WO2024250923A1 - Chemically modified nucleoside, chemically modified nucleoside triphosphate and use thereof - Google Patents

Abstract

A chemically modified nucleoside, a chemically modified nucleoside triphosphate, a chemical synthesis means and the use thereof, and a synthesized chemically modified nucleoside and a synthesized chemically modified nucleoside triphosphate. An mRNA obtained by replacing a natural nucleoside triphosphate in an mRNA with the chemically modified nucleoside triphosphate globally or in combination with other chemically modified nucleoside triphosphates has higher translation efficiency and stability, can reduce the immunogenicity, and provides supplementation and selection for mRNA modification technique.

Description

A chemically modified nucleoside, a chemically modified nucleoside triphosphate and its application Technical Field

The present application belongs to the field of nucleic acid modification technology, and specifically relates to a chemically modified nucleoside, a chemically modified nucleoside triphosphate and applications thereof.

Background Art

mRNA, also known as messenger RNA, is transcribed from DNA as a template and is responsible for guiding the synthesis of intracellular proteins. mRNA technology uses this rule to introduce in vitro artificially synthesized mRNA into specific cells, making cells into small factories for the production of protein drugs, and exerting therapeutic effects through the proteins produced by these cells. mRNA technology has great application potential in infectious disease prevention and control, tumor vaccines, tumor immunotherapy, protein replacement therapy, etc., but natural mRNA has poor stability and is prone to immunogenicity, which seriously hinders the application of mRNA. In the mainstream mRNA technology, naturally occurring modified nucleosides are used to replace natural nucleosides, for example, pseudouridine and methyl pseudouridine are used to replace uridine to solve the problems of stability and immunogenicity of mRNA drugs. However, the number of naturally occurring modified nucleosides is scarce, which cannot meet the needs of improving the properties of mRNA, nor is it conducive to establishing structure-activity relationships to reveal its mechanism of action at the molecular level. Therefore, it is of great research and application significance to prepare more chemically modified nucleosides using organic synthesis methods to improve the drugability of mRNA from different levels.

Studies have found that modifications in different regions of the eukaryotic mRNA structure have completely different effects on the translation activity and immunogenicity of mRNA. Therefore, in addition to the overall modification strategy of mRNA, it is very necessary to study the chemical modification of specific regions of mRNA. For example, by modifying the 3' tail structure, a single modification of mRNA can be achieved without affecting other regions of mRNA. At present, the research on mRNA tailing is still mainly focused on using natural adenosine to adjust the length of the poly A tail structure of mRNA, while the introduction of chemically modified non-natural adenosine into the poly A tail (poly A tail) structure and the control of its length at the same time are relatively scarce. Since the poly A tail structure is also an important control factor for translation initiation, and it can also regulate the stability and turnover rate of mRNA, thereby affecting the translation efficiency of mRNA, therefore, the development of a method using new chemically modified nucleotides and modifying the poly A tail structure of mRNA is of great significance and has broad application prospects in the relevant applications of mRNA.

The mRNA modification methods used in existing patents mainly use modified nucleosides existing in nature to replace conventional nucleosides, such as using pseudouridine and methyl pseudouridine to replace uridine. CN107090436B discloses a non-therapeutic method or in vitro method for reducing the immunogenicity of an in vitro synthesized RNA molecule obtained by in vitro transcription or a preparation of a gene therapy vector containing the RNA molecule to mammalian cells, wherein the replacement uridine contained in the RNA molecule is selected from a modified nucleoside of pseudouridine, 1-methyl pseudouridine, 5-methyluridine, 5-methoxyuridine and 2-thiouridine. The types of naturally occurring modified bases are very limited, so the means available for improving the properties of mRNA are limited.

The use of chemical means to synthesize non-natural modified nucleosides can greatly enrich the types and range of non-natural nucleosides and is also suitable for establishing structure-activity relationships, which is not available with naturally occurring modified bases.

CN112673106A discloses a click-modified mRNA, which relates to alkyne and/or azide-modified mRNA, and stabilizes RNA by introducing alkyne and/or azide-modified nucleotides. This modification can not only stabilize the target mRNA for in vitro application and subsequent administration to human patients, animals or plants, but also can easily attach detectable markers or functional groups. CN114807154A discloses a modified nucleic acid and its application, the modified nucleic acid includes uridine, cytosine, adenine, guanosine and chemically modified nucleosides; the chemically modified Nucleosides include one or more of chemically modified uridine nucleosides, chemically modified cytosine nucleosides, chemically modified adenine nucleosides, and chemically modified guanine nucleosides; the modified nucleic acids described in this application have high stability, low immunogenicity, and long in vivo half-life. However, there are still relatively few types of modified nucleic acids. Therefore, it is necessary to develop new non-natural modified nucleotides with novel structures and introduce them into mRNA to improve the stability of mRNA and enhance its translation activity, so as to achieve the goal of replacing, enriching, or supplementing existing mRNA chemical modifications, thereby providing support for new chemical modifications and applications of mRNA.

Summary of the invention

The present application provides a chemically modified nucleoside, a chemically modified nucleoside triphosphate and an application thereof. The present application uses chemical synthesis means to chemically modify natural nucleosides, wherein the chemically modified nucleosides include adenine nucleosides and cytosine nucleosides; the chemically modified nucleosides are triphosphorylated to obtain chemically modified adenine nucleoside triphosphates and chemically modified cytosine nucleoside triphosphates, and the chemically modified nucleoside triphosphates are used to globally replace the natural nucleoside triphosphates to obtain modified mRNA, and the modified mRNA exhibits enhanced translation efficiency and higher stability both in vitro and in cells.

In a first aspect, the present application provides a chemically modified nucleoside, wherein the chemically modified nucleoside is selected from a compound having a structure as shown in Formula 1, a salt thereof, or an isomer thereof:

Wherein, R ₃ is selected from hydroxyl, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether;

Y is selected from a cytosine ring containing a substituent or an adenine ring containing a substituent, wherein the cytosine ring containing a substituent is as shown in Formula 2, and the adenine ring containing a substituent is as shown in Formula 3. Indicates the connection location;

wherein R ₁ and R _1′ are each independently selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl;

R ₂ and R _2' are each independently selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo or nitrile; and

X is selected from nitrogen or carbon.

Preferably, the chemically modified nucleoside is a chemically modified cytosine nucleoside, and the chemically modified cytosine nucleoside is selected from a compound having a structure as shown in Formula 4, a salt thereof, or an isomer thereof:

wherein R ₁ is selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl;

_R2 is selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo or nitrile; and

_R3 is selected from hydroxy, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether.

In the present application, the chemically modified cytosine nucleoside is selected from: N4-propionyl cytidine, N4-butyryl cytidine, N4-pentanoyl cytidine, N4-hexanoyl cytidine, N4-octanoyl cytidine, N4-decanoyl cytidine, N4-p-nitrobenzoyl cytidine, 5-methyl-N4-butyryl cytidine, 5-methyl-N4-hexanoyl cytidine, 5-methyl-N4-benzoyl cytidine, 2'-fluoro-N4-acetyl cytidine, 2'-fluoro-N4-butyryl cytidine, 2'-fluoro-N4-hexanoyl cytidine, 2'-methoxy cytidine or 2'-ethoxy cytidine.

Preferably, the chemically modified nucleoside is a chemically modified adenine nucleoside, and the chemically modified adenine nucleoside is selected from a compound having a structure as shown in Formula 5, a salt thereof, or an isomer thereof:

wherein R _1′ is selected from acetyl, propionyl, butyryl, valeryl, hexanoyl or benzoyl;

R _2' is hydrogen;

_R3 is selected from hydrogen, hydroxy, fluoro or methoxy; and

X is selected from nitrogen or carbon.

In the present application, the use of chemical means to synthesize non-natural modified nucleosides can greatly enrich the types and ranges of non-natural nucleosides, is also conducive to the establishment of structure-activity relationships, and can also perform regional modifications on mRNA, which are all not available in the current use of naturally occurring modified bases to improve mRNA.

In a second aspect, the present application provides a chemically modified nucleoside triphosphate, wherein the chemically modified nucleoside triphosphate is selected from a compound having a structure as shown in Formula 6, a salt thereof, or an isomer thereof:

wherein _Rn is selected from hydroxy, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether;

W is selected from a cytosine ring containing a substituent or an adenine ring containing a substituent, wherein the cytosine ring containing a substituent is as shown in Formula 7, and the adenine ring containing a substituent is as shown in Formula 8. Indicates the connection location;

wherein R ₄ and R _4′ are each independently selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl;

R ₅ and R _5' are each independently selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo or nitrile; and

M is selected from nitrogen or carbon.

Preferably, the chemically modified nucleoside triphosphate is a chemically modified cytosine triphosphate, and the chemically modified cytosine triphosphate is selected from a compound having a structure as shown in Formula 9, a salt thereof, or an isomer thereof:

wherein _R4 is selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl;

R ₅ is selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo, nitrile; and

R _n is selected from hydroxy, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether.

In the present application, the chemically modified cytosine triphosphate is selected from: N4-acetyl cytidine triphosphate, N4-propionyl cytidine triphosphate, N4-butyryl cytidine triphosphate, N4-pentanoyl cytidine triphosphate, N4-hexanoyl cytidine triphosphate, N4-octanoyl cytidine triphosphate, cytidine triphosphate, 5-methyl-N4-acetyl cytidine triphosphate, 5-methyl-N4-butyryl cytidine triphosphate, 5-methyl-N4-hexanoyl cytidine triphosphate, 5-methyl-N4-benzoyl cytidine triphosphate, 2'-fluoro-N4-acetyl cytidine triphosphate, 2'-fluoro-N4-butyryl cytidine triphosphate, 2'-fluoro-N4-hexanoyl cytidine triphosphate, 2'-methoxy cytidine triphosphate, 2'-ethoxy cytidine triphosphate.

The present application provides a new chemically modified cytosine triphosphate, which provides more available cytosine triphosphates for mRNA. The chemically modified cytosine triphosphates synthesized in the present application can be used for the synthesis of mRNA, can replace natural cytosine triphosphate, alone or in combination with other modified nucleotides, and can play a corresponding translation function, can enhance the translation efficiency and stability of the target mRNA, and reduce immunogenicity, providing a supplement and new choice for mRNA technology beyond the current patented technology.

Preferably, the chemically modified nucleoside triphosphate is a chemically modified adenine nucleoside triphosphate, and the chemically modified adenine nucleoside triphosphate is selected from a compound having a structure as shown in Formula 10, a salt thereof, or an isomer thereof:

wherein R _4′ is selected from acetyl, propionyl, butyryl, valeryl, hexanoyl or benzoyl;

R _5' is selected from hydrogen;

_Rn is selected from hydrogen, hydroxy, fluoro or methoxy; and

M is selected from nitrogen or carbon.

The present application provides a new chemically modified adenine nucleoside and a chemically modified adenine nucleoside triphosphate, which provide more optional raw materials for mRNA technology. The natural adenine nucleoside triphosphate in mRNA is completely replaced by the new chemically modified adenine nucleoside triphosphate in the present application, or the mRNA prepared by in vitro transcription is polyadenylated with the chemically modified adenine nucleoside triphosphate, which can enhance the stability and translation efficiency of the target mRNA.

Preferably, the chemically modified nucleoside triphosphate is prepared by the following preparation method:

(1) subjecting the hydroxyl group of the compound of formula 11 to a hydroxyl protection reaction to obtain a compound of formula 12;

(2) reacting the compound of formula 12 with an acid anhydride or an acid chloride to obtain a compound of formula 13;

(3) performing a complete deprotection reaction on the protecting group of the compound of formula 13 to obtain a compound of formula 14;

(4) reacting the compound of formula 14 with a chlorinating agent to obtain a compound of formula 15;

(5) reacting the compound of formula 15 with tributylammonium pyrophosphate to obtain a compound of formula 16;

(6) subjecting the compound of formula 16 to a quenching reaction to obtain the compound of formula 9.

Preferably, in step (1), tert-butyldimethylsilyl chloride is used to carry out the hydroxyl protection reaction.

Preferably, in step (2), the acid anhydride is selected from acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, caproic anhydride, caprylic anhydride, capric anhydride or benzoic anhydride.

Preferably, in step (2), the acyl chloride is selected from 4-methoxybenzoyl chloride or 4-nitrobenzoyl chloride.

Preferably, in step (3), tetrabutylammonium fluoride is used to carry out a complete deprotection reaction.

Preferably, in step (4), the chlorination agent is selected from phosphorus oxychloride.

Preferably, the chemically modified nucleoside triphosphate is prepared by the following preparation method:

(1") subjecting the hydroxyl group of the compound of formula 17 to a hydroxyl protection reaction to obtain a compound of formula 18;

(2") reacting the compound of formula 18 with an acid anhydride or an acid chloride to obtain a compound of formula 19;

(3") performing a complete deprotection reaction on the protecting group of the compound of formula 19 to obtain a compound of formula 20;

(4") reacting the compound of formula 20 with a chlorinating agent to obtain a compound of formula 21;

(5") reacting the compound of formula 21 with tributylammonium pyrophosphate to obtain a compound of formula 22;

(6") The compound of formula 22 is subjected to a quenching reaction to obtain a compound of formula 23.

Preferably, in step (1"), tert-butyldimethylsilyl chloride is used to carry out the hydroxyl protection reaction.

Preferably, in step (2"), the acid anhydride is selected from acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, caproic anhydride or benzoic anhydride.

Preferably, in step (2"), the acyl chloride is selected from acetyl chloride, propionyl chloride, butyryl chloride, valeryl chloride, hexanoyl chloride or benzoyl chloride.

In the present application, compared with acid anhydride, the reaction effect of acid chloride is better.

Preferably, in step (3"), tetrabutylammonium fluoride is used to carry out a complete deprotection reaction.

Preferably, in step (4"), the chlorinating agent is selected from phosphorus oxychloride.

In a third aspect, the present application provides a nucleic acid containing chemically modified cytosine nucleosides, wherein during the preparation process of the nucleic acid, the chemically modified cytosine nucleoside triphosphates described in the second aspect are used to completely replace the natural cytosine nucleoside triphosphates in the mRNA, or the chemically modified cytosine nucleoside triphosphates and N1-methylpseudouridine triphosphate are used in combination to replace the natural cytosine nucleoside triphosphates in the mRNA.

Preferably, the nucleic acid further contains pseudouridine, N-methylpseudouridine, 5-methoxyuridine or 2-thiouridine.

The present application synthesizes non-natural modified nucleotides with novel structures and introduces them into mRNA, thereby improving the stability of mRNA and enhancing its translation activity, achieving the goal of replacing, enriching or supplementing existing mRNA chemical modifications, thereby providing assistance for novel chemical modifications and applications of mRNA.

Preferably, the nucleic acid comprises at least:

A) a promoter sequence;

B) a 5’UTR containing at least one Kozak sequence;

C) 3’UTR;

D) a coding sequence consisting of linked nucleotides;

E) Poly A tail.

