Novel capping strategies for mRNA 5'cap

Abstract

The present invention relates to synthetic cap analogs useful for the functionalization of mRNAs, which can replace canonical cap structures. Compounds disclosed herein may be functionalized with a polynucleotide using chemical synthesis approaches.

Description

NOVEL CAPPING STRATEGIES FOR MRNA 5'CAPPRIORITY CLAIM

[0001] The present application claims priority of US provisional application 63 / 614,889 dated December 26, 2023.FIELD OF THE INVENTION

[0002] The present invention pertains to synthetic cap analogs designed for capping polynucleotides, particularly mRNAs. These analogs serve as substitutes for endogenous cap structures, enhancing the translatability of mRNAs while also protecting them from degradation by enzymes and other factors, thereby stabilizing the molecule.BACKGROUND OF THE INVENTION

[0003] The 5'-cap is a defining feature of eukaryotic mRNA, playing a crucial role in various cellular functions. Chemically, the 5'-cap comprises an inverted 7-methylguanosine linked to the rest of the mRNA through a 5'-5' triphosphate bridge. This structure, known as capO, is essential for ensuring proper mRNA processing and helps stabilize mRNA during splicing, nuclear export, translation initiation, and decay. Key interaction partners for the 5'-cap include the cap-binding complex (CBC) in the nucleus, which is necessary for nuclear export, and the eukaryotic translation initiation factor 4E (el F4E) in the cytoplasm, crucial for cap-dependent translation. Additionally, capped RNA serves as a signal for the innate immune system, allowing it to differentiate between triphosphorylated viral RNAs and cellular RNAs. The antiviral response is partly mediated by the cytosolic receptor RIG-I, which detects short single- and double-stranded triphosphorylated RNAs, as well as MDA-5, which recognizes long triphosphorylated RNAs and those lacking the 2'-OH methylation at the first nucleotide (cap1), a modification commonly found in eukaryotes. Since natural cap structures are crucial for stability, translatability, and immunomodulation, synthetic mRNAs as used for instance for medical applications such as mRNA therapeutics require a cap structure, which requires enzymatic assembly of the cap structure at the 5' end of the synthetic mRNA following / during in vitro transcription (I VT).

[0004] Traditional enzymatic approaches for mRNA capping exhibit limitations due to their selectivity towards specific substrates and chemical modifications for the mRNA 5’ end. For instance, Ohno et al. (Nucleic Acid Research, 2023) has considered over two dozen cap analogs for mRNA using a vaccinia virus-capping enzyme. However, a significant number of their analogs were not able to be incorporated into the mRNA due to the selectivity of the vaccinia virus-capping, especially caps with modifications to the canonical N7-methyl, which is hallmarkof naturally occurring mRNA cap in mammalian systems. Therefore, it is desirable to develop cap analogs able to be chemically incorporated into the synthetic polynucleotides thus overcoming the need of substrate specific enzymes such as vaccinia virus-capping enzyme.DEFINITIONS

[0034] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0005] As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0006] As used herein, "Me" means "CH3 ", and "OCH3" or "OMe" denotes an oxygen atom bound to a methyl group, "CHO" denotes a carbon atom, C, bonded to a hydrogen atom, H, and double-bonded to an oxygen atom, O, (0=CH — ) and "Et" denotes "C2H5".

[0007] As used herein, a "locked nucleic acid" (LNA) means a ribonucleotide in which there is a bridge between the 2'0 and 4’C methylene bicyclonucleotide monomers.

[0008] 2-(2-Nitrophenyl)-propyloxycarbonyl (NPPOC), benzoyl-2-(2- nitrophenyl)propoxycarbonyl (Bz-NPPOC), and thiophenyl-2-(2-nitrophenyl)propoxycarbonyl (SPh-NPPOC) are commonly used photolabile protecting group in organic synthesis. It can be efficiently removed by photolysis, allowing for controlled deprotection of functional groups.

[0009] "Pmyoc" refers to pyrenylmethyloxycarbonyl, a commonly used photolabile protecting group in organic synthesis. It can be efficiently removed by photolysis, allowing for controlled deprotection of functional groups.

[0010] A "modified nucleobase" refers to a functional group capable of substituting a nucleobase while retaining the hydrogen-bonding properties required for sequence-specific binding between nucleic acid strands. These modified nucleobases can mimic the spatial arrangement and electronic properties of natural bases, such as uracil, thymine, adenine, cytosine, and guanine, without significantly affecting melting behavior, recognition by intracellular enzymes, or duplex activity. On the other hand, "modified nucleoside" or "modified nucleotide" encompasses nucleosides or nucleotides containing modified nucleobases, as well as other chemical modifications like modified sugars, modified phosphorus atom bridges, or modified internucleoside linkages. Examples of modified nucleobase, but not limited to these examples include 5-methylcytosine (m5C), 5 methyl uridine (m5U), N1 -Methylpseudouridine (rnl MJ), N6-Methyladenosine (m6A) and 8-Oxoguanine (oxoG).

