10.06 (d, J = 19.0, 1 P), -10.18 (d, J = 18.4 Hz, 1 P), -21.52 (dd, J = 19.0, 18.4 Hz) ppm;
Example 7: Preparation of capped mRNA by in vitro transcription method mRNAs encoding Gaussia luciferase were generated on template of pJET_T7_Gluc_128A plasmid digested with restriction enzyme Aarl (ThermoFisher Scientifics). The plasmid was obtained by cloning the T7 promoter sequence and coding sequence of Gaussia luciferase into pJET_luc_128A.[13] Typical in vitro transcription reaction (20 μl) was incubated at 37 °C for 2 h and contained: RNA Pol buffer (40 mM Tris-HCI pH 7.9, 10 mM MgCI2, 1 mM DTT, 2 mM spermidine), 10 U/mI T7 RNA polymerase, 1 U/mI RiboLock RNase Inhibitor, 2 mM ATP/CTP/UTP, 0.5 mM GTP, 3 mM cap analog of interest and 50 ng/mI digested plasmid as a template. Following 2 h incubation, 1 U/mI DNase I was added and incubation was continued for 30 min at 37 °C. The crude mRNAs were purified with NucleoSpin RNA Clean-up XS (Macherey-Nagel). Quality of transcripts was checked on native 1.2% 1xTBE agarose gel, whereas concentration was determined spectrophotometrically. To remove uncapped RNA, transcripts were treated with 5’-polyphosphatase (Epicentre) and Xrn1 (New England Biolabs). Briefly, mRNAs were incubated with 5’-polyphosphatase (20U / 5 μg of mRNA) in dedicated buffer for 30 min at 37 °C, then mRNAs were purified with NucleoSpin RNA Clean-up XS. Purified mRNAs were subjected to incubation with Xrn-1 (1 U / 1 μg of mRNA) in dedicated buffer for 60 min at 37 °C, then mRNAs were purified with NucleoSpin RNA Clean-up XS.
Example 8: Purification of capped mRNA using HPLC
Transcripts were purified using Agilent Technologies Series 1200 HPLC, for which the RNASep™ Prep - RNA Purification Column (ADS Biotec) was used. The separation was carried out at 55 °C as described in [14]. A linear gradient of 35% to 55% buffer B (0.1 M triethylammonium acetate pH 7.0 and 25% acetonitrile) in buffer A (0.1 M triethylammonium acetate pH 7.0) at a flow rate of 0.9 ml/min for 22.1 min (gradient A) or 17.5% to 25.8% buffer C (0.1 M triethylammonium acetate, pH 7.0, and 50% acetonitrile) in buffer A (0.1 M triethylammonium acetate, pH 7.0) at a flow rate of 0.9 ml/min over 20 min (gradient B). The retention times of the mRNA according to the invention compared with uncapped mRNA of the same sequence or mRNA capped with reference analogs are summarized in Table 1. After purification, the mRNA molecules were recovered by precipitation with isopropanol from the collected fractions. The quality of the transcripts was checked on a native 1 .2% 1xTBE agarose gel, and their concentration was determined spectrophotometrically.
Importantly, the use of the above-described purification procedure allowed in each case the separation of mRNAs encoding Gaussia luciferase possessing novel cap analogs (m7GpppA*pG-RNA) from non-capped RNA of the same sequence (ppp-RNA). The retention time at which mRNA elution occurred depended on the type of substituent at the N6-adenosine position (Table 1 ). For example, using gradient A, the retention times of the m7GpppBn6AmpG- RNAGluc and ppp-RNAGluc transcripts analyzed were 19.4 min for and 18.2 min, respectively.
Figure imgf000029_0001
Example 9: Protein expression analysis
3T3-L1 (murine embryo fibroblast-like cells, ATCC CL-173) were grown in DMEM (Gibco) supplemented with 10% FBS (Sigma), GlutaMAX (Gibco) and 1 % penicillin/streptomycin (Gibco) at 5% CO2 and 37 °C. Murine immature dendritic cell line JAWS II (ATCC CRL-11904) was grown in RPMI 1640 (Gibco) supplemented with 10% FBS, sodium pyruvate (Gibco), 1% penicillin/streptomycin and 5 ng/ml GM-CSF (PeproTech) at 5% CO2 and 37 °C. In a typical experiment, 104 of JAWS II cells and 4.103 of 3T3-L1 cells were seeded at the day of transfection in 100 μl medium without antibiotics per well of 96-well plate. Cells in each well were transfected for 16 h using a mixture of 0.3 mI Lipofectamine MessengerMAX Transfection Reagent (Invitrogen) and 25 ng mRNA in 10 mI Opti-MEM (Gibco). In order to assess Gaussia luciferase expression at multiple time points, medium was fully removed and replaced with the fresh one at each time point. To detect luminescence from Gaussia luciferase, 50 μI of 10 ng/ml h-coelenterazine (NanoLight) in PBS was added to 10 μI of cell cultured medium and the luminescence was measured on Synergy H1 (BioTek) microplate reader.
Conclusions
Examples 1-6 decribe methods for preparing tri- and tetranucleotide cap analogs according to the invention. Realizations of the invention, the synthesis of which has not been described in examples, can be executed by methods identical or very similar to those exemplified. Examples 7 and 8 describe a method for the preparation and purification of mRNAs obtained with the use of compounds according to the invention, in conditions enabling separation of capped mRNAs from uncapped mRNAs.
Fig. 1 depicts representative chromatograms demonstrating such separation in comparison with corresponding results obtained for mRNAs obtained with an unmodified reference trinucleotide, which as demonstrated, does not enable separation of capped and uncapped mRNAs under the same conditions. Table 1 compares the chromatographic properties of mRNAs modified with different cap analogs. mRNAs modified with cap analogs according to the invention were eluted from the HPLC column at retention times longer than mRNAs not capped or terminated with reference cap analogs known from the state of the art. This indicates that the introduction of a suitably hydrophobic substituent at the N6-adenosine position facilitates purification of mRNA from uncapped contaminants. Moreover, analysis of the data shown in Fig. 1 (integration of the corresponding signals), enabled calculation of capping efficiency for mRNAs obtained with compound according to the invention, which was 91.5%. %. This indicates that when using the trinucleotide analogues of the cap according to the invention, it is possible to obtain capping efficiencies comparable to the trinucleotides without the modification according to the invention, for which the capping efficiencies obtained in the literature under similar conditions were about 90% [12].
