Polynucleotide and medicinal composition

Abstract

The present invention relates to a polynucleotide including a translation region from a start codon to a stop codon, a 5' untranslated region, and a poly-A tail, 65% or more of the nucleotides that constitute the poly-A tail being sugar-modified nucleotides.

Description

Polynucleotides and Pharmaceutical Compositions

[0001] The present invention relates to polynucleotides and pharmaceutical compositions comprising said polynucleotides.

[0002] Genetic information in cells is transmitted by the transcription of messenger RNA (hereinafter referred to as "mRNA") by RNA polymerase using DNA as a template, followed by the binding of ribosomes to the transcribed single-stranded mRNA and the synthesis of proteins through translation. This mode of transmission is called the "central dogma" in molecular biology and is a fundamental principle common to both prokaryotes and eukaryotes. mRNA, an intermediate substance in genetic information transmission, has the base sequence information and structure required for direct recognition by ribosomes and translation into proteins.

[0003] Nucleic acid drugs have been gaining increasing attention as next-generation medicines in recent years. Artificial polynucleotides such as mRNA (hereinafter referred to as "artificial mRNA" in the Background Art section) can produce desired peptides and proteins through enhanced or increased expression, and can be used as nucleic acid drugs for protein replacement therapy or vaccine therapy. However, it is known that when artificial mRNA composed only of natural bases is introduced into cells from the outside, it binds to intracellular Toll-like receptors (TLR3, TLR7, TLR8, RIG-I, etc.) to rapidly induce an immune response, resulting in an inflammatory reaction and a decrease in protein translation (Non-Patent Document 1). To express proteins intracellularly, some method is required to reduce the immune reactivity of the artificial mRNA itself without reducing translation. Furthermore, because RNA composed only of natural bases is vulnerable to nucleases, the introduction of modified nucleotides is also necessary to enhance stability (Non-Patent Document 2). Among modified nucleotides, polynucleotides containing sugar-modified nucleotides such as 2'-O-methyl modified RNA, 2'-F-modified RNA, 2'-O-methoxyethyl modified RNA, and cross-linked nucleic acids such as LNA have been shown to be effective in both reducing the immune reactivity of nucleic acid drugs and conferring resistance to nucleases (Non-Patent Document 3).

[0004] In recent years, there has been a growing movement to use artificial mRNAs produced by in vitro transcription (hereinafter referred to as "IVT") as pharmaceuticals (Non-Patent Document 4). For example, Non-Patent Document 5 reports that in a clinical trial of an artificial mRNA cancer vaccine in melanoma patients, the incidence of metastasis significantly decreased after the start of administration of the cancer vaccine, and certain successes have been reported in using artificial mRNAs as pharmaceuticals. However, these artificial mRNAs in clinical applications are produced by IVT. Artificial mRNAs produced by IVT have the following two problems. First, the introduction position of modified nucleotides, which are introduced for the purposes of reducing immune reactivity or imparting stability to nucleases, cannot be controlled. Patent Document 1 discloses a case in which artificial mRNAs into which 2'-F-RNA was introduced by IVT exhibited reduced or lost peptide translation ability. Second, modified nucleotides other than those recognized as substrates by the RNA synthetase used in IVT cannot be introduced. Furthermore, Patent Document 1 discloses that it is difficult to prepare artificial mRNA containing 2'-O-methyl modified RNA by IVT reaction using a general RNA polymerase. Therefore, it is difficult to say that sufficient consideration has been given to the position and type of modified nucleotide in artificial mRNA prepared by introducing modified nucleotides by IVT.