The present application uses chemical synthesis to synthesize chemically modified cytosine triphosphates, and then uses transcriptase and/or tailing enzyme to synthesize modified mRNA with global replacement of modified cytosine. The modified mRNA exhibits enhanced translation efficiency and higher stability both in vitro and in cells.

In a fourth aspect, the present application provides a nucleic acid containing chemically modified adenine nucleosides, wherein during the preparation process of the nucleic acid, the chemically modified adenine nucleoside triphosphate described in the second aspect is used to completely replace the natural adenine nucleoside triphosphate in the mRNA, or the chemically modified adenine nucleoside triphosphate described in the second aspect is used to perform polyadenylation modification on the mRNA prepared by in vitro transcription.

In the present application, natural adenine is chemically modified by chemical synthesis, and these modified adenines are triphosphorylated, and then in vitro transcription is used to synthesize mRNA in which the modified adenines replace the natural adenines globally, or tailing enzymes are used to modify adenosines to perform polyadenylation modification on mRNA. These modified mRNAs show enhanced translation efficiency and higher stability both in vitro and in cells.

Preferably, the chemically modified nucleic acid comprises at least:

A') a promoter sequence;

B’) 5’UTR containing at least one Kozak sequence;

C’) 3’UTR;

D') a coding sequence consisting of linked nucleosides;

E') Poly A tail.

In a fifth aspect, the present application provides a method for preparing a nucleic acid containing a chemically modified cytosine nucleoside according to the third aspect, the preparation method comprising:

Using four ribonucleotides, adenosine triphosphate, guanosine triphosphate, uridine triphosphate and chemically modified cytosine triphosphate, as raw materials, PCR reaction is carried out with DNA as a template under the catalysis of RNA polymerase to synthesize chemically modified nucleic acids.

Preferably, the RNA polymerase is T7 RNA polymerase.

Preferably, the PCR reaction system includes: a DNA template, an RNase inhibitor, T7 RNA polymerase, an RNA polymerase buffer, adenosine triphosphate, guanosine triphosphate, uridine triphosphate, chemically modified cytosine triphosphate and a 5' cap structure.

In the present application, the chemically modified nucleic acid is a modified mRNA in which chemically modified cytidine triphosphate is used to globally replace natural cytidine, and the mRNA has higher translation efficiency and is more stable both in a cell-free system and in cells. The technical route for the synthesis and translation of mRNA containing chemically modified pyrimidine nucleosides is shown in Figure 1.

In a sixth aspect, the present application provides a method for preparing a nucleic acid containing chemically modified adenine nucleosides according to the fourth aspect, the preparation method comprising:

Using cytosine triphosphate, uridine triphosphate, guanine triphosphate and chemically modified adenine triphosphate as raw materials, PCR reaction is carried out with DNA as template under the catalysis of RNA polymerase to synthesize chemically modified nucleic acids;

Alternatively, using in vitro transcribed mRNA as a template and chemically modified adenine nucleoside triphosphate as a substrate, the mRNA is tailed using E. coli poly (A) polymerase or Yeast poly (A) polymerase to obtain chemically modified nucleic acid.

Preferably, the RNA polymerase is T7 RNA polymerase.

Preferably, the PCR reaction system includes: a DNA template, an RNase inhibitor, T7 RNA polymerase, an RNA polymerase buffer, chemically modified adenosine triphosphate, guanosine triphosphate, uridine triphosphate, cytosine triphosphate and a 5' cap structure.

In the present application, the chemically modified nucleic acid is a modified mRNA in which chemically modified adenosine triphosphate is used to replace natural adenosine triphosphate globally, and the mRNA has higher translation efficiency and is more stable in both cell-free systems and cells. The technical route for the synthesis and translation of mRNA containing chemically modified adenosine nucleosides is shown in Figure 1.

In the seventh aspect, the present application provides the use of any one or a combination of at least two of the chemically modified nucleosides described in the first aspect, the chemically modified nucleoside triphosphates described in the second aspect, the nucleic acids containing chemically modified cytosine nucleosides described in the third aspect, the nucleic acids containing chemically modified adenine nucleosides described in the fourth aspect, the method for preparing nucleic acids containing chemically modified cytosine nucleosides described in the fifth aspect, or the method for preparing nucleic acids containing chemically modified adenine nucleosides described in the sixth aspect in the preparation of mRNA drugs.

Compared with the prior art, this application has the following beneficial effects:

The present application provides completely new chemically modified nucleosides and chemically modified nucleoside triphosphates, including chemically modified cytosine nucleosides, chemically modified cytosine nucleoside triphosphates, chemically modified adenine nucleosides and chemically modified adenine nucleoside triphosphates; more available cytosine nucleoside triphosphates are provided for mRNA; the chemically modified nucleoside triphosphates synthesized in the present application can all be used for the synthesis of mRNA, can replace the corresponding natural nucleosides, and can exert corresponding translation functions, can enhance the translation efficiency and stability of the target mRNA, reduce immunogenicity, and provide a supplement and new option for mRNA technology.

In cell-free system and cell-level reaction experiments, the mRNA replaced with the chemically modified cytosine triphosphate provided in the present application showed that the translation effect of the mRNA replaced with modified cytosine reached or exceeded the modification method of replacing uridine with pseudouridine and methylpseudouridine used in the currently reported technology for mRNA modification, and at the same time had good biological stability.

The mRNA replaced with the chemically modified adenine nucleoside triphosphate provided in the present application has good translation effects at the in vitro and cellular levels, which is better than the modification method using pseudouridine to replace uridine. The mRNA replaced with the chemically modified adenine nucleoside and pseudouridine provided in the present application has a better translation effect at the in vitro and cellular levels, which is also better than the modification method using pseudouridine to replace uridine. The mRNA polyadenylated and tailed with chemically modified adenosine has a better translation effect at the cellular level than the mRNA with a poly-natural adenosine tail, and at the same time has higher stability at the single nucleoside level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a technical roadmap for the synthesis and translation of mRNA containing chemically modified nucleosides.

FIG. 2 is a technical roadmap for the preparation of chemically modified cytidine triphosphate.

FIG3 is a ¹ H NMR spectrum of N4-propionylcytidine triphosphate.

FIG4 is a ³¹ P NMR spectrum of N4-propionylcytidine triphosphate.

FIG5 is a mass spectrum of N4-propionylcytidine triphosphate.

FIG6 is a ¹ H NMR spectrum of N4-butyrylcytidine triphosphate.

FIG. 7 is a ³¹ P NMR spectrum of N4-butyrylcytidine triphosphate.

FIG8 is a ¹ H NMR spectrum of N4-pentanoyl cytidine triphosphate.

FIG. 9 is a ³¹ P NMR spectrum of N4-pentanoyl cytidine triphosphate.

FIG. 10 is a ¹ H NMR spectrum of N4-hexanoyl cytidine triphosphate.

FIG. 11 is a ³¹ P NMR spectrum of N4-hexanoyl cytidine triphosphate.

FIG12 is a ¹ H NMR spectrum of 5-methyl-N4-acetylcytidine triphosphate.

FIG13 is a ³¹ P NMR spectrum of 5-methyl-N4-acetylcytidine triphosphate.

FIG14 is a ¹ H NMR spectrum of 5-methyl-N4-butyryl cytidine triphosphate.

FIG. 15 is a ³¹ P NMR spectrum of 5-methyl-N4-butyryl cytidine triphosphate.

FIG. 16 is a ¹ H NMR spectrum of N4-benzoylcytidine triphosphate.

FIG. 17 is a ³¹ P NMR spectrum of N4-benzoylcytidine triphosphate.

FIG. 18 is a ¹ H NMR spectrum of N4-acetylcytidine triphosphate.

FIG. 19 is a ³¹ P NMR spectrum of N4-acetylcytidine triphosphate.

FIG. 20 is a ¹ H NMR spectrum of 5-methylcytidine triphosphate.

FIG. 21 is a ³¹ P NMR spectrum of 5-methylcytidine triphosphate.

FIG. 22 is a ¹ H NMR spectrum of 5-methyl-N4-hexanoyl cytidine triphosphate.

FIG. 23 is a ³¹ P NMR spectrum of 5-methyl-N4-hexanoyl cytidine triphosphate.

FIG. 24 is a ¹ H NMR spectrum of N4-octanoylcytidine triphosphate.

FIG. 25 is a ¹ H NMR spectrum of N4-decanoylcytidine triphosphate.

FIG26 is a ¹ H NMR spectrum of 5-methyl-N4-benzoylcytidine triphosphate.

FIG27 is a ¹ H NMR spectrum of 2'-fluoro-N4-acetylcytidine triphosphate.

FIG. 28 is a ¹ H NMR spectrum of 2'-fluoro-N4-butyrylcytidine triphosphate.

FIG. 29 is a ¹ H NMR spectrum of 2'-fluoro-N4-hexanoyl cytidine triphosphate.

FIG30 is a mass spectrum of N4-butyrylcytidine triphosphate.

FIG31 is a mass spectrum of N4-pentanoylcytidine triphosphate.

FIG32 is a mass spectrum of N4-hexanoyl cytidine triphosphate.

FIG33 is a mass spectrum of 5-methyl-N4-acetylcytidine triphosphate.

FIG34 is a mass spectrum of 5-methyl-N4-butyryl cytidine triphosphate.

FIG35 is a mass spectrum of N4-acetylcytidine triphosphate.

FIG36 is a mass spectrum of 5-methylcytidine triphosphate.

FIG37 is a mass spectrum of 5-methyl-N4-hexanoyl cytidine triphosphate.

FIG38 is a schematic diagram of the in vitro transcription results of chemically modified C-series nucleoside triphosphates.

Figure 39 shows the expression efficiency of transcribed mRNA after transfection into HEK 293T cells for 24 hours.

Figure 40 shows the expression efficiency of mRNA transcribed using N1-methylpseudouridine and modified cytidine 24 hours after transfection into HEK 293T cells.

Figure 41 shows the expression efficiency of transcribed mRNA after transfection into HEK 293T cells for 24 hours.

Figure 42 is a graph showing the expression efficiency of mRNA transcribed using a combination of N1-methylpseudouridine and modified cytidine 24 hours after transfection into HeLa cells.

Figure 43 is the qPCR results of the stability of chemically modified mRNA in HEK 293T cells.

FIG. 44 is a graph showing the qPCR results of the stability test of chemically modified mRNA in HeLa cells.

Figure 45 shows the protein blotting results of the immunogenicity test of chemically modified mRNA in RAW264.7 cells.

FIG. 46 is a ¹ H NMR characterization chart of N6-propionyl adenosine.

FIG. 47 is a ¹³ C NMR characterization chart of N6-propionyl adenosine.

FIG. 48 is a HRMS characterization chart of N6-propionyl adenosine.

FIG. 49 is a ¹ H NMR characterization chart of N6-propionyl adenosine triphosphate.

FIG. 50 is a ¹³ C NMR characterization chart of N6-propionyl adenosine triphosphate.

FIG. 51 is a HRMS characterization chart of N6-propionyl ATP.

FIG. 52 is a ¹ H NMR characterization chart of N6-butyryladenosine.

FIG. 53 is a ¹³ C NMR characterization chart of N6-butyryladenosine.

Figure 54 is a HRMS characterization of N6-butyryladenosine.

FIG. 55 is a ¹ H NMR characterization chart of N6-butyryl adenosine triphosphate.

FIG. 56 is a ¹³ C NMR characterization chart of N6-butyryl adenosine triphosphate.

Figure 57 is a HRMS characterization of N6-butyryl adenosine triphosphate.

FIG. 58 is a ¹ H NMR characterization chart of N6-pentanoyl adenosine.

FIG. 59 is a ¹³ C NMR characterization chart of N6-pentanoyl adenosine.

Figure 60 is a HRMS characterization chart of N6-pentanoyl adenosine.

FIG. 61 is a ¹ H NMR characterization chart of N6-pentanoyl adenosine triphosphate.

FIG. 62 is a ¹³ C NMR characterization chart of N6-pentanoyl adenosine triphosphate.

FIG. 63 is a HRMS characterization chart of N6-pentanoyl adenosine triphosphate.

FIG. 64 is a ¹ H NMR characterization chart of N6-hexanoyl adenosine.

FIG. 65 is a ¹³ C NMR characterization chart of N6-hexanoyl adenosine.

Figure 66 is a HRMS characterization chart of N6-hexanoyl adenosine.

FIG. 67 is a ¹ H NMR characterization chart of N6-hexanoyl adenosine triphosphate.

FIG. 68 is a ¹³ C NMR characterization chart of N6-hexanoyl adenosine triphosphate.

FIG. 69 is a HRMS characterization chart of N6-hexanoyl adenosine triphosphate.

FIG. 70 is a HRMS characterization chart of N6-benzoyl adenosine triphosphate.

Figure 71 is the HRMS characterization chart of N6-methyladenosine triphosphate.

Figure 72 is a HRMS characterization chart of N6-acetyl adenosine triphosphate.

FIG. 73 is a HRMS characterization chart of 2-aminoadenosine triphosphate.

FIG. 74 is a HRMS characterization chart of 7-deaza-ATP.

Figure 75 is a diagram showing the effect of polyadenylation modification of eGFP mRNA by E. coli polyA polymerase.

Figure 76 shows the effect of polyadenylation modification of eGFP mRNA by Yeast polyA polymerase.

Figure 77 is a graph showing the expression of luciferase mRNA in combination with chemically modified adenosine and pseudouridine after incubation in rabbit reticulocyte lysate for 90 minutes.

FIG78 is a graph showing the expression effect of luciferase mRNA in which chemically modified adenosine globally replaces natural adenosine after incubation in rabbit reticulocyte lysate for 90 minutes.

Figure 79 is a graph showing the expression effect of luciferase mRNA in which chemically modified adenosine globally replaces natural adenosine 24 hours after being transfected into HEK 293T cells.

Figure 80 is a graph showing the expression effect of luciferase mRNA combined with chemically modified adenosine and pseudouridine 24 hours after transfection into HeLa cells.

Figure 81 is a fluorescence imaging image of the expression effect of chemically modified adenosine polyadenylation-modified eGFP mRNA in HEK 293T cells.