Example 9 describes the method of analyzing protein expression in mammalian cells from mRNAs according to the invention obtained with compounds according to the invention. The analysis was performed in two cell lines representing cells from different origins (fibroblasts - 3T3-L1 and dendritic cells - JAWS II). In the case of mRNAs obtained with the use of compounds according to the invention, enzymatic treatment was not performed because it was redundant. mRNA obtained with the use of compounds according to the invention showed increased protein expression levels in comparison with mRNA obtained with cap analogs representing the state-of-the-art in at least one of the tested experimental variants.
Achieving increased protein expression has a lot of applications in biotechnology and production of biopharmaceutics (production of human recombinant proteins) as well as in mRNA-based gene therapies. Increased protein expression in dendritic cells is particularly beneficial in the case of application in therapeutic anti-cancer vaccines. [15] Increased protein expression in cells derived from other tissues (e.g. lungs, liver, other organs) is particularly beneficial in the case of application in gene replacement therapies. [16] One can expect that achieving the therapetic effect for the mRNAs according to the invention will be possible at lower doses compared to mRNAs obtained with state-of-the-art methods. Lowering the mRNA dose entails lower risk of side effects associated with toxicity of therapeutic mRNA, and thereby, increases the probability of therapeutic succes. Moreover, one can expect that preparation of mRNA with the use of compounds according to the invention will enable production of mRNAs devoid of undesired mRNA triphosphate impurities.
REFERENCES
1. Moore, M., From birth to death: The complex lives of eukaryotic mRNAs. Science 2005, 309 (5740), 1514-1518.
2. Ziemniak, M.; Strenkowska, M.; Kowalska, J.; Jemielity, J., Potential therapeutic applications of RNA cap analogs. Future Medicinal Chemistry 2013, 5 (10), 1141-1172.
3. Grudzien-Nogalska, E.; Stepinski, J.; Jemielity, J.; Zuberek, J.; Stolarski, R.; Rhoads, R. E.; Darzynkiewicz, E., Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability. Translation Initiation: Cell Biology, High-Throughput Methods, and Chemical-Based Approaches 2007, 431, 203-227.
4. Sahin, U.; Kariko, K.; Tureci, O., mRNA-based therapeutics - developing a new class of drugs. Nature Reviews Drug Discovery 2014, 13 (10), 759-780.
5. Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E., Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3 '-O- methyl)GpppG and 7-methyl(3 '-deoxy)GpppG. Rna-a Publication of the Rna Society 2001, 7 (10), 1486-1495.
6. Jemielity, J.; Fowler, T.; Zuberek, J.; Stepinski, J.; Lewdorowicz, M.; Niedzwiecka, A.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E., Novel "anti-reverse" cap analogs with superior translational properties. Rna-a Publication of the Rna Society 2003, 9 (9), 1108-1122.
7. Jemielity, J.; Kowalska, J.; Rydzik, A. M.; Darzynkiewicz, E., Synthetic mRNA cap analogs with a modified triphosphate bridge - synthesis, applications and prospects. New Journal of Chemistry 2010, 34 (5), 829-844. 8. Grudzien-Nogalska, E.; Jemielity, J.; Kowalska, J.; Darzynkiewicz, E.; Rhoads, R. E., Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. Rna-a Publication of the Rna Society 2007 , 13 (10), 1745-1755.
9. Kuhn, A. N.; Diken, M.; Kreiter, S.; Selmi, A.; Kowalska, J.; Jemielity, J.; Darzynkiewicz, E.; Huber, C.; Tureci, O.; Sahin, U., Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Therapy 2010, 17 (8), 961-971.
10. Grudzien, E.; Kalek, M.; Jemielity, J.; Darzynkiewicz, E.; Rhoads, R. E., Differential inhibition of mRNA degradation pathways by novel cap analogs. Journal of Biological Chemistry 2006, 281 (4), 1857-1867.
11. Sikorski, Pawel J; Warminski, Marcin; Kubacka, Dorota; Ratajczak, Tomasz; Nowis, Dominika; Kowalska, Joanna; Jemielity, Jacek; The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells. Nucleic Acids Research 2020, 305-1048.
12. J. Kowalska, M. Lewdorowicz, J. Zuberek, E. Grudzien-Nogalska, E. Bojarska, J. Stepinski, R. E. Rhoads, E. Darzynkiewicz, R. E. Davis, J. Jemielity, RNA 2008, 14, 1119- 1131.
13. M. Warminski, P. J. Sikorski, Z. Warminska, M. Lukaszewicz, A. Kropiwnicka, J. Zuberek, E. Darzynkiewicz, J. Kowalska, J. Jemielity, Bioconjugate Chemistry 2017, 28, 1978- 1992.
14. D. Weissman, N. Pardi, H. Muramatsu, K. Kariko, Methods Mol Biol 2013, 969, 43-54.
15. Norbert Pardi, Michael J. Hogan, Frederick W. Porter, Drew Weissman, N. Nature
Reviews Drug Discovery vol. 17, 261-279 (2018).
16. Berraondo P, Martini PGV, Avila MA, et al Messenger RNA therapy for rare genetic metabolic diseases Gut 2019;68:1323-1330.
17. Anna Maria Rydzik et. al., Organic & Biomolecular Chemistry, Issue 22, 2009.
18. M. Kalek et al., Bioorganic & Medicinal Chemistry, Vol. 14, Issue 9, 1 May 2006, Pages
3223-3230
19. J. Kowalska et.al. (2009), Phosphoroselenoate Dinucleotides for Modification of mRNA 5' End. ChemBioChem, 10: 2469-2473.
20. J. Kowalska, et. al., Nucleic Acids Research, Volume 42, Issue 16, 15 September 2014, Pages 10245-10264,
21. A. Rydzik et.al., Nucleic Acids Research, Volume 45, Issue 15, 6 September 2017, Pages 8661-8675,