[0005] Methods for artificially synthesizing mRNA using techniques for chemically linking multiple RNAs have been reported (Non-Patent Documents 6 and 7). Using this method, it is possible to introduce sugar-modified nucleotides into any position of an artificial mRNA, including the translated and untranslated regions. Furthermore, Patent Documents 2 and 3 disclose the concept of stabilizing an mRNA by introducing sugar-modified nucleotides into the untranslated region of the mRNA using a method for synthesizing an artificial mRNA using techniques for chemically linking multiple RNAs. Non-Patent Documents 6 and 7 disclose that the peptide translation ability of an artificial mRNA in which a 2'-O-methyl-modified RNA has been introduced into a single position in the translated region of the mRNA has been confirmed. On the other hand, it has also been disclosed that peptide translation ability can be significantly attenuated depending on the position at which the sugar-modified nucleotide is introduced (Non-Patent Documents 6 and 7). Thus, further knowledge regarding the modification rate, position, and type of modified nucleotides is needed to achieve sufficiently low immunoreactivity, high stability, and excellent translation ability as an artificial mRNA nucleic acid medicine.

[0006] International Publication No. WO 2014 / 093574 International Publication No. WO 1999 / 014346 International Publication No. WO 2016 / 022914

[0007] Nature Reviews Drug Discovery, Vol. 13, pp. 759-780 (2014); Nature Biotechnology, Vol. 35, No. 3, pp. 238-248 (2017); Drug Discovery Today, Vol. 13, No. 19 / 20, pp. 842-855 (2008); Nature Biotechnology, Vol. 35, No. 3, pp. 193-197 (2017); Nature, Vol. 547, No. 7662, pp. 222-226 (2017); Nucleic Acids Research, Vol. 44, No. 2, pp. 852-862 (2015); Genes, Vol. 10, No. 2, p. 84 (2019)

[0008] An object of the present invention is to provide a polynucleotide having excellent translation ability.

[0009] As a result of extensive investigations, the present inventors have found that excellent translation ability is exhibited when 65% or more of the nucleotides constituting the poly(A) tail in the 3'-untranslated region are sugar-modified nucleotides.

[0010] The present invention includes the following embodiments: [1] A polynucleotide comprising a translated region from an initiation codon to a termination codon, a 5'-side untranslated region, and a poly-A tail, wherein 65% or more of the nucleotides constituting the poly-A tail are sugar-modified nucleotides. [2] The polynucleotide according to [1], wherein all of the nucleotides constituting the poly-A tail are sugar-modified nucleotides. [3] The modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures: [4] The polynucleotide according to [1] or [2], wherein the modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures: [5] The polynucleotide according to any one of [1] to [3]. [5] The polynucleotide according to any one of [1] to [4], wherein the polyA tail contains at least one phosphate-modified nucleotide. [6] The polynucleotide according to any one of [1] to [5], wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the polyA tail are linked by phosphorothioate. [7] The polynucleotide according to any one of [1] to [6], wherein all nucleotides constituting the polyA tail are linked by phosphorothioate. [8] The polynucleotide according to any one of [1] to [7], wherein the polyA tail is 2 to 40 bases long. [9] The polynucleotide according to any one of [1] to [8], wherein the nucleotides in the 5'-untranslated region are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, or sugar-modified nucleotides.

[10] The first to sixth nucleotides from the 5' end of the 5'-untranslated region are sugar-modified nucleotides, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of [1] to [9].

[11] The polynucleotide according to

[10] , further comprising a portion consisting of 1 to 10 sugar-unmodified nucleotides on the 5' side of the 5' end of the 5' untranslated region.

[12] The polynucleotide according to any one of [1] to

[11] , wherein nucleotides excluding the first to sixth nucleotides from the 5' end of the 5' untranslated region comprise 2'-deoxyribonucleotides and / or spacer modifications.

[13] The 5' untranslated region and / or the 3' untranslated region comprise a spacer modification, preferably the 5' untranslated region and / or the 3' untranslated region comprise a spacer modification, and the spacer modifications are each independently selected from any one of the following structures: [Wherein, Rx is ethynyl, a hydrogen atom, or OH, M is a hydrogen atom or OH, n1 is 1, 2, or 5, and n2 is 1, 2, or 3.] The polynucleotide according to any one of [1] to

[12] . Note that, as a spacer modification in

[13] , the oxygen atom of the five-membered ring in the leftmost structure may be substituted with NH.