DETAILED DESCRIPTION

The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

Example 1 Preparation method of chemically modified cytidine triphosphate

This embodiment provides a method for preparing chemically modified cytidine triphosphate, and the preparation technology route of chemically modified cytidine triphosphate is shown in Figure 2. The reaction conditions of each step are as follows, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry:

a. 3 mmol (1 eq) of cytidine was dissolved in 15 mL of N,N-dimethylformamide, and then 12 mmol (4 eq) of tert-butyldimethylsilyl chloride and 15 mmol (5 eq) of imidazole were added in sequence, and stirred at 25°C overnight; after the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times; the organic phase was washed with water, and the extraction was repeated 3 times; the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol = 100/1) to obtain a white solid compound 1 (i.e., the compound of formula 12); the structure of compound 1 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

b. 2 mmol (1 eq) of compound 1 was dissolved in 15 mL of anhydrous pyridine, and then 1.2 eq of the corresponding acid anhydride was added dropwise, followed by 10 mg of 4-dimethylaminopyridine. The mixture was stirred at 25°C overnight. After the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times. The organic phase was washed with water, and the extraction was repeated 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol = 200/1) to obtain a white colloidal compound 2 (i.e., the compound of formula 13). The structure of compound 2 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

c. Dissolve 1.5 mmol (1 eq) of compound 2 in 15 mL of anhydrous tetrahydrofuran, then dropwise add 4.5 mL (3 eq) of tetrabutylammonium fluoride solution (1 M tetrahydrofuran solution), stir at room temperature for 0.5 h, then concentrate under reduced pressure, and purify by silica gel column chromatography (dichloromethane/methanol=20/1 to 10/1) to obtain a white solid compound 3 (i.e., compound of formula 14). The structure of compound 3 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

d. Dissolve 0.1 mmol of compound 3 in 2 mL of anhydrous pyridine and remove water under reduced pressure three times; inject 1 mL of dry trimethyl phosphate under nitrogen protection, and after the solid is completely dissolved, place the test tube in an ice bath to cool for 2 min, and add 1.5 eq of phosphorus oxychloride dropwise with a syringe; stir and react in an ice bath for 2.5 h.

e. Then, inject 1.5 eq of dry tributylamine and 1.5 mL (8 eq) of tributylammonium pyrophosphate solution (0.5 M N,N-dimethylformamide solution) in sequence under ice bath conditions, and move to room temperature to react for 0.5 h.

f. After the reaction is completed, the reaction is quenched with 5 mL of 2.0 M saturated triethylamine-carbonic acid aqueous solution under ice bath, and then the reaction solution is extracted with 10 mL of dichloromethane, and the aqueous phase is collected, and repeated 4 times; the collected aqueous phase is separated and purified by a rapid preparative liquid chromatograph C18 column, and 50 mM saturated triethylamine-carbonic acid aqueous solution and chromatographic grade acetonitrile are used as mobile phases; finally, the collected and purified solution is freeze-dried to obtain a white solid 4 (compound of formula 9). The structure of compound 4 is confirmed by ¹ H NMR, ³¹ P NMR and HRMS.

In the method for preparing chemically modified cytidine triphosphate in this embodiment, only step a, step b and step c are performed to obtain the method for preparing chemically modified cytidine.

Example 2 N4-Propionylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-propionyl cytidine triphosphate. The reaction conditions of each step of the preparation are as follows, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry:

a. Dissolve 3 mmol (1 eq) of cytidine in 15 mL of N,N-dimethylformamide, then add 12 mmol (4 eq) of tert-butyldimethylsilyl chloride and 15 mmol (5 eq) of imidazole, and stir overnight at 25 °C. After the reaction, add 10 mL of acetic acid. The reaction solution was extracted with ethyl ester for 3 times, and the organic phase was washed with water for 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=100/1) to obtain white solid compound 1 (i.e. compound 12) with a yield of 95%. The structure of compound 1 has been confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

b. 2 mmol (1 eq) of compound 1 was dissolved in 15 mL of anhydrous pyridine, and then 1.2 eq of propionic anhydride was added dropwise, followed by 10 mg of 4-dimethylaminopyridine. The mixture was stirred at 25°C overnight to react. After the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times. The organic phase was washed with water, and the extraction was repeated 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=200/1) to obtain a white colloidal compound 2 (i.e., the compound of formula 13) with a yield of 92%. The structure of compound 2 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

c. Dissolve 1.5 mmol (1 eq) of compound 2 in 15 mL of anhydrous tetrahydrofuran, then dropwise add 4.5 mL (3 eq) of tetrabutylammonium fluoride solution (1 M tetrahydrofuran solution), stir and react at room temperature for 0.5 h, then concentrate under reduced pressure, and purify by silica gel column chromatography (dichloromethane/methanol=20/1 to 10/1) to obtain a white solid compound 3 (i.e., the compound of formula 14). The structure of compound 3 has been confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

d. Dissolve 0.1 mmol of compound 3 in 2 mL of anhydrous pyridine and remove water under reduced pressure three times; inject 1 mL of dry trimethyl phosphate under nitrogen protection, and after the solid is completely dissolved, place the test tube in an ice bath to cool for 2 min, and add 1.5 eq of phosphorus oxychloride dropwise with a syringe; stir and react in an ice bath for 2.5 h.

e. Then, inject 1.5 eq of dry tributylamine and 1.5 mL (8 eq) of tributylammonium pyrophosphate solution (0.5 M N,N-dimethylformamide solution) in sequence under ice bath conditions, and move to room temperature to react for 0.5 h.

f. After the reaction is completed, the reaction is quenched with 5 mL of 2.0 M saturated triethylamine-carbonic acid aqueous solution under ice bath, and then the reaction solution is extracted with 10 mL of dichloromethane, and the aqueous phase is collected, and repeated 4 times; the collected aqueous phase is separated and purified by a rapid preparative liquid chromatograph C18 column, and 50 mM saturated triethylamine-carbonic acid aqueous solution and chromatographic grade acetonitrile are used as mobile phases; finally, the collected and purified solution is freeze-dried to obtain a white solid 4 (compound of formula 9). The structure of compound 4 has been confirmed by ¹ H NMR, ³¹ P NMR and HRMS.

Figure 3 is the ¹ H NMR spectrum of N4-propionylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 1.07, 1.09, 1.11; 2.44, 2.46, 2.48, 2.49; 4.27, 4.28, 4.30, 4.31, 4.32, 4.38, 4.40, 4.41; 5.92, 5.93; 7.29, 7.31; 8.39, 8.40. Figure 4 is a ³¹ P NMR spectrum of N4-propionyl cytidine triphosphate, from which it can be seen that the peak positions (unit: ppm) are: -22.70, -22.57, -22.45; -11.48, -11.46, -11.35, -11.34; -6.53, -6.40; 19.45, 19.51, 19.57. Figure 5 is a mass spectrum of N4-propionyl cytidine triphosphate.

Example 3 N4-butyryl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-butyryl cytidine triphosphate. The reaction conditions of each step of the preparation are as follows, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry:

a. 3 mmol (1 eq) of cytidine was dissolved in 15 mL of N,N-dimethylformamide, and then 12 mmol (4 eq) of tert-butyldimethylsilyl chloride and 15 mmol (5 eq) of imidazole were added in sequence, and stirred at 25°C overnight; after the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times; the organic phase was washed with water, and the extraction was repeated 3 times; the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=100/1) to obtain a white solid compound 1 (i.e., the compound of formula 12) with a yield of 93%; the structure of compound 1 has been confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

b. 2 mmol (1 eq) of compound 1 was dissolved in 15 mL of anhydrous pyridine, and then 1.2 eq of butyric anhydride was added dropwise, followed by 10 mg of 4-dimethylaminopyridine. The mixture was stirred at 25°C overnight to react. After the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times. The organic phase was washed with water, and the extraction was repeated 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=200/1) to obtain a white colloidal compound 2 (i.e., the compound of formula 13) with a yield of 92%. The structure of compound 2 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

c. Dissolve 1.5 mmol (1 eq) of compound 2 in 15 mL of anhydrous tetrahydrofuran, then dropwise add 4.5 mL (3 eq) of tetrabutylammonium fluoride solution (1 M tetrahydrofuran solution), stir and react at room temperature for 0.5 h, then concentrate under reduced pressure, and purify by silica gel column chromatography (dichloromethane/methanol=20/1 to 10/1) to obtain a white solid compound 3 (i.e., the compound of formula 14). The structure of compound 3 has been confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

d. Dissolve 0.1 mmol of compound 3 in 2 mL of anhydrous pyridine and remove water under reduced pressure three times; inject 1 mL of dry trimethyl phosphate under nitrogen protection, and after the solid is completely dissolved, place the test tube in an ice bath to cool for 2 min, and add 1.5 eq of phosphorus oxychloride dropwise with a syringe; stir and react in an ice bath for 2.5 h.

e. Then, inject 1.5 eq of dry tributylamine and 1.5 mL (8 eq) of tributylammonium pyrophosphate solution (0.5 M N,N-dimethylformamide solution) in sequence under ice bath conditions, and move to room temperature to react for 0.5 h.

f. After the reaction is completed, the reaction is quenched with 5 mL of 2.0 M saturated triethylamine-carbonic acid aqueous solution under ice bath, and then the reaction solution is extracted with 10 mL of dichloromethane, and the aqueous phase is collected, and repeated 4 times; the collected aqueous phase is separated and purified by a rapid preparative liquid chromatograph C18 column, and 50 mM saturated triethylamine-carbonic acid aqueous solution and chromatographic grade acetonitrile are used as mobile phases; finally, the collected and purified solution is freeze-dried to obtain a white solid 4 (compound of formula 9). The structure of compound 4 has been confirmed by ¹ H NMR, ³¹ P NMR and HRMS.

Figure 6 is the ¹ H NMR spectrum of N4-butyrylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.87, 0.89, 0.91; 1.59, 1.61, 1.63, 1.65; 2.41, 2.42, 2.44; 4.24, 4.27, 4.28, 4.30, 4.31, 4.32, 4.39, 4.40, 4.42; 5.92, 5.93; 7.28, 7.30; 8.39, 8.41. Figure 7 is a ³¹ P NMR spectrum of N4-butyryl cytidine triphosphate, from which it can be seen that the peak positions (unit: ppm) are: -22.68, -22.56, -22.43; -11.47, -11.45, -11.34, -11.33; -6.46, -6.33; 19.52, 19.58. Figure 30 is a mass spectrum of N4-butyryl cytidine triphosphate.

Example 4 N4-Valerylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-pentanoyl cytidine triphosphate. The reaction conditions of each step of the preparation are as described in Example 1. The anhydride used in step b is valeric anhydride.

Figure 8 is the ¹ H NMR spectrum of N4-pentanoylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.80, 0.82, 0.84; 1.26, 1.28; 1.52, 1.53, 1.55, 1.57, 1.59; 2.40, 2.42, 2.44; 4.21, 4.24, 4.25, 4.27, 4.28, 4.29, 4.35, 4.37, 4.38; 5.89, 5.90; 7.25, 7.27; 8.36, 8.38. Figure 9 is a ³¹ P NMR spectrum of N4-pentanoyl cytidine triphosphate, from which it can be seen that the peak positions (unit: ppm) are: -22.69, -22.57, -22.44; -11.51, -11.49, -11.39, -11.36, -11.35, -11.32; -6.54, -6.41. Figure 31 is a mass spectrum of N4-pentanoyl cytidine triphosphate.

Example 5 N4-hexanoyl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-hexanoyl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1. The anhydride used in step b is hexanoic anhydride.

Figure 10 is a ¹ H NMR spectrum of N4-hexanoyl cytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.77, 0.79, 0.80; 1.23, 1.24; 1.56, 1.58, 1.60; 2.39, 2.41, 2.43; 4.24, 4.25, 4.28, 4.35, 4.37, 4.38; 5.90; 7.26, 7.28; 8.36, 8.38. Figure 11 is a ³¹ P NMR spectrum of N4-hexanoyl cytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: -22.64, -22.51, -22.38; -11.45, -11.33; -6.50, -6.37. FIG32 is a mass spectrum of N4-hexanoyl cytidine triphosphate.

Example 6 5-Methyl-N4-acetylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 5-methyl-N4-acetyl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 5-methyl-N4-acetyl cytidine triphosphate. Cytidine, the acid anhydride used in step b is acetic anhydride.

Figure 12 is the ¹ H NMR spectrum of 5-methyl-N4-acetylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 2.06; 2.24, 2.25; 4.24, 4.25, 4.27, 4.28; 4.36, 4.37, 4.39; 5.89, 5.90, 5.91; 8.22, 8.30. Figure 13 is the ³¹ P NMR spectrum of 5-methyl-N4-acetylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: -22.99, -22.86, -22.74; -11.77, -11.75, -11.65, -11.63; -8.19, -8.05; 19.42, 19.47, 19.53. FIG33 is a mass spectrum of 5-methyl-N4-acetylcytidine triphosphate.

Example 7 5-Methyl-N4-butyryl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 5-methyl-N4-butyryl cytidine triphosphate. The reaction conditions of each step of the preparation refer to those of Example 1, except that the reaction starting material used is 5-methylcytidine, and the anhydride used in step b is butyric anhydride.

Figure 14 is the ¹ H NMR spectrum of 5-methyl-N4-butyryl cytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.84, 0.86, 0.88; 1.56, 1.57, 1.59, 1.61; 2.03; 2.43, 2.45, 2.47; 4.19, 4.21, 4.24, 4.25, 4.25, 4.36, 4.37, 4.39; 5.87, 5.88; 8.22. Figure 15 is a ³¹ P NMR spectrum of 5-methyl-N4-butyryl cytidine triphosphate. From the figure, it can be seen that the peak positions (unit: ppm) are: -22.74, -22.61, -22.48; -11.77, -11.76, -11.65, -11.63; -6.52, -6.39. Figure 34 is a mass spectrum of 5-methyl-N4-butyryl cytidine triphosphate.

Example 8 N4-benzoylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-benzoyl cytidine triphosphate. The reaction conditions of each step of the preparation are as in Example 1. The anhydride used in step b is benzoic anhydride.

Figure 16 is the ¹ H NMR spectrum of N4-benzoylcytidine triphosphate; it can be seen from the figure that the peak positions (unit: ppm) are: 4.28, 4.28, 4.29, 4.30, 4.31, 4.33, 4.34, 4.34, 4.35, 4.39, 4.41, 4.42; 5.96, 5.97; 7.43, 7.45, 7.51, 7.53, 7.55, 7.63, 7.65, 7.67; 7.87, 7.87, 7.89; 8.45, 8.47. Figure 17 is the ³¹ P NMR spectrum of N4-benzoylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: -22.88, -22.75, -22.63; -11.51, -11.48, -11.46, -11.43, -11.38, -11.35, -11.34, -11.31; -7.88, -7.77.

Example 9 N4-acetylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-acetyl cytidine triphosphate. The reaction conditions of each step of the preparation are as described in Example 1. The anhydride used in step b is acetic anhydride.

Figure 18 is a ¹ H NMR spectrum of N4-acetylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 2.18; 4.24, 4.27; 4.28; 4.31, 4.31, 4.32, 4.39, 4.40, 4.41; 5.92, 5.93; 7.26, 7.27; 8.39, 8.41. Figure 19 is a ³¹ P NMR spectrum of N4-acetylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: -22.68, -22.56, -22.43; -11.46, -11.34; -6.46, -6.33. Figure 35 is a mass spectrum of N4-acetylcytidine triphosphate.

Example 10 N4-p-Anisyl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-p-methoxybenzoyl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1. The acyl chloride used in step b is p-methoxybenzoyl chloride (4-methoxybenzoyl chloride).

The obtained N4-p-methoxybenzoylcytidine triphosphate was detected, and the nuclear magnetic resonance data were: ¹ H NMR (400 MHz, D ₂ O) δ9.93 (s, 1H), 8.94 (d, J=0.8 Hz, 1H), 7.95 (d, J=2.4 Hz, 2H), 7.03 (d, J=2.8 Hz, 2H), 5.85 (d, J=2.0 Hz, 1H), 5.53 (d, J=4.4 Hz, 1H), 4.04-4.57 (m, 7H), 4.20 (m, 4H), 3.81 (s, 3H).