Claims

1. A compound of formula:
Figure imgf000033_0001
wherein:
R1, R2, R3 and R4 are selected from the group consisting of: H, CH3 and alkyl, where R substituents with different numbers may be the same or different, n is 0 or 1 ,
R5 is selected from the group consisting of: benzyl, substituted benzyl, especially mono- or disubstituted benzyl, preferably wherein benzyl is substituted with one of the following substituents: chlorine, fluorine, bromine, iodine, methyl, alkyl, nitro group, carboxyl group, azide group, amine group, hydroxyl group or a combination thereof, 1-naphthylmethyl, 2- nphtylmethyl, substituted naphthylmethyl, alkylaryl, aryl,
Basei and Base2 are independently selected from the group consisting of:
Figure imgf000033_0002
X1, X3, are selected from a group including: O, S, Se, whereby substituents X with different numbers may be the same or different
X2, X4 and X5 are selected from the group consisting of: O, S, Se, BH3, wherein X substituents with different numbers can be the same or different,
X6 is selected from the group consisting of: O, CH2, CF2, CCI2, wherein preferably R5 is benzyl, monosubstituted benzyl, disubstituted benzyl, 1-napthylmethyl or 2-naphtylmethyl, X1, , X4, X5, X6 are O, X2, X3 are O or S, R3, R4 are H.
2. A compound according to claim 1 , wherein said compound is selected from the group consisting of: compound m7GpppBn6AmpG of formula:
Figure imgf000034_0001
compound m7Gppp2MeBn6 ’AmpG of formula:
Figure imgf000034_0002
compound m7Gppp3MeBn6AmpG of formula:
Figure imgf000034_0003
compound m7Gppp4MeBn6AmpG of formula:
Figure imgf000035_0001
compound m7Gppp4FBn6AmpG of formula:
Figure imgf000035_0002
compound m7Gppp , 3,4diFBn6 AmpG of formula:
Figure imgf000035_0003
compound rn7Gppp1Naphm6AmpG of formula:
Figure imgf000036_0003
compound m7Gppp2Naphm6AmpG of formula:
Figure imgf000036_0001
compound m7GppspBn6AmpG of formula:
Figure imgf000036_0002
compound m7Gppp5 S,Bn6AmpG of formula:
Figure imgf000037_0002
compound m7GpppBn6AmpGpG of formula:
Figure imgf000037_0001
3. A compound according to claim 1 , wherein said compound consists essentially of a single stereoisomer or comprises a mixture of at least two stereoisomers, the first diastereomer and the second diastereomer, wherein the diastereomers are otherwise identical, except that they have different stereochemical configurations around the stereogenic phosphorus atom, wherein a stereogenic phosphorus atom is attached to a sulfur atom, selenium atom, or borane group.
4. An RNA molecule whose 5' end incorporates a compound as defined in any one of claims 1-3.
5. A method for synthesizing an RNA molecule as defined in claim 4 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a compound as defined in claims 1-3, and a polynucleotide template in the presence of RNA polymerase, under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; wherein some of the RNA copies will incorporate the compound as defined in any of claims 1-3 to make an RNA molecule as defined in claim 4.
6. An in vitro protein or peptide synthesis method, said method comprising translating the RNA molecule according to claim 4, in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.
7. A method for synthesizing a protein or peptide in living cell, said method comprising introducing an RNA molecule as defined in claim 4 into cells, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame, wherein said cell is not contained in patent’s body.
8. A method of purifying a molecule as defined in claim 4, wherein said method comprises using a chromatographic method, preferably a reversed-phase HPLC method, whereby the column is equilibrated, a sample containing a molecule as defined in claim 4 is introduced onto the chromatographic column, the components of the sample are separated in a buffered aqueous solution/organic solvent system, the fractions containing the molecule as defined in claim 4 are collected, wherein mRNA molecules defined in claim 4 are separated from other RNA molecules that do not have at the 5' end structures as defined in claims 1-3..
9. The use of a compound as defined in any one of claims 1-3 in in-vitro synthesis of RNA molecules.
10. The use of an RNA molecule as defined in claim 4 in in-vitro synthesis of protein or peptide.
11 . A compound as defined in claims 1-3 or the RNA molecule as defined in claim 4 for use in medicine, pharmacy or diagnostics.
PCT/PL2021/050007 2020-02-12 2021-02-12 Novel mrna 5'-end cap analogs, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide Ceased WO2021162567A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2021219563A AU2021219563A1 (en) 2020-02-12 2021-02-12 Novel mRNA 5'-end cap analogs, RNA molecule incorporating the same, uses thereof and method of synthesizing RNA molecule or peptide
CA3167570A CA3167570A1 (en) 2020-02-12 2021-02-12 Novel mrna 5'-end cap analogs, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide
EP21754142.4A EP4103577A4 (en) 2020-02-12 2021-02-12 NOVEL 5' END CAP ANALOGS OF MRNA, RNA MOLECULE INCORPORATING THEM, USES THEREOF AND METHODS OF SYNTHESIS OF RNA MOLECULE OR PEPTIDE
US17/798,099 US12606590B2 (en) 2020-02-12 2021-02-12 MRNA 5′-end cap analogs, RNA molecule incorporating the same, uses thereof and method of synthesizing RNA molecule or peptide
JP2022548990A JP7805938B2 (en) 2020-02-12 2021-02-12 Novel mRNA 5'-end cap analogs, RNA molecules incorporating same, their uses, and methods for synthesizing RNA molecules or peptides
KR1020227030072A KR20220163359A (en) 2020-02-12 2021-02-12 Novel mRNA 5'-end cap analogues, RNA molecules incorporating them, uses thereof and methods for synthesizing RNA molecules or peptides
JP2025162162A JP2026012182A (en) 2020-02-12 2025-09-29 Novel mRNA 5'-end cap analogs, RNA molecules incorporating same, their uses, and methods for synthesizing RNA molecules or peptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL432883A PL248416B1 (en) 2020-02-12 2020-02-12 New analogs of 5' mRNA end cap, RNA molecule that contains them, their applications and method for synthesis of the RNA molecule and peptide
PLP.432883 2020-02-12

Publications (1)

Publication Number Publication Date
WO2021162567A1 true WO2021162567A1 (en) 2021-08-19

Family

ID=77292947

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/PL2021/050007 Ceased WO2021162567A1 (en) 2020-02-12 2021-02-12 Novel mrna 5'-end cap analogs, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide

Country Status (8)