[0011] A "nucleobase" refers to a nitrogen-containing heterocyclic group that plays a crucial role in the hydrogen-bonding interactions between nucleic acid strands, enabling sequencespecific binding. Non-limiting examples of suitable nucleobases include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine.

[0012] While the following detailed description of the present disclosure is provided, it should be noted that the disclosure is not restricted to the specific methodologies, protocols, and reagents described herein, as they may vary. Furthermore, the terminology employed in this disclosure is solely intended to describe specific embodiments and should not be construed as limiting the scope of the present disclosure, which will be determined solely by the appended claims. Unless otherwise defined, all technical and scientific terms utilized in this disclosure hold their customary meanings as understood by a person skilled in the relevant field.DETAILED DESCRIPTION OF THE INVENTION

[0013] This patent application reports a novel approach to 5' capping of mRNA molecules, with a strong emphasis on chemical strategies, offering a wider range of cap analogs. Our research has uncovered a significant inverse correlation between the cap-elF4e dissociation constant (Kd) and mRNA translation efficiency. Building upon this observation, we conducted multiple lines of analysis of the crystal structure of human elF4E bound with GTP-guanosine cap, leading to the identification of specific changes that enhance capped-mRNA. Furthermore, we explored modifications to the ribosugar component and the addition of hydrophobic photolabile groups, which provide increased resistance against decapping enzymes and facilitate isolation of capped products. Finally, we suggest cap analogues that are amenable to conjugate with chemically synthesized mRNA by imidazole-driven reactions and click-driven reactions among others.

[0014] To overcome these limitations, one strategy among others of this application is leveraging a chemical reaction for the capping procedure. Such chemical reactions mitigate some of the structural constraints found in traditional caps by circumventing the binding requirements between the cap and the capping enzyme. Therefore, they enable the utilisation of a broader range of cap analogs. For example, such chemical strategies can utilise a reaction between (a) a cap that consists of a guanosine 5' diphosphate-imidazolide with the required chemical modifications and (b) the mRNA molecule. In other example of such chemical strategy, the reaction can utilise click chemistry as was described in Walczak et al. Chem Sci. 2017 and / or by Kozarski et al., RSC Adv., 2023.

[0015] As such, a broader range of cap analogs can be considered with primary emphasis of the ability of such cap analogs to promote translations, resist decapping enzymes, and enable purification of the capped product. Overall, these caps analogs have the potential for increased efficacy of therapeutic mRNA beyond the standard caps.

[0016] By analysis of previous studies data, we have discovered a strong inverse correlation between the cap-elF4e dissociation constant and mRNA translation efficiency (Fig. 1 A). The Kd dissociation constant explained over 40% of the variance log-transformed translation efficiency, which was statistically significant (p<0.006). For each 10x improvement in the cap- elF4e binding, there is about a 10x increase in translation efficiency. We also found a correlation between compounds that can inhibit elF4e in in-vitro experiments and stronger translationwhen these compounds are bound to mRNA (Fig. 1 B). The statistical trend was significant (p<0.05) but weaker. The log-IC50 of the inhibition levels of various compounds as independent cap analogs explained 24% of the variance in translation of the proteins when these analogs were incorporated into mRNA.

[0017] Next, we used a machine learning approach to build four nested linear models and measure the ability of each model to predict the translation increase. We compared the mean square error (MSE) and the Bayesian information criterion (BIC), which balances between model complexity and explanatory power of the model. The results are below in Table 1 :Table 1

[0018] The results above show that cap binding to elF4e is the strongest predictor for translation increase under strict model with parsimonious requirements (Model I). However, if allowing for a slightly more complex model, the analysis also suggests that cap structures that are strong binders and have some resistance to decapping susceptibility could better increase translation efficiency (Model IV).

[0019] By leveraging these insights and the broad flexibility of chemical capping, we studied the crystal structure of human elF4E bound with a short mRNA-guanosine cap to rationally identify cap structures that are likely to be strong binders. To this end, we modelled a third nucleotide onto the experimentally determined structures to better understand the binding interaction of mRNA with a 5' cap to el F4E and DPS. We introduced various molecular changes and employed energy minimization techniques under Amber10-EHT. This process identified key changes that enhance cap binding, specifically through the introduction of N7 modifications in addition to the standard methyl group.