Example 11 N4-p-nitrobenzoylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-p-nitrobenzoyl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1. The acyl chloride used in step b is p-nitrobenzoyl chloride (4-nitrobenzoyl chloride).

The obtained N4-p-nitrobenzoylcytidine triphosphate was detected, and the nuclear magnetic resonance data were: ¹ H NMR (400 MHz, D ₂ O) δ9.91 (s, 1H), 8.96 (d, J=0.8 Hz, 1H), 8.33 (d, J=2.4 Hz, 2H), 8.20 (d, J=3.2 Hz, 2H), 5.84 (d, J=2.0 Hz, 1H), 5.54 (d, J=4.8 Hz, 1H), 4.03-4.57 (m, 7H), 4.21 (m, 4H), 3.81 (s, 3H).

Example 12 5-Methylcytidine triphosphate

This example provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 5-methylcytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that 5-methylcytidine is used as the starting reactant and the triphosphorylation reaction is directly carried out under the conditions of step d.

Figure 20 is a ¹ H NMR spectrum of 5-methylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 1.91; 4.15, 4.16, 4.18; 4.23, 4.25, 4.26; 4.36, 4.37, 4.38; 5.91, 5.92; 7.73. Figure 21 is a ³¹ P NMR spectrum of 5-methylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: -23.09, -22.66, -22.54, -22.41; -11.68, -11.56; -7.53, -6.49, -6.36. Figure 36 is a mass spectrum of 5-methylcytidine triphosphate.

Example 13 5-Methyl-N4-hexanoyl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 5-methyl-N4-hexanoyl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 5-methylcytidine, and the acid anhydride used in step b is hexanoic anhydride.

Figure 22 is the ¹ H NMR spectrum of 5-methyl-N4-hexanoyl cytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.79, 0.80, 0.80, 0.82; 1.24, 1.25, 1.26; 1.60, 1.62; 2.06, 2.06; 2.49, 2.51, 2.52; 4.24, 4.25, 4.28, 4.28, 4.34, 4.35, 4.37; 5.90, 5.91; 8.22. Figure 23 is a ³¹ P NMR spectrum of 5-methyl-N4-hexanoyl cytidine triphosphate, from which it can be seen that the peak positions (unit: ppm) are: -22.68, -22.56, -22.43; -11.73, -11.61; -6.49, -6.36. Figure 37 is a mass spectrum of 5-methyl-N4-hexanoyl cytidine triphosphate.

Example 14 N4-octanoyl cytidine triphosphate

This example provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-octanoyl cytidine triphosphate, and the reaction conditions of each step of the preparation are referred to Example 1. The anhydride used in step b is octanoic anhydride.

Figure 24 is the ¹ H NMR spectrum of N4-octanoylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.74, 0.76, 0.78; 1.23, 1.32, 1.34, 1.56, 1.57, 1.59; 2.39, 2.41, 2.43; 4.24, 4.25, 4.26, 4.28, 4.29, 4.30; 5.89, 5.90; 7.24, 7.26, 7.27; 8.32, 8.34.

Example 15 N4-decanoylcytidine triphosphate

This example provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is N4-decanoyl cytidine triphosphate, and the reaction conditions of each step of the preparation are as described in Example 1. The anhydride used in step b is decanoic anhydride.

Figure 25 is the ¹ H NMR spectrum of N4-decanoylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.73, 0.75, 0.76; 1.20, 1.22; 1.56, 1.57, 1.59; 2.40, 2.42, 2.44; 4.24, 4.26, 4.27, 4.28, 4.30; 5.89, 5.90; 7.19, 7.21; 8.36, 8.38.

Example 16 5-Methyl-N4-benzoylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 5-methyl-N4-benzoyl The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 5-methylcytidine, and the acid anhydride used in step b is benzoic anhydride.

Figure 26 is the ¹ H NMR spectrum of 5-methyl-N4-benzoylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 2.10; 4.24, 4.25, 4.27, 4.28, 4.31, 4.32, 4.33, 4.41, 4.42, 4.43; 5.92, 5.93; 7.47, 7.49, 7.51, 7.59, 7.61, 7.63; 7.86, 7.88; 8.24.

Example 17 2'-Fluoro-N4-acetylcytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 2'-fluoro-N4-acetyl cytidine triphosphate, and the reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 2'-fluorocytidine, and the acid anhydride used in step b is acetic anhydride.

Figure 27 is the ¹ H NMR spectrum of 2'-fluoro-N4-acetylcytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 2.14; 4.22, 4.24, 4.25, 4.28, 4.30, 4.33, 4.36, 4.40, 4.41, 4.42, 4.44, 4.47, 4.48, 4.49, 4.50; 4.98, 4.99; 5.11, 5.12; 5.98, 6.02; 7.23, 7.25; 8.35, 8.37.

Example 18 2'-Fluoro-N4-butyryl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 2'-fluoro-N4-butyryl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 2'-fluorocytidine, and the anhydride used in step b is butyric anhydride.

Figure 28 is the ¹ H NMR spectrum of 2'-fluoro-N4-butyryl cytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.83, 0.84, 0.86; 1.55, 1.56, 1.58, 1.60; 2.36, 2.38, 2.40; 4.22, 4.24, 4.27, 4.28, 4.30, 4.33, 4.36, 4.40, 4.41, 4.43, 4.43, 4.47, 4.48, 4.49, 4.50; 4.98, 4.99; 5.11, 5.12; 5.98, 6.02; 7.25, 7.27; 8.34, 8.36.

Example 19 2'-Fluoro-N4-hexanoyl cytidine triphosphate

This embodiment provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 2'-fluoro-N4-hexanoyl cytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 2'-fluorocytidine, and the acid anhydride used in step b is hexanoic anhydride.

Figure 29 is the ¹ H NMR spectrum of 2'-fluoro-N4-hexanoyl cytidine triphosphate. It can be seen from the figure that the peak positions (unit: ppm) are: 0.76, 0.77, 0.79; 1.22, 1.22; 1.54, 1.56, 1.58; 2.38, 2.40, 2.41; 4.22, 4.25, 4.27, 4.34, 4.37, 4.39, 4.40, 4.41, 4.42, 4.46, 4.47, 4.48, 4.49; 4.98, 4.99; 5.11, 5.12; 5.98, 6.02; 7.26, 7.27; 8.33, 8.36.

Example 20 2'-Methoxycytidine triphosphate

This example provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 2'-methoxycytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 2'-methoxycytidine.

The obtained 2'-methoxycytidine triphosphate was detected, and the nuclear magnetic resonance data were: ¹ H NMR (400 MHz, D ₂ O) δ7.96 (d, J=0.8 Hz, 1H), 6.05 (d, J=0.8 Hz, 1H), 5.96 (d, J=1.2 Hz, 1H), 5.88 (d, J=3.6 Hz, 1H), 4.72 (t, 1H), 4.03-4.51 (m, 5H), 3.40 (s, 3H).

Example 21 2'-ethoxycytidine triphosphate

This example provides a chemically modified cytidine triphosphate, wherein the chemically modified cytidine triphosphate is 2'-ethoxycytidine triphosphate. The reaction conditions of each step of the preparation are referred to Example 1, except that the reaction starting material used is 2'-ethoxycytidine.

The obtained 2'-ethoxycytidine triphosphate was detected, and the nuclear magnetic resonance data were: ¹ H NMR (400 MHz, D ₂ O) δ7.97 (d, J=0.8 Hz, 1H), 6.67 (s, 2H), 6.03 (d, J=0.8 Hz, 1H), 5.53 (d, J=3.6 Hz, 1H), 4.70 (t, 1H), 4.02-4.52 (m, 5H), 3.87 (q, 2H), 1.19 (t, 3H).

Example 22 DNA template preparation

The plasmid encoding luciferase is used as a template and DNA polymerase is used in a PCR tube. The specific steps are as follows:

The luciferase DNA template was prepared by PCR and carried out in a 96-well PCR instrument.

The PCR product includes at least: A) a promoter sequence (T7 promoter); B) a 5'UTR containing at least one Kozak sequence; C) a 3'UTR; D) a luciferase coding sequence; E) a poly A tail. The specific sequences involved in this embodiment are shown in Table 1.

Table 1

The DNA template was amplified according to the following reaction system: reaction volume, 50 μL (reaction volume of a single tube, multiple tubes were reacted at one time), and the specific reaction system is shown in Table 2.

Table 2

The reaction program was as follows: pre-denaturation at 95°C for 1 min, denaturation at 95°C for 15 s, annealing at 52°C for 5 s, extension at 72°C for 2 min, for a total of 32 cycles; and final extension at 72°C for 5 min.

After the reaction is completed, the reaction solutions are combined in a 1.5 mL tube, and 2 μL is taken for DNA agarose gel electrophoresis to confirm the success of the reaction (agarose gel electrophoresis detection conditions: 2% agarose, 5 V/min, 30 min).

The reaction product was purified using HiPure Gel Pure DNA Mini Kit. The purification process is as follows:

(1) Briefly centrifuge the PCR product.

(2) Add an equal volume of GDP buffer and mix by inversion.

(3) Place the HiPure DNA column in a collection tube, transfer the mixture to the DNA column, and centrifuge at 12,000 g for 30-60 seconds. Discard the filtrate and place the column back in a 2 mL centrifuge tube. Add 600 μL of buffer DW2 (diluted with anhydrous ethanol) to the column and centrifuge at 12,000 g for 30-60 seconds.

(4) Discard the filtrate and put the column back into a 2 mL centrifuge tube. Add 600 μL of buffer DW2 (diluted with anhydrous ethanol) to the column and centrifuge at 12,000 g for 30-60 seconds. Discard the filtrate and put the column back into a 2 mL centrifuge tube and centrifuge at 12,000 g for 2 minutes.

(5) Put the column back into a 1.5 mL centrifuge tube, add 15-30 μL of elution buffer to the center of the column membrane, leave at room temperature for 2 minutes, and centrifuge at 12,000 g for 1 minute. Discard the column, use a NanoDrop Spectrophotometer to detect the concentration of the template DNA, as well as the ratios of 260/280 and 260/230, and then store the DNA at -20°C.

Example 23 In vitro transcription synthesis of mRNA with chemically modified nucleosides replacing natural nucleosides

The prepared chemically modified nucleosides are used to globally replace natural nucleosides to synthesize chemically modified mRNA. The chemically modified cytidine triphosphate includes modifications as follows: N4-acetyl, N4-propionyl, N4-butyryl, N4-pentanoyl, N4-hexanoyl, N4-benzoyl, 5-methyl-N4-acetyl, 5-methyl-N4-butyryl, 5-methyl-N4-hexanoyl, 5-methyl.

The synthesis of chemically modified mRNA was achieved by in vitro transcription using T7 RNA polymerase. Taking a 50 μL reaction system as an example, the components listed in Table 3 were added sequentially into a 0.2 mL PCR reaction tube.

Table 3

CTP* is cytosine triphosphate or chemically modified cytosine triphosphate. Cap Analogue# is a commercially available cap structure analog; in this example, the one used is 3'-O-Me-m ⁷ -G(5')ppp(5')G RNA Cap Structure Analog provided by New England Biolabs, with the catalog number S1411S.

The reaction procedure is as follows: Turn on the thermostat's hot cover and set it to 60°C. Click the thermostat reaction system, 37°C, 2-4h. After the incubation is completed, the mRNA purification method is as follows:

(1) Add 1 μL DNase I (RNase-free) to each 50 μL reaction system and incubate at 37°C for 15 minutes to digest the DNA template in the system.

(2) Add 2.5 μL of ethylenediaminetetraacetic acid disodium salt solution (500 mM) to each 50 μL reaction system and incubate at 65°C for 10 minutes.

(3) Suspend the cellulose solution in the elution solution to prepare an elution solution with a concentration of 0.2 g cellulose/mL (reference for preparation method: A Facile Method for the Removal of dsRNA Contaminants from In Vitro Transcribed mRNA, Molecular Therapy Nucleic Acids, 2019, 15, 26-35). Put the DNA adsorption column in a 2 mL receiving tube (Guangzhou Meiji Biotechnology, DNA/RNA small-volume adsorption column, item number M021), then take 630 μL of the suspension into the DNA column and vortex for 2 h for activation.

(4) The activated cellulose suspension was centrifuged at 13,000 g for 1 minute, the filtrate was discarded, and 500 μL of the elution solution was added to resuspend. The in vitro transcription reaction mixture solution was then added to the DNA column and vortexed for 30 minutes.

(5) Centrifuge the DNA column at 13,000 g for 1 min, collect the filtrate, add it to a new activated DNA column (activation step as described in step 3), and vortex for 30 min.

(6) Centrifuge the DNA column at 13,000 g for 1 minute, collect the filtrate, and then transfer the filtrate to a new 1.5 mL nuclease-free centrifuge tube pre-chilled on ice.

(7) Pre-cool anhydrous isopropanol and 3 M sodium acetate solution on ice, then add 500 μL of isopropanol and 50 μL of sodium acetate to the centrifuge tube in step (6), gently invert to mix, and then centrifuge at 14,000 g for 10 minutes at 4°C.

(8) Remove the supernatant solution, then add 300 μL of 4°C precooled anhydrous ethanol into the centrifuge tube, and then centrifuge at 14,000 g at 4°C for 10 minutes.

(9) Remove the supernatant solution and air-dry the precipitate at room temperature for 30 minutes.

(10) The obtained mRNA product was dissolved in 20-50 μL of nuclease-free water, and the concentration of the purified mRNA was measured using an ultra-micro spectrophotometer, and the ratios of 260/230 and 260/280 were also measured simultaneously.

(11) Take 500 ng of purified mRNA and perform agarose gel electrophoresis to detect the integrity of its fragments.

(12) Store the mRNA solution at -80°C.

Figure 38 is a schematic diagram of the in vitro transcription results of chemically modified C-series nucleoside triphosphates. In the figure, lane 1 is natural cytosine, lane 2 is 5-methylcytosine, lane 3 is 5-methyl-N4-acetylcytosine, lane 4 is 5-methyl-N4-butyrylcytosine, lane 5 is 5-methyl-N4-hexanoyl, lane 6 is 5-methyl-N4-benzoylcytosine, lane 7 is N4-acetylcytosine, lane 8 is N4-propionylcytosine, lane 9 is N4-butyrylcytosine, and lane 10 is the mRNA of N4-hexanoylcytosine; lane M is the RNA molecular weight standard.

If the DNA template obtained by PCR does not contain polyadenylic acid series (polyA), a tailing reaction is required here. The specific steps of the tailing reaction are as follows: prepare the reaction system as shown in Table 7 in a 0.2mL PCR Tube at room temperature; Table 4 is the tailing reaction system, and the total volume is 20μL.

Table 4

The reaction procedure is as follows: set the thermostatic reactor with a lid temperature of 60°C and incubate at 37°C for 30 minutes. After the incubation is completed, the tailed mRNA is purified by the cellulose method described above, and then the mRNA tail length and the integrity of the tailed mRNA fragment are detected by agarose gel electrophoresis.