Country Link
US (1) US12606590B2 (en)
EP (1) EP4103577A4 (en)
JP (2) JP7805938B2 (en)
KR (1) KR20220163359A (en)
AU (1) AU2021219563A1 (en)
CA (1) CA3167570A1 (en)
PL (1) PL248416B1 (en)
WO (1) WO2021162567A1 (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021162566A1 (en) 2020-02-12 2021-08-19 Uniwersytet Warszawski Novel mrna 5'-end cap analogs modified within phosphate residues, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide
WO2022006368A3 (en) * 2020-07-02 2022-03-17 Life Technologies Corporation Trinucleotide cap analogs, preparation and uses thereof
US11414453B2 (en) 2015-09-21 2022-08-16 Trilink Biotechnologies, Llc Compositions and methods for synthesizing 5′-capped RNAs
WO2024075022A2 (en) 2022-10-04 2024-04-11 BioNTech SE Rna constructs and uses thereof
WO2024044741A3 (en) * 2022-08-26 2024-04-18 Trilink Biotechnologies, Llc Efficient method for making highly purified 5'-capped oligonucleotides
EP4464713A1 (en) 2023-05-18 2024-11-20 Siec Badawcza Lukasiewicz - PORT Polski Osrodek Rozwoju Technologii Circular rna analogs, preparation and application thereof
US12264175B2 (en) 2020-09-04 2025-04-01 Verve Therapeutics, Inc. Compositions and methods for capping RNAS
WO2025054401A3 (en) * 2023-09-06 2025-05-08 Trilink Biotechnologies, Llc Cap analogs and methods of use thereof
WO2025147660A2 (en) 2024-01-04 2025-07-10 Trilink Biotechnologies, Llc Modified rna for increasing protein expression
EP4606900A1 (en) * 2024-02-21 2025-08-27 ExploRNA Therapeutics Sp. z o. o. Modified rna for the treatment of cfdna-associated diseases
WO2025187713A1 (en) * 2024-03-07 2025-09-12 Eisai R&D Management Co., Ltd. Polynucleotide and producing method thereof
WO2026010855A1 (en) 2024-07-02 2026-01-08 Trilink Biotechnologies, Llc Modified rna for increasing protein expression
EP4486355A4 (en) * 2022-03-01 2026-04-22 Verve Therapeutics Inc COMPOSITIONS AND METHODS FOR CAPPING RNAS

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017053297A1 (en) * 2015-09-21 2017-03-30 Trilink Biotechnologies, Inc. Compositions and methods for synthesizing 5'-capped rnas

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11866754B2 (en) 2015-10-16 2024-01-09 Modernatx, Inc. Trinucleotide mRNA cap analogs
KR20200143391A (en) 2018-03-15 2020-12-23 비온테크 알엔에이 파마슈티컬스 게엠베하 5'-cap-trinucleotide- or higher order oligonucleotide compounds and their use in RNA stabilization, protein expression and therapy
PL432884A1 (en) 2020-02-12 2021-08-16 Uniwersytet Warszawski New analogs of the 5 'end cap of mRNA, the RNA molecule containing them, their applications and the method of synthesizing RNA and peptide molecules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017053297A1 (en) * 2015-09-21 2017-03-30 Trilink Biotechnologies, Inc. Compositions and methods for synthesizing 5'-capped rnas