[14] The polynucleotide according to any one of [1] to

[13] , wherein the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate.

[15] The 5' untranslated region contains a base-modified nucleotide, and the modified base moiety of the base-modified nucleotide has the following structure: [In the formula, R is an alkyl group having 1 to 6 carbon atoms.] The polynucleotide according to any of [1] to

[14] .

[16] The polynucleotide according to any of [1] to

[15] , wherein the translation region comprises at least two codons whose first nucleotide is a sugar-modified nucleotide.

[17] The polynucleotide according to any of [1] to

[16] , wherein the translation region comprises four or more codons, and the first nucleotide of all of the codons is a sugar-modified nucleotide.

[18] The polynucleotide according to any of [1] to

[16] , wherein the translation region comprises a first nucleotide of all of the codons, excluding the stop codon, which is a sugar-modified nucleotide, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of [1] to

[17] .

[19] The polynucleotide according to any one of [1] to

[18] , wherein the translation region contains 2000 or less codons. [19-1] The polynucleotide according to any one of [1] to

[19] , wherein the translation region contains 4 to 2000 (4 to 2000) codons.

[20] The polynucleotide according to any one of [1] to [19-1], wherein all nucleotides of the stop codon are sugar-modified nucleotides.

[21] The polynucleotide comprising the following structure: [In the formula, R 1 and R 2 are each independently H, OH, F, OCH2CH2OCH3 or OCH3, B1 and B 2 are each independently a base moiety, 1 is O, S or NH, X 2 is O, S, NH or the following structure: X 3 is OH, SH or a salt thereof, provided that X 1 and X 2 and are not simultaneously O. The polynucleotide according to any one of [1] to

[20] .

[22] A pharmaceutical composition comprising the polynucleotide according to any one of [1] to

[21] .

[0011] The present invention further includes the following embodiments.

[101] A polynucleotide comprising a translated region from an initiation codon to a termination codon, a 5'-untranslated region, and a polyA tail, wherein the nucleotides in the 5'-untranslated region are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, or sugar-modified nucleotides. [101-1] The polynucleotide according to

[101] , wherein the nucleotides in the 5'-untranslated region comprise at least one sugar-modified nucleotide.

[102] The polynucleotide according to

[101] or [101-1], wherein 65% or more of the nucleotides constituting the polyA tail are sugar-modified nucleotides, preferably all of the nucleotides constituting the polyA tail are sugar-modified nucleotides.

[103] The modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures:

[104] The polynucleotide according to any one of

[101] to

[102] .

[104] The modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures:

[105] The polynucleotide according to any one of

[101] to

[104] , wherein the polyA strand contains at least one phosphate-modified nucleotide.

[106] The polynucleotide according to any one of

[101] to

[105] , wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3'-end of the polyA strand are linked by phosphorothioate.

[107] The polynucleotide according to any one of

[101] to

[106] , wherein all nucleotides constituting the polyA strand are linked by phosphorothioate.

[108] The polynucleotide according to any one of

[101] to

[107] , wherein the polyA strand is 2 to 40 bases long.

[109] The first to sixth nucleotides from the 5'-end of the 5'-untranslated region are sugar-modified nucleotides, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of

[101] to

[108] .

[110] The polynucleotide according to

[109] , further comprising a portion consisting of 1 to 10 sugar-unmodified nucleotides on the 5' side of the 5' end of the 5' untranslated region.

[111] The polynucleotide according to any one of

[101] to

[110] , wherein nucleotides excluding the first to sixth nucleotides from the 5' end of the 5' untranslated region comprise 2'-deoxyribonucleotides and / or spacer modifications.