Example 24 In vitro transcription synthesis of mRNA using chemically modified nucleosides and N1-methylpseudouridine to globally replace natural nucleosides

The same method as in Example 23 was used, except that in this example, chemically modified cytosine triphosphate and N1-methyl pseudouridine triphosphate were mixed in a 50/50 ratio, and natural cytosine triphosphate was replaced globally to perform in vitro transcription synthesis of luciferase mRNA. The combinations used included N4-acetylcytosine triphosphate/N1-methyl pseudouridine triphosphate=50/50, N4-butyrylcytosine triphosphate/N1-methyl pseudouridine triphosphate=50/50, and N4-hexanoylcytosine triphosphate/N1-methyl pseudouridine triphosphate=50/50.

Example 25 Expression efficiency of chemically modified mRNA in cells

Synthesize mRNA containing a single type of cytidine modification or mRNA modified by a single type of modified cytidine combined with N1 methyl pseudouridine encoding luciferase, and detect its expression efficiency in cells; after transfecting it into cells, translate it into the target protein luciferase, and then use luciferin as a substrate to use the translated luciferase to oxidize luciferin chemiluminescence, and use a microplate reader to detect its luminescence intensity, so as to determine the translation efficiency of natural and chemically modified mRNA. In this example, the chemically modified mRNA is transfected into HEK 293T cells and HeLa cells, respectively, and the luminescence intensity is detected.

The specific steps for transfecting HEK 293T cells with chemically modified mRNA are as follows:

(1) Cell transfection

About 24 hours after inoculation of human embryonic kidney 293T cells (purchased from the Chinese Academy of Sciences Cell Bank), observe the cell status in the 24-well plate, and the confluence is 70%. In the biological safety cabinet, prepare 90% (volume percentage) DMEM + 10% (volume percentage) FBS culture medium. Discard the culture medium in the well plate 30 minutes before transfection, and add 500 μL of fresh culture medium, i.e. 90% (volume percentage) DMEM + 10% (volume percentage) FBS culture medium, to each well.

(2) Preparation of transfection system

Take 50μL Opti-MEM, add 800ng mRNA, and mix well. Also prepare a mixed solution of 2μL Lipofectamine 2000 and 50μL Opti-MEM, and incubate at room temperature for 10 minutes. Then add the Lipofectamine 2000 mixture to the mRNA mixture, mix well, and incubate for 20 minutes. Add the mixed solution to the well plate. Change the medium 6h after transfection, aspirate the old culture medium, and replace each well with 500μL fresh culture medium, that is, 90% (volume percentage) DMEM + 10% (volume percentage) FBS culture medium. Harvest 24h/48h after transfection. Aspirate the old culture medium and wash once with 1mL PBS. Aspirate the PBS, digest the cells with 200 μL 1× Cell Lysis Buffer (Abcam, ab152163) for 5 minutes, then blow down the cells with a pipette, collect them in a 1.5 mL centrifuge tube, centrifuge at 14000 g for 10 minutes at 4°C, and then take 170 μL of the supernatant solution in a new 1.5 mL centrifuge tube for luciferin luminescence detection.

The experimental steps for detecting luminescence with an ELISA reader are as follows:

Add 50 μL of the above-extracted protein solution containing luciferase to a 96-well plate, then incubate at room temperature for 10 minutes, then add 50 μL Luciferase Assay Reagent (Promega, E1483), pipette evenly, and complete the luminescence intensity detection on the microplate reader within 5 minutes. Set up two replicate wells for each sample.

Figure 39 is a graph showing the expression efficiency of transcribed mRNA transfected into HEK 293T cells 24 hours after transfection. The horizontal axis in the figure is the sample type. In the figure:

1 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

2 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate instead of natural cytidine triphosphate as substrate;

3 indicates luciferase mRNA transcribed using N4-propionyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

4 indicates luciferase mRNA transcribed using N4-butyryl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

5 indicates luciferase mRNA transcribed using N4-pentanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

6 represents luciferase mRNA transcribed using N4-hexanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

7 represents luciferase mRNA transcribed using N4-benzoylcytidine triphosphate instead of natural cytidine triphosphate as substrate.

As can be seen from Figure 39, the translation efficiency of luciferase mRNA with global replacement of N4-propionylcytosine, N4-butyrylcytosine, N4-pentanoylcytosine and N4-hexanoylcytosine in HEK 293T cells is higher than that of luciferase mRNA transcribed from natural cytosine, among which the translation efficiency of luciferase mRNA with global replacement of N4-butyrylcytosine can be enhanced to 2.91 times, proving that chemically modified cytosine can effectively enhance the translation efficiency of transcribed mRNA.

The steps of chemically modified mRNA transfection of HeLa cells refer to the method of transfecting HEK 293T cells. Figure 41 is the expression efficiency diagram of transcribed mRNA transfected HEK 293T cells 24 hours after transfection. In the figure:

1 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

2 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate instead of natural cytidine triphosphate as substrate;

3 indicates luciferase mRNA transcribed using N4-propionyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

4 indicates luciferase mRNA transcribed using N4-butyryl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

5 indicates luciferase mRNA transcribed using N4-pentanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

6 represents luciferase mRNA transcribed using N4-hexanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

7 represents luciferase mRNA transcribed using N4-benzoylcytidine triphosphate instead of natural cytidine triphosphate as substrate.

As can be seen from Figure 41, the translation efficiency of luciferase mRNA with global replacement of N4-propionylcytosine, N4-butyrylcytosine, N4-pentanoylcytosine and N4-hexanoylcytosine in HeLa cells is higher than that of luciferase mRNA transcribed from natural cytosine, among which the translation efficiency of luciferase mRNA with global replacement of N4-propionylcytosine, N4-butyrylcytosine and N4-pentanoylcytosine can be enhanced by more than 10 times, proving that chemically modified cytosine can effectively enhance the translation efficiency of transcribed mRNA.

Figure 40 is a graph showing the expression efficiency of mRNA transcribed by N1-methyl pseudouridine and modified cytidine after transfection into HEK 293T cells for 24 hours. In the figure:

1 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

2 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate as substrates;

3 indicates luciferase mRNA transcribed using N4-propionylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate as substrates;

4 represents luciferase mRNA transcribed with N4-butyrylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate instead of natural cytidine triphosphate as substrates;

5 indicates luciferase mRNA transcribed with N4-pentanoylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate instead of natural cytidine triphosphate as substrates;

6 represents luciferase mRNA transcribed with N4-hexanoylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate instead of natural cytidine triphosphate as substrates;

7 represents luciferase mRNA transcribed using N1-methylpseudouridine triphosphate instead of uridine triphosphate as substrate.

As can be seen from the figure, compared with natural cytosine, the translation efficiency of luciferase mRNA transcribed by combining N4-acetylcytidine triphosphate with N1-methyl pseudouridine triphosphate, N4-propionylcytidine triphosphate with N1-methyl pseudouridine triphosphate in HEK 293T cells was enhanced by 2.63-3.32 times. The mRNA with global replacement of N1-methyl pseudouridine triphosphate alone can achieve an enhanced translation efficiency of 1.87 times.

FIG42 is a graph showing the expression efficiency of mRNA transcribed by N1-methyl pseudouridine and modified cytidine after transfection into HeLa cells for 24 hours, in which:

1 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

2 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate as substrates;

3 indicates luciferase mRNA transcribed using N4-propionylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate as substrates;

4 represents luciferase mRNA transcribed with N4-butyrylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate instead of natural cytidine triphosphate as substrates;

5 indicates luciferase mRNA transcribed with N4-pentanoylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate instead of natural cytidine triphosphate as substrates;

6 represents luciferase mRNA transcribed with N4-hexanoylcytidine triphosphate and N1-methylpseudouridine triphosphate instead of natural cytidine triphosphate and uridine triphosphate instead of natural cytidine triphosphate as substrates;

7 represents luciferase mRNA transcribed using N1-methylpseudouridine triphosphate instead of uridine triphosphate as substrate.

As can be seen from the figure, compared with natural cytosine, the translation efficiency of luciferase mRNA transcribed by N4-acetylcytidine triphosphate, N4-propionylcytidine triphosphate, N4-butyrylcytidine triphosphate, and N4-pentanoylcytidine triphosphate in HeLa cells was enhanced by 1.66 to 10.82 times after the combination of N4-acetylcytidine triphosphate, N4-propionylcytidine triphosphate, N4-butyrylcytidine triphosphate and N1-methyl pseudouridine. The translation efficiency of mRNA replaced by N1-methyl pseudouridine alone was enhanced by 9.5 times.

Example 26 Stability of chemically modified mRNA in cells

An mRNA encoding luciferase containing a single type of cytidine modification was synthesized and its stability in cells was tested; its stability refers to the relative amount of residual intact mRNA extracted after the mRNA was transfected into the cells for a certain period of time.

In this example, the chemically modified mRNA was transfected into HEK 293T cells and HeLa cells, and qPCR detection was performed. The specific steps of chemically modified mRNA transfection into HEK 293T cells and qPCR detection are as follows:

Cell inoculation and cell transfection steps are described in Example 25. 3h after transfection, the culture medium was discarded, the cells were rinsed once with 1×PBS, and then the PBS washing solution was aspirated. 200μL TransZol Up lysis solution (Quanshi Jin Bio, ET1111) was added, and it was placed horizontally for a moment to evenly distribute the lysis solution on the cell surface and lyse the cells, and then the cells were blown off with a pipette. After that, the lysis solution was transferred to a centrifuge tube, and it was blown repeatedly with a pipette until there was no obvious precipitation in the lysate, and it was left to stand at room temperature for 5 minutes. 40μL chloroform was added, and it was shaken violently for 30 seconds, and then incubated at room temperature for 3 minutes. Centrifuged at 10000g for 15 minutes at 4°C, at which time the sample was divided into 3 layers, the upper layer was a colorless aqueous phase, and the middle layer and the lower layer were pink organic phases. At this time, RNA was in the aqueous phase, and 90μL of colorless aqueous phase was extracted with a pipette, transferred to a new centrifuge tube, 90μL of anhydrous ethanol was added, and gently inverted to mix evenly. The obtained precipitate and solution were transferred to the centrifuge column and centrifuged at 12000g for 30 seconds at room temperature. Elute. Add 100 μL CB9, centrifuge at 12000g for 30 seconds at room temperature, discard the effluent, and repeat once. Add 100 μL WB9, centrifuge at 12000g for 30 seconds at room temperature, discard the effluent, and repeat once. Then centrifuge at 12000g for 2 minutes at room temperature to completely remove the residual anhydrous ethanol. Place the centrifuge column in a nuclease-free tube, add 20 μL nuclease-free water to the center of the centrifuge column, and let it stand at room temperature for 1 minute. Then centrifuge at 12000g for 1 minute at room temperature to elute the RNA. The total RNA obtained was quantified using NanoDrop. Then take 1 μg of total RNA and synthesize cDNA using Hifair 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yishen Bio, 11139ES10). The obtained cDNA was quantified by NanoDrop, and 500 ng of cDNA was taken and then fluorescent quantitative amplification was performed using Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (Yisheng Bio, 11184ES08). The fluorescent quantitative PCR instrument used was qTower. The stability of mRNA was expressed by calculating the relative value of the obtained Ct value using the 2 ^-Δ method.

FIG43 is a graph showing the qPCR results of the stability of chemically modified mRNA in HEK 293T cells, in which:

1 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

2 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate instead of natural cytidine triphosphate as substrate;

3 indicates luciferase mRNA transcribed using N4-propionyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

4 indicates luciferase mRNA transcribed using N4-butyryl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

5 indicates luciferase mRNA transcribed using N4-pentanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

6 represents luciferase mRNA transcribed using N4-hexanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

7 represents luciferase mRNA transcribed using N4-benzoylcytidine triphosphate instead of natural cytidine triphosphate as substrate.

As can be seen from Figure 43, the stability of luciferase mRNA with global replacement of N4-propionylcytosine, N4-butyrylcytosine, N4-pentanoylcytosine, and N4-hexanoylcytosine in HEK 293T cells is higher than that of luciferase mRNA transcribed from natural cytosine, proving that global replacement of chemically modified cytosine can enhance the stability of transcribed mRNA in cells.

The steps of chemically modified mRNA transfection into HeLa cells and qPCR detection are carried out in accordance with the method of transfecting HEK 293T cells. Figure 44 is a graph showing the qPCR results of the stability test of chemically modified mRNA in HeLa cells, in which:

1 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

2 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate instead of natural cytidine triphosphate as substrate;

3 indicates luciferase mRNA transcribed using N4-propionyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

4 indicates luciferase mRNA transcribed using N4-butyryl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

5 indicates luciferase mRNA transcribed using N4-pentanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

6 represents luciferase mRNA transcribed using N4-hexanoyl cytidine triphosphate instead of natural cytidine triphosphate as a substrate.

As can be seen from Figure 44, the stability of luciferase mRNA with global replacement of N4-propionylcytosine, N4-pentanoylcytosine and N4-hexanoylcytosine in HEK 293T cells is higher than that of luciferase mRNA transcribed from natural cytosine, proving that global replacement of chemically modified cytosine can enhance the stability of transcribed mRNA in cells.

Example 27 Immunogenicity of chemically modified mRNA in cells

Synthesize mRNA encoding luciferase containing a single type of cytidine modification and test its immunogenicity. Transfect the mRNA into macrophages RAW264.7, and then detect the immune factors produced by RAW264.7 cells 24 hours after transfection, including tumor necrosis factor α and interleukin 6. The specific steps are as follows:

The cell inoculation and cell transfection steps refer to those described in Example 25. The cells used in this example are macrophages RAW264.7. 24 hours after cell transfection, digest the cells with 200 μL 1×Cell Lysis Buffer (Abcam, ab152163) for 5 minutes, then blow down the cells with a pipette and collect them in a 1.5 mL centrifuge tube. Centrifuge at 14000g for 10 minutes at 4°C, and then take 170 μL of the supernatant solution in a new 1.5 mL centrifuge tube. Take 50 μL of the extracted protein solution, add 100 μL of BCA protein quantification kit solution (Yishen Bio, 20201ES76), and then blow down with a pipette. Beat evenly, incubate at 30°C for 30 minutes, and then detect its absorbance at 562nm using Epoch Microplate Reader. Calculate the total protein concentration extracted according to the standard curve. The immunogenicity of luciferase mRMA is expressed by the amount of cytokines secreted by it after stimulating RAW264.7 cells, including tumor necrosis factor α and interleukin 6.