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
A. RYDZIK, NUCLEIC ACIDS RESEARCH, vol. 45, 6 September 2017 (2017-09-06), pages 8661 - 8675
ANNA MARIA RYDZIK, ORGANIC & BIOMOLECULAR CHEMISTRY, 2009
BERRAONDO PMARTINI PGVAVILA MA ET AL., MESSENGER RNA THERAPY FOR RARE GENETIC METABOLIC DISEASES GUT, vol. 68, 2019, pages 1323 - 1330
D. WEISSMANN. PARDIH. MURAMATSUK. KARIKÓ, METHODS MOLBIOL, vol. 969, 2013, pages 43 - 54
E. GRUDZIEN: "RNA", vol. 10, 1 September 2004, COLD SPRING HARBOR LABORATORY PRESS, pages: 1479 - 1487
GARCIA A.: "Use of N*1-Benzyladenosine as an Analog of InternalMethylation (N^-Methyladenosine) in Studies of Post-Transcriptional Regulation of RNA in Human CellsInfected by Herpes Simplex Virus", METABOLISM AND MOLECULAR ACTIVITIES OF CYTOKINS, 1 January 1981 (1981-01-01), pages 329 - 330, XP093254062
GRUDZIEN EWA ET AL: "Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency", RNA, vol. 10, no. 9, 1 September 2004 (2004-09-01), US , pages 1479 - 1487, XP002452227, ISSN: 1355-8382, DOI: 10.1261/rna.7380904
GRUDZIEN, E.KALEK, M.JEMIELITY, J.DARZYNKIEWICZ, E.RHOADS, R. E.: "Differential inhibition of mRNA degradation pathways by novel cap analogs", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 281, no. 4, 2006, pages 1857 - 1867, XP002452226, DOI: 10.1074/jbc.M509121200
GRUDZIEN-NOGALSKA, E.JEMIELITY, J.KOWALSKA, J.DARZYNKIEWICZ, E.RHOADS, R. E: "Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells", RNA-A PUBLICATION OF THE RNA SOCIETY, vol. 13, no. 10, 2007, pages 1745 - 1755, XP002554207, DOI: 10.1261/rna.701307
GRUDZIEN-NOGALSKA, E.STEPINSKI, J.JEMIELITY, J.ZUBEREK, J.STOLARSKI, R.RHOADS, R. E.DARZYNKIEWICZ, E.: "Synthesis of anti-reverse cap analogs (ARCAs) and their applications in mRNA translation and stability", TRANSLATION INITIATION: CELL BIOLOGY, HIGH-THROUGHPUT METHODS, AND CHEMICAL-BASED APPROACHES, vol. 431, 2007, pages 203 - 227
ISHIKAWA MASAHIDE ET AL.: "Preparation of eukaryotic mRNA having differently methylated adenosine at the 5'-terminus and the effect of the methyl group in translation", NUCLEIC ACIDS SYMPOSIUM SERIES, vol. 53, no. 1, 27 September 2009 (2009-09-27), pages 129 - 130, XP055331388, DOI: 10.1093/nass/nrp065 *
J. KOWALSKA, NUCLEIC ACIDS RESEARCH, vol. 42, 15 September 2014 (2014-09-15), pages 10245 - 10264
J. KOWALSKA: "Phosphoroselenoate Dinucleotides for Modification of mRNA 5' End", CHEMBIOCHEM, vol. 10, 2009, pages 2469 - 2473, XP055367433, DOI: 10.1002/cbic.200900522
J. KOWALSKAM. LEWDOROWICZJ. ZUBEREKE. GRUDZIEN-NOGALSKAE. BOJARSKAJ. STEPINSKIR. E. RHOADSE. DARZYNKIEWICZR. E. DAVISJ. JEMIELITY, RNA, vol. 14, 2008, pages 1119 - 1131
JEMIELITY, J.FOWLER, T.ZUBEREK, J.STEPINSKI, J.LEWDOROWICZ, M.NIEDZWIECKA, A.STOLARSKI, R.DARZYNKIEWICZ, E.RHOADS, R. E.: "Novel ''anti-reverse'' cap analogs with superior translational properties", RNA-A PUBLICATION OF THE RNA SOCIETY, vol. 9, no. 9, 2003, pages 1108 - 1122, XP002466761, DOI: 10.1261/rna.5430403
JEMIELITY, J.KOWALSKA, J.RYDZIK, A. M.DARZYNKIEWICZ, E.: "Synthetic mRNA cap analogs with a modified triphosphate bridge - synthesis, applications and prospects", NEW JOURNAL OF CHEMISTRY, vol. 34, no. 5, 2010, pages 829 - 844, XP055747726, DOI: 10.1039/c0nj00041h