[0020] Our investigation into the crystal structure of elF4E also unveiled that the ribosugar component plays a minimal role in cap binding. Inspired by this finding, we explored various sugar modifications to the cap, aiming to enhance resistance against decapping enzymes. To this end, we considered the 2.5A resolution crystal structure of human DcpS bound to m7GDP(PDB: 1XMM) and the 2.5A resolution crystal structure of human Dcp2 bound to GMP. DcpS cleaves between the y and p of the phosphates that bridge between the cap and the mRNA, whereas Dcp2 cleaves the bridge between the p and a phosphates. Thus, interfering with the binding of these enzymes is desired. Through a comparative analysis of these crystal structures, we identified potential sugar modifications, in particular alkylation of the ribose hydroxyl groups, and other types of cap modifications that interfere with their binding. As such, these cap structures should permit an increase in protein expression from therapeutic mRNA.

[0021] In addition to modifications enhancing cap binding and decapping resistance, we considered the inclusion of highly hydrophobic photolabile groups at either N2 or 2'0 or 3'0 position. These groups facilitate the isolation of capped products, allowing for easy removal and separation from the uncapped mRNA. By incorporating these photolabile groups, our chemical capping strategy offers improved control and versatility for downstream applications.

[0022] In summary, our novel chemical capping approach for mRNA 5’ cap broadens the range of cap analogs available, overcoming the limitations associated with enzymatic methods. Our research reveals key insights into cap-elF4e interactions, enabling the design of caps with enhanced binding affinity. Moreover, modifications to the ribosugar component and the incorporation of hydrophobic photolabile groups enhance resistance against decapping enzymes and facilitate product isolation. The proposed chemical capping strategy holds significant potential for advancing mRNA-based technologies in various fields, including drug development and biotechnology.BRIEF DESCRIPTION OF THE FIGURES

[0023] Figure 1 A shows the correlation between cap binding and translation activity, B shows the correlation between Inhibition concentration and translation activity.

Claims

WHAT CLAIMED IS1. A compound of formula (I):Formula (I) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I;R2 is selected from H, methoxy (OMe), methoxyethyl (MOE), F, locked nucleic acid (LNA), and 2’-O-(2-(2-Methoxyethoxy) ethyl) (O-MOEOE);R3 is selected from H, OMe, MOE, F, -O-MOEOE, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC- NPPOC, PyMoc and OMe.R4 is selected from Diphosphate imidazolide (Im-DP) or [p*]k[N], i is a positive integer; k is selected from an integer ranging from 0 to 4; p* is either a phosphate, phosphorothioate, phosphorothiolate, or a combination thereof; N is a nucleotide chain consists of nucleotides or modified nucleotides, including 2'-0Me and m6A and connected to each other using covalent links from the 3' of one nucleotide to 5' of the next nucleotide;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me.

2. A compound of formula (VII):Formula (VII) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I;R2 is selected from H, methoxy (OMe), methoxyethyl (MOE), F, locked nucleic acid (LNA), and 2'-O-(2-(2-Methoxyethoxy) ethyl) (O-MOEOE);R3 is selected from H, OMe, MOE, F, -O-MOEOE, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC- NPPOC, PyMoc and OMe.R4 is selected from Diphosphate imidazolide (Im-DP) or [p*]k[N], i is a positive integer; k is selected from an integer ranging from 0 to 4; p* is either a phosphate, phosphorothioate, phosphorothiolate, or a combination thereof; N is a nucleotide chain consists of nucleotides or modified nucleotides, including 2'-0Me and m6A and connected to each other using covalent links from the 3' of one nucleotide to 5' of the next nucleotide;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;The compound according to claim 1, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; n is selected from an integer ranging from 0 to 3;R2 is selected from OMe, MOE, F, O-R6 and R6 forms a methylene bridge with the 4'C such it creates an LNA, -O-MOEOE; andR3 is OH.

4. The compound according to claim 2, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; n is selected from an integer ranging from 0 to 3;R2 is selected from OMe, MOE, F, O-R6 and R6 forms a methylene bridge with the 4'C such it creates an LNA, -O-MOEOE; and R3 is OH.

5. The compound according to claim 1, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; n is selected from an integer ranging from 0 to 3;R2 is selected from H and Me; andR3 is OH.

6. The compound according to claim 2 wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; n is selected from an integer ranging from 0 to 3;R2 is selected from H and Me; andR3 is OH.