The amount of cytokines was quantified by western blotting, and the specific steps were as follows: 20 μg of total protein sample was taken, 1/5 volume of 6× Protein Loading Buffer (All-Gold Bio, DL101) was added, and then boiled at 95°C for 10 minutes, and then cooled to 4°C on ice. After a brief centrifugation, all samples were separated by 12% SDS-PAGE gel. The sample was then transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk at room temperature for 2 hours, and then rinsed with 1× TBST Buffer 3 times, 20 mL each time. Then, the corresponding primary antibodies (the primary antibodies are the primary antibodies of the target protein and the primary antibodies of the internal reference protein, so there are two types, including: recombinant Anti-TNF alpha antibody, EPR19147, Abcam and Antib-beta Actin antibody, AC-15, Abcam) diluted 1:3000 were incubated at 4°C overnight. Then, the membrane was rinsed 3 times with 1× TBST Buffer, 20 mL each time. After that, the membrane was incubated with the corresponding secondary antibody (Goat Anti-Rabbit IgG H&L HRP, ab6721, Abcam and Goat Anti-Mouse IgG H&L HRP, ab6728, Abcam) diluted 1:5000 for 1 hour, and then the membrane was rinsed 3 times with 1× TBST Buffer, 10 mL each time. Then, the enhanced ECL chemiluminescence exposure reagent (Yisheng Bio, 36222ES60) was prepared for chemical exposure. The relative content of the produced cytokines was expressed as the intensity of the extracted bands.

Figure 45 is a Western blot result of the immunogenicity test of chemically modified mRNA in RAW264.7 cells, in which:

1 represents buffer solution PBS;

2 indicates luciferase mRNA transcribed using natural nucleoside triphosphates as substrates;

3 indicates luciferase mRNA transcribed using N4-acetylcytidine triphosphate instead of natural cytidine triphosphate as substrate;

4 indicates luciferase mRNA transcribed using N4-propionylcytidine triphosphate instead of natural cytidine triphosphate as substrate;

5 indicates luciferase mRNA transcribed using N4-butyryl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

6 indicates luciferase mRNA transcribed using N4-pentanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

7 indicates luciferase mRNA transcribed using N4-hexanoyl cytidine triphosphate instead of natural cytidine triphosphate as substrate;

8 represents luciferase mRNA transcribed using N4-benzoylcytidine triphosphate instead of natural cytidine triphosphate as substrate.

As can be seen from the figure, compared with the luciferase mRNA transcribed from natural cytosine, the luciferase mRNA with global replacement of chemically modified cytosine did not show weakened immunogenicity in RAW264.7 cells.

Example 28 Synthesis of chemically modified adenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine triphosphate, and the preparation technology route is as follows. In this embodiment, R _5' of the chemically modified adenosine triphosphate is hydrogen, R _n is hydroxyl, and M is nitrogen. The structure of the compound is characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry.

a. 3 mmol (1 eq) of adenosine was dissolved in 15 mL of N,N-dimethylformamide, and then 12 mmol (4 eq) of tert-butyldimethylsilyl chloride and 15 mmol (5 eq) of imidazole were added in sequence, and stirred at 25°C overnight; after the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times. The organic phase was washed with water, and the extraction was repeated 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol = 100/1) to obtain a white solid compound 5 (i.e., the compound of formula 18); the structure of compound 5 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

b. 12 mmol (1 eq) of compound 5 was dissolved in 15 mL of anhydrous pyridine, and then 1.2 eq of the corresponding acid chloride was added dropwise, followed by 10 mg of 4-dimethylaminopyridine. The mixture was stirred at 25°C overnight. After the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times. The organic phase was washed with water, and the extraction was repeated 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol = 200/1) to obtain a white foamy solid compound 6 (i.e., compound 19). The structure of compound 6 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

c. Dissolve 1.5 mmol (1 eq) of compound 6 in 15 mL of anhydrous tetrahydrofuran, then dropwise add 4.5 mL (3 eq) of tetrabutylammonium fluoride solution (1 M tetrahydrofuran solution), stir at room temperature for 0.5 h, then concentrate under reduced pressure, and purify by silica gel column chromatography (dichloromethane/methanol=20/1 to 10/1) to obtain white solid compound 7 (i.e., compound of formula 20). The structure of compound 7 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

d. Dissolve 0.1 mmol of compound 7 in 2 mL of anhydrous pyridine and remove water under reduced pressure three times; inject 1 mL of dry trimethyl phosphate under nitrogen protection, and after the solid is completely dissolved, place the test tube in an ice bath for 2 min, and add 1.5 eq of phosphorus oxychloride dropwise with a syringe; stir and react in an ice bath for 2.5 h.

e. Then, inject 1.5 eq of dry tributylamine and 1.5 mL (8 eq) of tributylammonium pyrophosphate solution (0.5 M N,N-dimethylformamide solution) in sequence under ice bath conditions, and move to room temperature to react for 0.5 h.

f. After the reaction was completed, the reaction was quenched with 5 mL of 2.0 M saturated triethylamine-carbonic acid aqueous solution under ice bath, and then the reaction solution was extracted with 10 mL of dichloromethane, and the aqueous phase was collected, and repeated 4 times; the collected aqueous phase was separated and purified by rapid preparative liquid chromatography C18 column, and 50 mM saturated triethylamine-carbonic acid aqueous solution and chromatographic grade acetonitrile were used as mobile phases; finally, the collected purified solution was The solution was lyophilized to obtain a white solid compound 8 (ie, the compound of formula 23). The structure of compound 8 was confirmed by ¹ H NMR, ³¹ P NMR, ¹³ C NMR and HRMS.

In the method for preparing chemically modified adenosine triphosphate in this embodiment, only step a, step b and step c are performed to form the method for preparing chemically modified adenosine, which will not be described in detail here.

Example 29 Synthesis of N6-propionyl adenosine and N6-propionyl adenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine and chemically modified adenosine triphosphate, wherein the chemically modified adenosine is N6-propionyl adenosine, and the chemically modified adenosine triphosphate is N6-propionyl adenosine triphosphate, and the structures are shown below.

The reaction conditions of each step of the preparation are shown below, and the structure of the compound was characterized by nuclear magnetic resonance and mass spectrometry:

a. 3 mmol (1 eq) of adenosine was dissolved in 15 mL of N,N-dimethylformamide, and then 12 mmol (4 eq) of tert-butyldimethylsilyl chloride and 15 mmol (5 eq) of imidazole were added in sequence, and stirred at 25°C overnight; after the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, and the extraction was repeated 3 times; the organic phase was washed with water, and the extraction was repeated 3 times; the organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=100/1) to obtain a white solid compound 5 with a yield of 95%; the structure of compound 1 has been confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

b. 12 mmol (1 eq) of compound 5 was dissolved in 15 mL of anhydrous pyridine, and then 1.2 eq of propionyl chloride was added dropwise, followed by 10 mg of 4-dimethylaminopyridine. The mixture was stirred at 25°C overnight to react. After the reaction, the reaction solution was extracted with 10 mL of ethyl acetate, repeated 3 times, and the organic phase was washed with water, repeated 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (dichloromethane/methanol=200/1) to obtain compound 6 as a white foamy solid with a yield of 92%. The structure of compound 2 was confirmed by ¹ H NMR, ¹³ C NMR and HRMS.

c. Dissolve 1.5 mmol (1 eq) of compound 6 in 15 mL of anhydrous tetrahydrofuran, then dropwise add 4.5 mL (3 eq) of tetrabutylammonium fluoride solution (1 M tetrahydrofuran solution), stir the reaction at room temperature for 0.5 h, then concentrate under reduced pressure, and purify by silica gel column chromatography (dichloromethane/methanol=20/1 to 10/1) to obtain a white solid compound 7 (N6-propionyl adenosine). The structure of compound 7 has been confirmed by ¹ H NMR, ¹³ C NMR and HRMS. The ¹ H NMR characterization of N6-propionyl adenosine is shown in FIG46 , the ¹³ C NMR characterization of N6-propionyl adenosine is shown in FIG47 , and the HRMS characterization of N6-propionyl adenosine is shown in FIG48 .

d. Dissolve 0.1 mmol of compound 7 in 2 mL of anhydrous pyridine and remove water under reduced pressure three times; inject 1 mL of dry trimethyl phosphate under nitrogen protection, and after the solid is completely dissolved, place the test tube in an ice bath for 2 min, and add 1.5 eq of phosphorus oxychloride dropwise with a syringe; stir and react in an ice bath for 2.5 h.

e. Then, inject 1.5 eq of dry tributylamine and 1.5 mL (8 eq) of tributylammonium pyrophosphate solution (0.5 M N,N-dimethylformamide solution) in sequence under ice bath conditions, and move to room temperature to react for 0.5 h.

f. After the reaction is completed, quench the reaction with 5 mL of 2.0 M saturated triethylamine-carbonic acid aqueous solution under ice bath, then extract the reaction solution with 10 mL of dichloromethane, collect the aqueous phase, and repeat 4 times; separate and purify the collected aqueous phase with a rapid preparative liquid chromatograph C18 column, and use 50 mM saturated triethylamine-carbonic acid aqueous solution and chromatographic grade acetonitrile as mobile phases; finally, freeze-dry the collected and purified solution to obtain white solid compound 8 (N6-propionyl adenosine triphosphate). The structure of compound 8 has been confirmed by ¹ H NMR, ³¹ PNMR and HRMS. The ¹ H NMR characterization of N6-propionyl adenosine triphosphate is shown in Figure 49, the ¹³ C NMR characterization of N6-propionyl adenosine triphosphate is shown in Figure 50, and the HRMS characterization of N6-propionyl adenosine triphosphate is shown in Figure 51.

Example 30 Synthesis of N6-butyryladenosine and N6-butyryladenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine and chemically modified adenosine triphosphate, wherein the chemically modified adenosine is N6-butyryladenosine, and the chemically modified adenosine triphosphate is N6-butyryladenosine triphosphate, and the structures are shown below.

The difference between the preparation method and Example 29 is that butyryl chloride is used for the reaction in step b, the reaction conditions of each preparation step refer to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry.

The ¹ H NMR characterization chart of N6-butyryladenosine is shown in Figure 52, ^{the 13} C NMR characterization chart of N6-butyryladenosine is shown in Figure 53, and the HRMS characterization chart of N6-butyryladenosine is shown in Figure 54. The ¹ H NMR characterization chart of N6-butyryladenosine triphosphate is shown in Figure 55, the ¹³ C NMR characterization chart of N6-butyryladenosine triphosphate is shown in Figure 56, and the HRMS characterization chart of N6-butyryladenosine triphosphate is shown in Figure 57.

Example 31 Synthesis of N6-pentanoyl adenosine and N6-pentanoyl adenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine and chemically modified adenosine triphosphate, wherein the chemically modified adenosine is N6-pentanoyl adenosine, and the chemically modified adenosine triphosphate is N6-pentanoyl adenosine triphosphate, and the structures are shown below.

The difference between the preparation method and Example 29 is that valeryl chloride is used for the reaction in step b, the reaction conditions of each step of the preparation refer to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry.

The ¹ H NMR characterization chart of N6-pentanoyl adenosine is shown in Figure 58, ^{the 13} C NMR characterization chart of N6-pentanoyl adenosine is shown in Figure 59, and the HRMS characterization chart of N6-pentanoyl adenosine is shown in Figure 60. The ¹ H NMR characterization chart of N6-pentanoyl adenosine triphosphate is shown in Figure 61, the ¹³ C NMR characterization chart of N6-pentanoyl adenosine triphosphate is shown in Figure 62, and the HRMS characterization chart of N6-pentanoyl adenosine triphosphate is shown in Figure 63.

Example 32 Synthesis of N6-hexanoyl adenosine and N6-hexanoyl adenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine and chemically modified adenosine triphosphate, wherein the chemically modified adenosine is N6-hexanoyl adenosine, and the chemically modified adenosine triphosphate is N6-hexanoyl adenosine triphosphate, and the structures are shown below.

The difference between the preparation method and Example 29 is that hexanoyl chloride is used for the reaction in step b, the reaction conditions of each preparation step refer to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry.

The ¹ H NMR characterization chart of N6-hexanoyl adenosine is shown in Figure 64, ^{the 13} C NMR characterization chart of N6-hexanoyl adenosine is shown in Figure 65, and the HRMS characterization chart of N6-hexanoyl adenosine is shown in Figure 66. The ¹ H NMR characterization chart of N6-hexanoyl adenosine triphosphate is shown in Figure 67, the ¹³ C NMR characterization chart of N6-hexanoyl adenosine triphosphate is shown in Figure 68, and the HRMS characterization chart of N6-hexanoyl adenosine triphosphate is shown in Figure 69.

Example 33 N6-Benzoyl adenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine triphosphate, wherein the chemically modified adenosine triphosphate is N6-benzoyl adenosine triphosphate, and the structure is shown below.

The difference between the preparation method and Example 29 is that benzoic anhydride is used for the reaction in step b, the reaction conditions of each step of the preparation are referred to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry. The HRMS characterization chart of N6-benzoyl adenosine triphosphate is shown in Figure 70.

Example 34 N6-methyladenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine triphosphate, wherein the chemically modified adenosine triphosphate is N6-methyladenosine triphosphate, and the structure is shown below.

The difference between the preparation method and Example 29 is that commercial N6-methyladenosine (CAS No.: 1867-73-8) is used as a raw material for triphosphorylation reaction, the reaction conditions of each step of the preparation are referred to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry. The HRMS characterization chart of N6-methyladenosine triphosphate is shown in Figure 71.

Example 35 N6-acetyl adenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine triphosphate, wherein the chemically modified adenosine triphosphate is N6-acetyl adenosine triphosphate, and the structure is shown below.

The difference between the preparation method and Example 29 is that acetyl chloride is used for reaction in step b, and the reaction of each step of the preparation is The conditions were similar to those in Example 29, and the structure of the compound was characterized by nuclear magnetic resonance and mass spectrometry. The HRMS characterization graph of N6-acetyl adenosine triphosphate is shown in FIG72 .

Example 36 2-aminoadenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine triphosphate, wherein the chemically modified adenosine triphosphate is 2-aminoadenosine triphosphate, and the structure is shown below.

The difference between the preparation method and Example 29 is that commercial 2-aminoadenosine (CAS No.: 2096-10-8) is directly used as a raw material for the reaction, the reaction conditions of each step of the preparation are referred to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry. The HRMS characterization chart of 2-aminoadenosine triphosphate is shown in Figure 73.

Example 37 7-Deazaadenosine triphosphate

This embodiment provides a method for preparing chemically modified adenosine triphosphate, wherein the chemically modified adenosine triphosphate is 7-deazaadenosine triphosphate, and the structure is shown below.

The difference between the preparation method and Example 29 is that commercial 7-deazaadenosine (CAS No.: 69-33-0) is directly used as a raw material for the reaction, the reaction conditions of each step of the preparation are referred to Example 29, and the structure of the compound is characterized by nuclear magnetic resonance and mass spectrometry. The HRMS characterization chart of 7-deazaadenosine triphosphate is shown in Figure 74.

Example 38 Preparation of DNA Template for Compiling Enhanced Green Fluorescent Protein

The DNA template for compiling enhanced green fluorescent protein is prepared by linearizing the plasmid containing the target gene with an endonuclease.

1) Enzyme digestion was used to prepare an enhanced green fluorescent protein linear DNA template in a 96-well PCR instrument. The linearized product at least includes: A) a promoter sequence (T7 promoter); B) a 5'UTR containing at least one Kozak sequence; C) a 3'UTR; D) an eGFP coding sequence. The specific sequences involved in this embodiment are shown in Table 5.