JOUANNEAU JEAN-PIERRE, TEYSSENDIER DE BERNARD, SERVE L A, PEAUD-LENOEL CLAUDE: "Frequency of N6-Benzyladenine Incorporation into Major RNA Fractions of Tobacco Cell Suspensions Grown at Stimulatory or Cytostatic Cytokinin Concentration1", PLANT PHYSIOL, vol. 74, 1 January 1984 (1984-01-01), pages 663 - 668, XP093254060
KUHN, A. N.; DIKEN, M.KREITER, S.SELMI, A.KOWALSKA, J.JEMIELITY, J.DARZYNKIEWICZ, E.HUBER, C.TURECI, O.SAHIN, U.: "Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo", GENE THERAPY, vol. 17, no. 8, 2010, pages 961 - 971
M. ISHIKAWA, R. MURAI, H. HAGIWARA, T. HOSHINO, K. SUYAMA: "Preparation of eukaryotic mRNA having differently methylated adenosine at the 5'-terminus and the effect of the methyl group in translation", NUCLEIC ACIDS SYMPOSIUM SERIES, vol. 53, no. 1, 1 September 2009 (2009-09-01), GB , pages 129 - 130, XP055331388, ISSN: 0261-3166, DOI: 10.1093/nass/nrp065
M. KALEK ET AL., BIOORGANIC & MEDICINAL CHEMISTRY, vol. 14, 1 May 2006 (2006-05-01), pages 3223 - 3230
M. WARMINSKIP. J. SIKORSKIZ. WARMINSKAM. LUKASZEWICZA. KROPIWNICKAJ. ZUBEREKE. DARZYNKIEWICZJ. KOWALSKAJ. JEMIELITY, BIOCONJUGATE CHEMISTRY, vol. 28, 2017, pages 1978 - 1992
MOORE, M.: "From birth to death: The complex lives of eukaryotic mRNAs", SCIENCE, vol. 309, no. 5740, 2005, pages 1514 - 1518, XP093191378
NORBERT PARDIMICHAEL J. HOGANFREDERICK W. PORTERDREW WEISSMAN, N. NATURE REVIEWS DRUG DISCOVERY, vol. 17, 2018, pages 261 - 279
PAWEL J SIKORSKI, WARMINSKI MARCIN, KUBACKA DOROTA, RATAJCZAK TOMASZ, NOWIS DOMINIKA, KOWALSKA JOANNA, JEMIELITY JACEK: "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5′ cap modulates protein expression in living cells", NUCLEIC ACIDS RESEARCH, vol. 48, no. 4, 28 February 2020 (2020-02-28), GB , pages 1607 - 1626, XP055747723, ISSN: 0305-1048, DOI: 10.1093/nar/gkaa032
PAWELJ. SIKORSKI ET AL.: "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5 cap modulates protein expression in living cells", NUCLEIC ACIDS RESEARCH, vol. 48, no. 4, 27 January 2020 (2020-01-27), pages 1607 - 1626, XP055747723, DOI: 10.1093/nar/gkaa032 *
SAHIN, U.KARIKO, K.TURECI, O.: "mRNA-based therapeutics - developing a new class of drugs", NATURE REVIEWS DRUG DISCOVERY, vol. 13, no. 10, 2014, pages 759 - 780, XP055544673, DOI: 10.1038/nrd4278
See also references of EP4103577A1
SIKORSKI, PAWEL JWARMINSKI, MARCINKUBACKA, DOROTARATAJCZAK, TOMASZNOWIS, DOMINIKAKOWALSKA, JOANNAJEMIELITY, JACEK: "The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells", NUCLEIC ACIDS RESEARCH, 2020, pages 305 - 1048
STEPINSKI, J.WADDELL, C.STOLARSKI, R.DARZYNKIEWICZ, E.RHOADS, R. E.: "Synthesis and properties of mRNAs containing the novel ''anti-reverse'' cap analogs 7-methyl(3 '-0-methyl)GpppG and 7-methyl(3 '-deoxy)GpppG", RNA-A PUBLICATION OF THE RNA SOCIETY, vol. 7, no. 10, 2001, pages 1486 - 1495, XP002466762
WARMINSKI MARCIN, TREPKOWSKA EDYTA, SMIETANSKI MIROSLAW, SIKORSKI PAWEL J., BARANOWSKI MAREK R., BEDNARCZYK MARCELINA, KEDZIERSKA : "Trinucleotide mRNA Cap Analogue N 6-Benzylated at the Site of Posttranscriptional m6 A m Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 146, no. 12, 27 March 2024 (2024-03-27), pages 8149 - 8163, XP093254059, ISSN: 0002-7863, DOI: 10.1021/jacs.3c12629
ZIEMNIAK, MSTRENKOWSKA, M.KOWALSKA, J.JEMIELITY, J.: "Potential therapeutic applications of RNA cap analogs", FUTURE MEDICINAL CHEMISTRY, vol. 5, no. 10, 2013, pages 1141 - 1172, XP055573135, DOI: 10.4155/fmc.13.96