7. The compound according to claim 1, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; n is selected from an integer ranging from 0 to 3;R2 is selected from H and Me; andR3 is selected from H, OH, OMe, F, MOE, OMOEOE and LNA.The compound according to claim 2, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; n is selected from an integer ranging from 0 to 3;R2 is selected from H and Me; andR3 is selected from H, OH, OMe, F, MOE, OMOEOE and LNA.The compound according to claim 1, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; and n is selected from an integer ranging from 0 to 3.

10. The compound according to claim 2, wherein the compound is selected from the following:whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; and n is selected from an integer ranging from 0 to 3.

11. A compound of formula (III):Formula (111) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I;R4 is selected from Diphosphate imidazolide (Im-DP) or [p*]k[N], i is a positive integer; k is selected from an integer ranging from 0 to 4; p* is either a phosphate, phosphorothioate, phosphorothiolate, or a combination thereof; N is a nucleotide chain consists of nucleotides or modified nucleotides, including 2'-0Me and m6A and connected to each other using covalent links from the 3' of one nucleotide to 5' of the next nucleotide;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;12. A compound of formula (VIII):Formula (VIII) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I;R4 is selected from Diphosphate imidazolide (Im-DP) or [p*]k[N], i is a positive integer; k is selected from an integer ranging from 0 to 4; p* is either a phosphate, phosphorothioate, phosphorothiolate, or a combination thereof; N is a nucleotide chain consists of nucleotides or modified nucleotides, including 2'-0Me and m6A and connected to each other using covalent links from the 3' of one nucleotide to 5' of the next nucleotide;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;The compound according to claim 11, wherein the compound is selected from the following:R1 is selected from Me and compound of formula (II)Formula (II);wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; and n is selected from an integer ranging from 0 to 3;14. The compound according to claim 12, wherein the compound is selected from the following:wherein R1 is selected from Me and compound of formula (II)Formula (II); wherein X is selected from F, Cl, Br, Me, CN, CF3 and I; and n is selected from an integer ranging from 0 to 3; 15. A compound of formula (IV):Formula (IV), or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R2, R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;R3 is selected from H, OMe, MOE, F, -O-MOEOE NPPOC, Bz-NPPOC, SPh-NPPOC, BOC- NPPOC, PyMoc and OMe;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

16. A compound of formulaor a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R2, R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;R3 is selected from H, OMe, MOE, F, -O-MOEOE NPPOC, Bz-NPPOC, SPh-NPPOC, BOC- NPPOC, PyMoc and OMe;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

17. A compound of the formula (IVa):Formula (IVa) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R2, R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;R3 is selected from H, OMe, MOE, F, -O-MOEOE NPPOC, Bz-NPPOC, SPh-NPPOC, BOC- NPPOC, PyMoc and OMe;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

18. A compound of the formula (IVai):Formula (IVai) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R2, R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;R3 is selected from H, OMe, MOE, F, -O-MOEOE NPPOC, Bz-NPPOC, SPh-NPPOC, BOC- NPPOC, PyMoc and OMe;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

19. A compound of a formula (V):Formula (V); or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S;andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

20. A compound of a formula (Vi):Formula (Vi) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

21. A compound of a formula (Va):Formula (Va) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S;andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

22. A compound of a formula (Vai):Formula (Vai) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S;andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

23. A compound of a formula (VI):or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

24. A compound of a formula (Vli):Formula (Vli) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

25. A compound of a formula (Via):Formula (Via) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II);wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

26. A compound of a formula (Vlai):Formula (Vlai) or a salt or stereoisomer thereof, whereinR1 is selected from Me and compound of formula (II)Formula (II); wherein Y is selected from F, Cl, Br, Me, CN, CF3 and I;R5 is selected from H, NPPOC, Bz-NPPOC, SPh-NPPOC, BOC-NPPOC, PyMoc and Me;R6, R7 and R8 each independently is selected from H, methoxy (OMe), methoxyethyl (MOE), F, LNA and 2'-O-(2-(2-Methoxyethoxy)ethyl) (O-MOEOE); n selected from an integer ranging from 0 to 3;X independently at any position is selected from O or S; andB1, B2 and B3 each independently is nucleobase or modified nucleobase.

27. The compound of any of claims 15 to 26, wherein said modified nucleobase is selected from 5-methylcytosine (m5C), 5 methyl uridine (m5U), N1 -Methylpseudouridine (rnl MJ), N6-Methyladenosine (m6A) and 8-Oxoguanine (oxoG).

28. The compound of claims 1 to 27, for the use of purifying mRNA molecules.

29. The compound of claims 1 to 27, for the use of stabilizing mRNA molecules.