Table 5

The following reaction system was prepared to linearize the DNA template: reaction volume, 20 μL (reaction volume of a single tube, multiple tubes were reacted at one time), and the specific reaction system is shown in Table 6.

Table 6

Turn on the thermostat's hot cover and set it to 60°C. Click the thermostat reaction system, 37°C, 15 min. The enzyme digestion product was purified using HiPure Gel Pure DNA Mini Kit (Guangzhou Meiji Biotechnology Co., Ltd., catalog number: D2111). The purification process is as follows:

(1) Briefly centrifuge the digestion reaction products.

(2) Add an equal volume of GDP buffer and mix by inversion.

(3) Place the HiPure DNA column in a collection tube, transfer the mixture to the DNA column, and centrifuge at 12,000 g for 30-60 seconds. Discard the filtrate and place the column back in a 2 mL centrifuge tube. Add 600 μL of buffer DW2 (diluted with anhydrous ethanol) to the column and centrifuge at 12,000 g for 30-60 seconds.

(4) Discard the filtrate and put the column back into a 2 mL centrifuge tube. Add 600 μL of buffer DW2 (diluted with anhydrous ethanol) to the column and centrifuge at 12,000 g for 30-60 seconds. Discard the filtrate and put the column back into a 2 mL centrifuge tube and centrifuge at 12,000 g for 2 minutes.

(5) Put the column back into a 1.5 mL centrifuge tube, add 15-30 μL of elution buffer to the center of the column membrane, leave at room temperature for 2 minutes, and centrifuge at 12,000 g for 1 minute. Discard the column, measure the concentration of the template DNA, and the ratios of 260/280 and 260/230 using a NanoDrop spectrophotometer, and then store the DNA at -20°C.

Example 39 Preparation of DNA template for encoding luciferase

Using the plasmid encoding luciferase as a template, DNA amplification was performed in a PCR tube using DNA polymerase. The specific steps are as follows:

The luciferase DNA template was prepared by PCR and carried out in a 96-well PCR instrument.

The PCR product contains at least: A) a promoter sequence (T7 promoter); B) at least one Kozak sequence 5'UTR; C) 3'UTR; D) luciferase coding sequence; E) poly A tail (polyA tail). The specific sequences involved in this example are shown in Table 7

Table 7

The following reaction system was prepared for amplification of the DNA template: reaction volume, 50 μL (reaction volume of a single tube, multiple tubes were reacted at one time), and the specific reaction system is shown in Table 8.

Table 8

The reaction program was as follows: pre-denaturation at 95°C for 1 minute, denaturation at 95°C for 15 seconds, annealing at 52°C for 5 seconds, extension at 72°C for 2 minutes, for a total of 32 cycles; and final extension at 72°C for 5 minutes.

After the reaction is completed, the reaction solution is combined in a 1.5 mL tube. 2 μL is taken for DNA agarose gel electrophoresis to confirm the success of the reaction (agarose gel electrophoresis detection conditions: 2% agarose, 5 V/min, electrophoresis for 30 minutes).

The reaction product was purified using HiPure Gel Pure DNA Mini Kit. The purification process is as follows:

(1) Briefly centrifuge the PCR product.

(2) Add an equal volume of GDP buffer and mix by inversion.

(3) Place the HiPure DNA column in a collection tube, transfer the mixture to the DNA column, and centrifuge at 12,000 g for 30-60 seconds. Discard the filtrate and place the column back in a 2 mL centrifuge tube. Add 600 μL of buffer DW2 (diluted with anhydrous ethanol) to the column and centrifuge at 12,000 g for 30-60 seconds.

(4) Discard the filtrate and put the column back into a 2 mL centrifuge tube. Add 600 μL of buffer DW2 (diluted with anhydrous ethanol) to the column and centrifuge at 12,000 g for 30-60 seconds. Discard the filtrate and put the column back into a 2 mL centrifuge tube and centrifuge at 12,000 g for 2 minutes.

(5) Put the column back into a 1.5 mL centrifuge tube, add 15-30 μL of elution buffer to the center of the column membrane, leave at room temperature for 2 minutes, and centrifuge at 12,000 g for 1 minute. Discard the column, measure the concentration of the template DNA, and the ratios of 260/280 and 260/230 using a NanoDrop spectrophotometer, and then store the DNA at -20°C.

Example 40 In vitro transcription synthesis of mRNA in which chemically modified adenosine globally replaces natural adenosine

The synthesis of mRNA in which chemically modified adenosine globally replaces natural adenosine is achieved by in vitro transcription using T7 RNA polymerase. Taking a 50 μL reaction system as an example, the components listed in Table 9 are added sequentially into a 0.2 mL PCR reaction tube.

Table 9

*When preparing eGFP mRNA by in vitro transcription, the template is an enhanced green fluorescent protein DNA template. **Adenosine triphosphate or chemically modified cytosine triphosphate. Cap Analogue# is a commercially available cap structure analog; in this example, the one used is 3’-O-Me-m7-G(5’)ppp(5’)G RNA Cap Structure Analog provided by New England Biolabs, with the catalog number S1411S.

Turn on the thermostat's hot cover and set it to 60°C. Click on the thermostat reaction system, 37°C, 2-4h. After the incubation is complete, the mRNA purification method is as follows:

1. Add 1 μL DNase I (RNase-free) to each 50 μL reaction system and incubate at 37°C for 15 minutes to digest the DNA template in the system.

2. Add 2.5 μL of ethylenediaminetetraacetic acid disodium salt solution (500 mM) to each 50 μL reaction system and incubate at 65°C for 10 minutes.

3. Suspend the cellulose solution in the elution solution to prepare a suspension of 0.2 g cellulose/mL (reference for preparation method: A Facile Method for the Removal of dsRNA Contaminants from In Vitro Transcribed mRNA, Molec μLar Therapy Nucleic Acids, 2019, 15, 26-35). Put the DNA adsorption column in a 2 mL receiving tube (Guangzhou Meiji Biotechnology, DNA/RNA small amount adsorption column, product number is M021), then take 630 μL of the suspension into the DNA column and vortex for 2 hours for activation.

4. Centrifuge the activated cellulose suspension at 13000g for 1 minute, discard the filtrate, and then resuspend it with 500μL of elution solution. Then add the in vitro transcription reaction mixture solution to the DNA column and vortex for 30 minutes.

5. Centrifuge the DNA column at 13000g for 1 minute, collect the filtrate, add it to a new activated DNA column (activation step as described in step 3), and vortex for 30 minutes.

6. Centrifuge the DNA column at 13,000 g for 1 minute, collect the filtrate, and transfer the filtrate to a new 1.5 mL nuclease-free centrifuge tube pre-chilled on ice.

7. Pre-cool anhydrous isopropanol and 3M sodium acetate solution on ice, then add 500 μL of anhydrous isopropanol and 50 μL of sodium acetate solution to the centrifuge tube in step 6, gently invert to mix, and then centrifuge at 14000g at 4°C for 10 minutes.

8. Remove the supernatant solution, then add 300 μL of 4°C pre-cooled anhydrous ethanol to the centrifuge tube, and then incubate at 4°C at 14000 Centrifuge at 4 °C for 10 min.

9. Remove the supernatant solution and air-dry the pellet at room temperature for 30 minutes.

10. Dissolve the obtained mRNA product in 20-50 μL of nuclease-free water, and measure the concentration of the purified mRNA using a NanoDrop spectrophotometer, and simultaneously measure the ratios of 260/230 and 260/280.

11. Take 500 ng of purified mRNA and perform agarose gel electrophoresis to detect the integrity of its fragments.

12. Store the mRNA solution at -80°C.

The chemically modified adenine nucleosides include the following: N6-methyladenosine, N6-acetyladenosine, N6-propionyladenosine, N6-butyryladenosine, N6-pentanoyladenosine, N6-hexanoyladenosine, N6-benzoyladenosine, 2-aminoadenosine or 7-deazaadenosine, etc.

Example 41 In vitro transcription and synthesis of mRNA using chemically modified adenosine and pseudouridine to globally replace natural adenosine and natural uridine

The conditions and operation methods of this example are exactly the same as those of Example 40, except that natural uridine is completely replaced by pseudouridine in the substrate for in vitro transcription. The rest of the purification and characterization methods are exactly the same.

Table 10

** is adenosine triphosphate or chemically modified adenosine triphosphate. Cap Analogue# is a commercially available cap structure analog; in this example, 3’-O-Me-m7-G(5’)ppp(5’)G RNA Cap Structure Analog provided by New England Biolabs was used, with the catalog number S1411S.

Example 42 Polyadenylation of in vitro transcribed eGFP mRNA

Chemically modified adenosine is used to perform polyadenylation modification on in vitro transcribed eGFP mRNA using E. coli polyA polymerase or Yeast polyA polymerase. Taking a 20 μL reaction system as an example, add the components shown in Tables 11 and 12 in a 0.2 mL PCR tube.

Table 11

* is adenosine triphosphate or chemically modified adenosine triphosphate. The components of buffer A (5×) are as follows: 500 mM potassium acetate, 100 mM Tris-HCl (pH 8.0), 10 mM magnesium acetate, 0.25% NP-40.

Table 12

* is adenosine triphosphate or chemically modified adenosine triphosphate. The components of buffer B (5×) are as follows: 50% glycerol, 125 mM Tris-HCl (pH 7.0), 0.5 mg/mL BSA, 3.5 mM manganese chloride, 250 mM potassium chloride, 0.05 mM EDTA, 2.5 mM dithiothreitol.

Turn on the thermostat's hot cover and set it to 60° C. Click on the thermostat reaction system, 37° C., 30 min. After the incubation is complete, the tailed mRNA is purified as described in Example 40.

500 ng of purified mRNA was taken and subjected to agarose gel electrophoresis to confirm the success of the reaction (agarose gel electrophoresis detection conditions: 2% agarose, 5 V/cm, electrophoresis for 30 minutes).

Figure 75 is a diagram showing the effect of polyadenylation modification of eGFP mRNA by E. coli polyA polymerase. In the figure, lanes 1-13 are: lane 1: eGFP mRNA template; lane 2: natural adenosine tailing product of eGFP mRNA; lane 3: 2'-methoxyadenosine tailing product of eGFP mRNA; lane 4: 2'-fluoroadenosine tailing product of eGFP mRNA; lane 5: N6-methyladenosine tailing product of eGFP mRNA; lane 6: N6-acetyladenosine tailing product of eGFP mRNA. Lane 7: N6-propionyladenosine tailing products of eGFP mRNA; Lane 8: N6-butyryladenosine tailing products of eGFP mRNA; Lane 9: N6-pentanoyladenosine tailing products of eGFP mRNA; Lane 10: N6-hexanoyladenosine tailing products of eGFP mRNA; Lane 11: N6-benzoyladenosine tailing products of eGFP mRNA; Lane 12: 2-aminoadenosine tailing products of eGFP mRNA; Lane 13: 7-deazaadenosine tailing products of eGFP mRNA.

Figure 76 is a diagram showing the effect of polyadenylation modification of eGFP mRNA by Yeast polyA polymerase. In the figure, lanes 1-13 are respectively: lane 1: eGFP mRNA template; lane 2: natural adenosine tailing product of eGFP mRNA; lane 3: 2'-methoxyadenosine tailing product of eGFP mRNA; lane 4: 2'-fluoroadenosine tailing product of eGFP mRNA; lane 5: N6-methyladenosine tailing product of eGFP mRNA; lane 6: N6-acetyladenosine tailing product of eGFP mRNA. Lane 10: N6-hexanoyladenosine tailing product of eGFP mRNA; Lane 11: N6-benzoyladenosine tailing product of eGFP mRNA; Lane 12: 2-aminoadenosine tailing product of eGFP mRNA; Lane 13: 7-deazaadenosine tailing product of eGFP mRNA.

Example 43 Translation of luciferase mRNA in which chemically modified adenosine globally replaces natural adenosine or luciferase mRNA in which chemically modified adenosine and pseudouridine are combined in rabbit reticulocyte lysate system

The rabbit reticulocyte lysate system is shown in Table 13.

Table 13

Turn on the thermostat cover and set it to 60°C. Click the thermostat reaction system, 30°C, 90min. The incubated product is directly used for the luminescence experiment of luciferin.

Example 44 Luminescence experiment of luciferase oxidized luciferin prepared by in vitro translation

The experimental steps for detecting luminescence by microplate reader are as follows: 2.5 μL of the incubation product in Example 43 was added to a 96-well plate, and then incubated at room temperature for 10 minutes, followed by adding 50 μL of luciferase detection reagent (Promega, E1483), pipetting evenly to remove bubbles in the system, and completing the luminescence intensity detection on the microplate reader within 5 minutes. Two replicate wells were set for each sample.

Figure 77 is a graph showing the expression of luciferase mRNA in combination with chemically modified adenosine and pseudouridine after incubation in rabbit reticulocyte lysate for 90 minutes. Figures 1-6 respectively represent: 1: expression effect graph of unmodified luciferase mRNA; 2: expression effect graph of luciferase mRNA replaced with pseudouridine; 3: expression effect graph of luciferase mRNA replaced with N6-acetyladenosine and pseudouridine; 4: expression effect graph of luciferase mRNA replaced with N6-propionyladenosine and pseudouridine; 5: expression effect graph of luciferase mRNA replaced with N6-butyryladenosine and pseudouridine; 6: expression effect graph of luciferase mRNA replaced with 2-aminoadenosine and pseudouridine. The results showed that when chemically modified adenosine and pseudouridine were used together, the expression efficiency of the modified mRNA in rabbit reticulocyte lysate was higher, exceeding the expression efficiency of mRNA modified with pseudouridine alone. Therefore, chemically modified adenosine can be used in combination with pseudouridine to further enhance the expression efficiency of the modified mRNA as a supplementary option to pseudouridine modification.

FIG78 is a graph showing the expression effect of luciferase mRNA in which chemically modified adenosine globally replaces natural adenosine after incubation in rabbit reticulocyte lysate for 90 minutes. 1-10 in the figure respectively represent, 1: the expression effect diagram of unmodified luciferase mRNA; 2: the expression effect diagram of luciferase mRNA replaced by N6-methyladenosine; 3: the expression effect diagram of luciferase mRNA replaced by N6-acetyladenosine; 4: the expression effect diagram of luciferase mRNA replaced by N6-propionyladenosine; 5: the expression effect diagram of luciferase mRNA replaced by N6-butyryladenosine; 6: the expression effect diagram of luciferase mRNA replaced by N6-pentanoyladenosine; 7: the expression effect diagram of luciferase mRNA replaced by N6-hexanoyladenosine; 8: the expression effect diagram of luciferase mRNA replaced by N6-benzoyladenosine; 9: the expression effect diagram of luciferase mRNA replaced by 2-aminoadenosine; 10: the expression effect diagram of luciferase mRNA replaced by 7-deazaadenosine. The results showed that luciferase mRNA in which chemically modified adenosine globally replaced natural adenosine had higher expression efficiency in rabbit reticulocyte lysate than unmodified mRNA, where the modification types included N6-acetyl modification, N6-propionyl modification, N6-butyryl modification and N6-benzoyl modification.