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878991B2 (en) 2015-09-21 2024-01-23 Trilink Biotechnologies, Llc Compositions and methods for synthesizing 5′-Capped RNAs
US12103944B2 (en) 2015-09-21 2024-10-01 Trilink Biotechnologies, Llc Compositions and methods for synthesizing 5′-capped RNAs
US11414453B2 (en) 2015-09-21 2022-08-16 Trilink Biotechnologies, Llc Compositions and methods for synthesizing 5′-capped RNAs
US11578095B2 (en) 2015-09-21 2023-02-14 Trilink Biotechnologies, Llc Compositions and methods for synthesizing 5'-Capped RNAs
EP3906789B1 (en) 2015-09-21 2023-11-22 TriLink BioTechnologies, LLC Initiating capped oligonucleotide primers for synthesizing 5'-capped rnas
US12258369B2 (en) 2015-09-21 2025-03-25 Trilink Biotechnologies, Llc Compositions and methods for synthesizing 5′-capped RNAs
WO2021162566A1 (en) 2020-02-12 2021-08-19 Uniwersytet Warszawski Novel mrna 5'-end cap analogs modified within phosphate residues, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide
EP4103578A4 (en) * 2020-02-12 2024-01-10 Uniwersytet Warszawski NOVEL 5' END CAP ANALOGS OF MRNA MODIFIED IN PHOSPHATE RESIDUES, RNA MOLECULE INCORPORATING THE SAME, THEIR USES AND METHOD FOR SYNTHESIS OF AN RNA MOLECULE OR A PEPTIDE
US12385039B2 (en) 2020-07-02 2025-08-12 Life Technologies Corporation Trinucleotide cap analogs, preparation and uses thereof
WO2022006368A3 (en) * 2020-07-02 2022-03-17 Life Technologies Corporation Trinucleotide cap analogs, preparation and uses thereof
US12264175B2 (en) 2020-09-04 2025-04-01 Verve Therapeutics, Inc. Compositions and methods for capping RNAS
EP4486355A4 (en) * 2022-03-01 2026-04-22 Verve Therapeutics Inc COMPOSITIONS AND METHODS FOR CAPPING RNAS
WO2024044741A3 (en) * 2022-08-26 2024-04-18 Trilink Biotechnologies, Llc Efficient method for making highly purified 5'-capped oligonucleotides
WO2024075022A2 (en) 2022-10-04 2024-04-11 BioNTech SE Rna constructs and uses thereof
EP4464713A1 (en) 2023-05-18 2024-11-20 Siec Badawcza Lukasiewicz - PORT Polski Osrodek Rozwoju Technologii Circular rna analogs, preparation and application thereof
WO2024236202A1 (en) 2023-05-18 2024-11-21 Sieć Badawcza Łukasiewicz Port Polski Ośrodek Rozwoju Technologii Circular rna analogs, preparation and application thereof
WO2024236203A1 (en) 2023-05-18 2024-11-21 Sieć Badawcza Łukasiewicz Port Polski Ośrodek Rozwoju Technologii Circular rna analogs comprising 7-methylguanosine cap, preparation, and application thereof
WO2025054401A3 (en) * 2023-09-06 2025-05-08 Trilink Biotechnologies, Llc Cap analogs and methods of use thereof
WO2025147660A2 (en) 2024-01-04 2025-07-10 Trilink Biotechnologies, Llc Modified rna for increasing protein expression
EP4606900A1 (en) * 2024-02-21 2025-08-27 ExploRNA Therapeutics Sp. z o. o. Modified rna for the treatment of cfdna-associated diseases
WO2025176838A1 (en) * 2024-02-21 2025-08-28 Explorna Therapeutics Sp. Z O. O. Modified rna for the treatment of cfdna-associated diseases
WO2025187713A1 (en) * 2024-03-07 2025-09-12 Eisai R&D Management Co., Ltd. Polynucleotide and producing method thereof
WO2026010855A1 (en) 2024-07-02 2026-01-08 Trilink Biotechnologies, Llc Modified rna for increasing protein expression

Also Published As

Publication number Publication date
JP2023513764A (en) 2023-04-03
JP2026012182A (en) 2026-01-23
KR20220163359A (en) 2022-12-09
PL432883A1 (en) 2021-08-16
AU2021219563A1 (en) 2022-09-08
US12606590B2 (en) 2026-04-21
US20230130423A1 (en) 2023-04-27
JP7805938B2 (en) 2026-01-26
CA3167570A1 (en) 2021-08-19
EP4103577A1 (en) 2022-12-21
PL248416B1 (en) 2025-12-08
EP4103577A4 (en) 2024-03-06

Similar Documents

Publication Publication Date Title
US12606590B2 (en) MRNA 5′-end cap analogs, RNA molecule incorporating the same, uses thereof and method of synthesizing RNA molecule or peptide
WO2021162566A1 (en) Novel mrna 5&#39;-end cap analogs modified within phosphate residues, rna molecule incorporating the same, uses thereof and method of synthesizing rna molecule or peptide
JP5677716B2 (en) Kits and methods comprising oligonucleotide analogues and use of oligonucleotide analogues
CA2735251C (en) Rna synthesis - phosphoramidites for synthetic rna in the reverse direction, and application in convenient introduction of ligands, chromophores and modifications of synthetic rna at the 3&#39; - end
US5512668A (en) Solid phase oligonucleotide synthesis using phospholane intermediates
EP1954707B1 (en) Polynucleotide containing a phosphate mimetic
DE60126762T2 (en) Combinatorial production of nucleotide and nucleoside (XITP) analogs
CN110023322B (en) Novel bicyclic nucleosides and oligomers prepared therefrom
JP2013520438A (en) Phosphoramidites for reverse synthetic RNA
EP3770257A1 (en) Bbb-passing lipid ligand of hetero nucleic acid
CN116003496A (en) Modified mRNA5&#39; -cap analogues
EP2961757B1 (en) Cell-penetrating oligonucleotides
EP0358657B1 (en) Poly(deoxyribonucleotides), pharmaceutical compositions, use and preparation of the poly(deoxyribonucleotides)
RU2041884C1 (en) 2,6-n,n′-bis[1-(dimethylamino)ethylidene]-4,4′-o-(2′- dimethoxytriphenylmethyl)-2-amino-3′-deoxyadenosine-n,n′- o-alkyl-n,n-diisopropylamidophosphites and process for preparing thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21754142

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022548990

Country of ref document: JP

Kind code of ref document: A

Ref document number: 3167570

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2021219563

Country of ref document: AU

Date of ref document: 20210212

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021754142

Country of ref document: EP

Effective date: 20220912