Example 45 Luminescence experiment of luciferase oxidized luciferin prepared by intracellular translation

The experimental steps for extracting the target protein luciferase from cells are as follows: cell culture and mRNA transfection are performed in a 24-well plate. 24 hours after the luciferase mRNA transfection cells, the 24-well plate is placed on ice, the culture medium is removed, and the plate is washed twice with cold PBS solution, and then 200 μL RIPA lysis buffer (Yisheng, catalog number 20115ES60, 1 mM PMSF is added before use). The cells were fully lysed by blowing with a pipette for 5 minutes, and the cell lysate was collected and transferred to a 1.5 mL EP tube. The tube was centrifuged at 15,000 rpm for 10 minutes at 4°C, and 150 μL of the supernatant was taken for subsequent detection.

The experimental steps of detecting luminescence by an ELISA instrument are as follows: The experimental steps of detecting luminescence by an ELISA instrument are as in Example 44, except that 50 μL of the protein solution extracted in this example is added to the 96-well plate, and then 50 μL of luciferase detection reagent (Promega, E1483) is added for detection. Two replicate wells are set for each sample.

Figure 79 shows the expression effect of luciferase mRNA in which chemically modified adenosine globally replaces natural adenosine 24 hours after transfection into HEK 293T cells. In the figure, 1-9 respectively represent, 1: expression effect of unmodified luciferase mRNA; 2: expression effect of luciferase mRNA replaced by N6-methyladenosine; 3: expression effect of luciferase mRNA replaced by N6-acetyladenosine; 4: expression effect of luciferase mRNA replaced by N6-propionyladenosine; 5: expression effect of luciferase mRNA replaced by N6-butyryladenosine; 6: expression effect of luciferase mRNA replaced by N6-pentanoyladenosine; 7: expression effect of luciferase mRNA replaced by N6-hexanoyladenosine; 8: expression effect of luciferase mRNA replaced by 2-aminoadenosine; 9: expression effect of luciferase mRNA replaced by 7-deazaadenosine. As can be seen from Figure 34, the translation efficiency of luciferase mRNA modified by replacing natural adenosine with N6-acetyladenosine in HEK 293T cells is nearly 3 times higher than that of unmodified mRNA.

Figure 80 is a graph showing the expression of chemically modified adenosine and pseudouridine combined luciferase mRNA 24h after transfection into HeLa cells. In the figure, 1-6 respectively represent, 1: expression effect graph of unmodified luciferase mRNA; 2: expression effect graph of luciferase mRNA replaced by pseudouridine; 3: expression effect graph of luciferase mRNA replaced by N6-acetyladenosine and pseudouridine; 4: expression effect graph of luciferase mRNA replaced by N6-propionyladenosine and pseudouridine; 5: expression effect graph of luciferase mRNA replaced by N6-butyryladenosine and pseudouridine; 6: expression effect graph of luciferase mRNA replaced by 2-aminoadenosine and pseudouridine. The results showed that when chemically modified adenosine and pseudouridine were used together, the expression efficiency of the modified mRNA in the cell system was higher, exceeding the expression efficiency of mRNA modified by pseudouridine alone. Therefore, chemically modified adenosine can be used in combination with pseudouridine to further enhance the expression efficiency of the modified mRNA as a supplementary option for pseudouridine modification.

Example 46 Expression efficiency of eGFP mRNA containing a chemically modified polyadenosine tail in cells

eGFP mRNA containing a chemically modified polyadenosine tail was synthesized and its expression efficiency in cells was tested. After being transfected into cells, it was translated into the target protein enhanced green fluorescent protein, and its fluorescence intensity was detected using a laser confocal fluorescence microscope to determine the translation efficiency of eGFP mRNA containing natural and chemically modified polyadenosine tails.

The specific steps for transfecting HEK 293T cells with chemically modified mRNA are as follows:

(1) Cell transfection

About 24 hours after inoculation of human embryonic kidney 293T cells (purchased from the Chinese Academy of Sciences Cell Bank), the cell state in the 35mm well plate was observed, and the confluence was 70%. In a biological safety cabinet, 90% (volume percentage) DMEM + 10% (volume percentage) FBS culture medium was prepared. 30 minutes before transfection, the culture medium in the well plate was discarded, and 3 mL of fresh culture medium, i.e. 90% (volume percentage) DMEM + 10% (volume percentage) FBS culture medium, was added to each well.

(2) Preparation of transfection system

Take 50μL Opti-MEM, add 800ng mRNA, and mix well. Also prepare a mixed solution of 2μL Lipofectamine 2000 (liposome 2000) and 50μL Opti-MEM, and incubate at room temperature for 10 minutes. Then add the liposome 2000 mixture to the mRNA mixture, mix well, and incubate for 20 minutes. Add the mixed solution to the well plate. Change the medium 6h after transfection, aspirate the old culture medium, and replace each well with 3mL of fresh culture medium, that is, 90% (volume percentage) DMEM + 10% (volume percentage) FBS culture medium. Replace the fresh culture medium 24h/48h after transfection, and detect the fluorescence intensity of the cells with a laser confocal fluorescence microscope.

Figure 81 is a fluorescence imaging diagram of the expression effect of chemically modified adenosine polyadenylation-modified eGFP mRNA in HEK 293T cells. In the figure, 1 represents the fluorescence imaging diagram of the expression effect of eGFP mRNA without poly (A) tail; 2 represents the fluorescence imaging diagram of the expression effect of eGFP mRNA with poly natural adenosine tail; 3 represents the fluorescence imaging diagram of the expression effect of eGFP mRNA with poly N6-acetyl adenosine tail; 4 represents the fluorescence imaging diagram of the expression effect of eGFP mRNA with poly N6-propionyl adenosine tail. The scale bar in the figure is 50 μm.

As can be seen from Table 81, the eGFP mRNA with a poly-N6-acetyladenosine tail has a higher fluorescence intensity after being transfected into HEK 293T cells, and the protein expression efficiency exceeds that of the mRNA with a poly-natural adenosine tail.

In summary, the present application provides a chemically modified nucleoside and a chemically modified nucleoside triphosphate, which provide supplements and new options for mRNA modification technology. The chemically modified cytosine triphosphate is used to globally replace the natural cytosine triphosphate to obtain a chemically modified mRNA, which has a higher translation efficiency in both the cell-free system and the cell, and is more stable. The natural adenine nucleosides in the mRNA are globally replaced with the chemically modified adenine triphosphate, or the mRNA is polyadenylated and tailed, which can enhance the translation efficiency and stability of the target mRNA, reduce immunogenicity, and provide supplements and new options for mRNA modification technology.

The applicant declares that the above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Technical personnel in the relevant technical field should understand that any changes or substitutions that can be easily thought of by technical personnel in the relevant technical field within the technical scope disclosed in the present application are within the protection scope and disclosure scope of the present application.

Claims (13)

A chemically modified nucleoside selected from a compound having a structure as shown in Formula 1, a salt thereof or an isomer thereof:
Wherein, R ₃ is selected from hydroxyl, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether; Y is selected from a cytosine ring containing a substituent or an adenine ring containing a substituent, wherein the cytosine ring containing a substituent is as shown in Formula 2, and the adenine ring containing a substituent is as shown in Formula 3. Indicates the connection location;
wherein R ₁ and R _1′ are each independently selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl; R ₂ and R _2' are each independently selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo or nitrile; and X is selected from nitrogen or carbon. The chemically modified nucleoside according to claim 1, wherein the chemically modified nucleoside is a chemically modified cytosine nucleoside, and the chemically modified cytosine nucleoside is selected from a compound having a structure as shown in Formula 4, a salt thereof, or an isomer thereof:
wherein R ₁ is selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl; _R2 is selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo or nitrile; and _R3 is selected from hydroxy, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether. The chemically modified nucleoside according to claim 1, wherein the chemically modified nucleoside is a chemically modified adenine nucleoside. The chemically modified adenine nucleoside is selected from a compound having a structure as shown in Formula 5, a salt thereof or an isomer thereof:
wherein R _1′ is selected from acetyl, propionyl, butyryl, valeryl, hexanoyl or benzoyl; R _2' is hydrogen; _R3 is selected from hydrogen, hydroxy, fluoro or methoxy; and X is selected from nitrogen or carbon. A chemically modified nucleoside triphosphate selected from a compound having a structure as shown in Formula 6, a salt thereof or an isomer thereof:
wherein _Rn is selected from hydroxy, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether; W is selected from a cytosine ring containing a substituent or an adenine ring containing a substituent, wherein the cytosine ring containing a substituent is as shown in Formula 7, and the adenine ring containing a substituent is as shown in Formula 8. Indicates the connection location;
wherein R ₄ and R _4′ are each independently selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl; R ₅ and R _5' are each independently selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo or nitrile; and M is selected from nitrogen or carbon. The chemically modified nucleoside triphosphate according to claim 4, wherein the chemically modified nucleoside triphosphate is a chemically modified cytosine triphosphate, and the chemically modified cytosine triphosphate is selected from a compound having a structure as shown in Formula 9, a salt thereof, or an isomer thereof:
wherein _R4 is selected from hydrogen, acetyl, halogen-substituted acetyl, propionyl, halogen-substituted propionyl, butyryl, halogen-substituted butyryl, valeryl, halogen-substituted valeryl, hexanoyl, halogen-substituted hexanoyl, octanoyl, decanoyl, benzoyl, 4-methoxybenzoyl or 4-nitrobenzoyl; R ₅ is selected from hydrogen, methyl, methoxy, fluoro, chloro, bromo, iodo, nitrile; and R _n is selected from hydroxy, hydrogen, fluorine, methoxy, ethoxy or methoxymethyl ether. The chemically modified nucleoside triphosphate according to claim 4, wherein the chemically modified nucleoside triphosphate is a chemically modified adenine nucleoside triphosphate, and the chemically modified adenine nucleoside triphosphate is selected from a compound having a structure as shown in Formula 10, a salt thereof, or an isomer thereof:
wherein R _4′ is selected from acetyl, propionyl, butyryl, valeryl, hexanoyl or benzoyl; R _5' is selected from hydrogen; _Rn is selected from hydrogen, hydroxy, fluoro or methoxy; and M is selected from nitrogen or carbon. The chemically modified nucleoside triphosphate according to claim 5, wherein the chemically modified nucleoside triphosphate is prepared by the following preparation method:
(1) subjecting the hydroxyl group of the compound of formula 11 to a hydroxyl protection reaction to obtain a compound of formula 12; (2) reacting the compound of formula 12 with an acid anhydride or an acid chloride to obtain a compound of formula 13; (3) performing a complete deprotection reaction on the protecting group of the compound of formula 13 to obtain a compound of formula 14; (4) reacting the compound of formula 14 with a chlorinating agent to obtain a compound of formula 15; (5) reacting the compound of formula 15 with tributylammonium pyrophosphate to obtain a compound of formula 16; (6) subjecting the compound of formula 16 to a quenching reaction to obtain a compound of formula 9; Preferably, in step (1), tert-butyldimethylsilyl chloride is used to carry out the hydroxyl protection reaction; Preferably, in step (2), the acid anhydride is selected from acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, caproic anhydride, caprylic anhydride, capric anhydride or benzoic anhydride; Preferably, in step (2), the acyl chloride is selected from 4-methoxybenzoyl chloride or 4-nitrobenzoyl chloride; Preferably, in step (3), tetrabutylammonium fluoride is used for complete deprotection reaction; Preferably, in step (4), the chlorination agent is selected from phosphorus oxychloride. The chemically modified nucleoside triphosphate according to claim 6, wherein the chemically modified nucleoside triphosphate is prepared by the following preparation method:
(1") subjecting the hydroxyl group of the compound of formula 17 to a hydroxyl protection reaction to obtain a compound of formula 18; (2") reacting the compound of formula 18 with an acid anhydride or an acid chloride to obtain a compound of formula 19; (3") performing a complete deprotection reaction on the protecting group of the compound of formula 19 to obtain a compound of formula 20; (4") reacting the compound of formula 20 with a chlorinating agent to obtain a compound of formula 21; (5") reacting the compound of formula 21 with tributylammonium pyrophosphate to obtain a compound of formula 22; (6") quenching the compound of formula 22 to obtain a compound of formula 23; Preferably, in step (1"), tert-butyldimethylsilyl chloride is used to carry out the hydroxyl protection reaction; Preferably, in step (2"), the acid anhydride is selected from acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, caproic anhydride or benzoic anhydride; Preferably, in step (2"), the acyl chloride is selected from acetyl chloride, propionyl chloride, butyryl chloride, valeryl chloride, hexanoyl chloride or benzoyl chloride; Preferably, in step (3"), tetrabutylammonium fluoride is used for complete deprotection reaction; Preferably, in step (4"), the chlorinating agent is selected from phosphorus oxychloride. A nucleic acid containing chemically modified cytosine nucleosides, wherein during the preparation process, the chemically modified cytosine nucleoside triphosphates described in claim 5 are used to completely replace the natural cytosine nucleoside triphosphates in mRNA, or the chemically modified cytosine nucleoside triphosphates and N1-methylpseudouridine triphosphate are used in combination to replace the natural cytosine nucleoside triphosphates in mRNA. A nucleic acid containing chemically modified adenine nucleosides, wherein the chemically modified adenine nucleoside triphosphate described in claim 6 is used to completely replace the natural adenine nucleoside triphosphate in mRNA during the preparation process, or the chemically modified adenine nucleoside triphosphate described in claim 6 is used to perform polyadenylation modification on mRNA prepared by in vitro transcription. A method for preparing a nucleic acid containing chemically modified cytosine nucleoside according to claim 9, comprising: Adenosine triphosphate, guanosine triphosphate, uridine triphosphate and chemically modified cytosine triphosphate are used as raw materials, and PCR reaction is carried out with DNA as template under the catalysis of RNA polymerase. Chemically modified nucleic acids. A method for preparing a nucleic acid containing chemically modified adenine nucleosides according to claim 10, comprising: Using cytosine triphosphate, uridine triphosphate, guanine triphosphate and chemically modified adenine triphosphate as raw materials, PCR reaction is carried out with DNA as template under the catalysis of RNA polymerase to synthesize chemically modified nucleic acids; Alternatively, using in vitro transcribed mRNA as a template and chemically modified adenine nucleoside triphosphate as a substrate, the mRNA is tailed using E. coli poly (A) polymerase or Yeast poly (A) polymerase to obtain chemically modified nucleic acid. Use of any one or a combination of at least two of the chemically modified nucleoside according to any one of claims 1 to 3, the chemically modified nucleoside triphosphate according to any one of claims 4 to 8, the nucleic acid containing chemically modified cytosine nucleoside according to claim 9, the nucleic acid containing chemically modified adenine nucleoside according to claim 10, the method for preparing a nucleic acid containing chemically modified cytosine nucleoside according to claim 11, or the method for preparing a nucleic acid containing chemically modified adenine nucleoside according to claim 12 in the preparation of an mRNA drug.