[112] The 5' untranslated region and / or the 3' untranslated region comprise a spacer modification, preferably the 5' untranslated region and / or the 3' untranslated region comprise a spacer modification, and the spacer modifications are each independently selected from any one of the following structures: [Wherein, Rx is ethynyl, a hydrogen atom, or OH, M is a hydrogen atom or OH, n1 is 1, 2, or 5, and n2 is 1, 2, or 3.] The polynucleotide according to any one of

[101] to

[111] . Note that, as a spacer modification in

[112] , the oxygen atom of the five-membered ring in the leftmost structure may be substituted with NH.

[113] The polynucleotide according to any one of

[101] to

[112] , wherein the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate.

[114] The 5' untranslated region contains a base-modified nucleotide, and the modified base moiety of the base-modified nucleotide has the following structure: [Wherein R is an alkyl group having 1 to 6 carbon atoms.] The polynucleotide according to any one of

[101] to

[113] .

[115] The polynucleotide according to any one of

[101] to

[114] , wherein the translation region comprises at least two codons whose first nucleotide is a sugar-modified nucleotide.

[116] The polynucleotide according to any one of

[101] to

[115] , wherein the translation region comprises four or more codons, and the first nucleotide of all of the codons is a sugar-modified nucleotide. [116-1] The polynucleotide according to any one of

[101] to

[116] , wherein the translation region comprises from 4 to 2000 (4 to 2000) codons.

[117] The polynucleotide according to any one of

[101] to

[116] , wherein the translation region comprises from 4 to 2000 codons, and the first nucleotide of all of the codons in the translation region, except for the stop codon, is a sugar-modified nucleotide, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of

[101] to [116-1].

[118] The polynucleotide according to any one of

[101] to

[117] , wherein the translation region contains 2000 or less codons. [118-1] The polynucleotide according to any one of

[101] to

[118] , wherein the translation region contains 4 to 2000 codons.

[119] The polynucleotide according to any one of

[101] to [118-1], wherein all nucleotides of the stop codon are sugar-modified nucleotides.

[120] The polynucleotide according to any one of

[101] to [118-1], wherein the polynucleotide comprises the following structure: [In the formula, R 1 and R 2 are each independently H, OH, F, OCH2CH2OCH3 or OCH3, B 1 and B 2 are each independently a base moiety, 1 is O, S or NH, X 2 is O, S, NH or the following structure: X 3 is OH, SH or a salt thereof, provided that X 1 and X 2 and are not simultaneously O].

[121] A pharmaceutical composition comprising the polynucleotide of any of

[101] to

[120] .

[0012] The present invention further includes the following embodiments as aspects different from the above [1] to

[22] .

[201] A polynucleotide comprising a translated region from an initiation codon to a termination codon, a 5'-side untranslated region, and a polyA tail, wherein the nucleotides constituting the polyA are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, or sugar-modified nucleotides. [201-1] The polynucleotide according to

[201] , wherein the nucleotides constituting the polyA tail include at least one sugar-modified nucleotide.

[202] The polynucleotide according to

[201] or [202-1], wherein 65% or more of the nucleotides constituting the polyA tail are sugar-modified nucleotides, preferably all of the nucleotides constituting the polyA tail are sugar-modified nucleotides.

[203] The modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures:

[204] The polynucleotide according to any one of

[201] to

[202] .

[204] The modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures: The polynucleotide according to any one of

[201] to

[203] .

[205] The polynucleotide according to any one of

[201] to

[204] , wherein the polyA strand contains at least one phosphate-modified nucleotide.

[206] The polynucleotide according to any one of

[201] to

[205] , wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3'-end of the polyA strand are linked by phosphorothioate.

[207] The polynucleotide according to any one of

[201] to

[206] , wherein all nucleotides constituting the polyA strand are linked by phosphorothioate.

[208] The polynucleotide according to any one of

[201] to

[207] , wherein the polyA strand is 2 to 40 bases long.

[209] The first to sixth nucleotides from the 5'-end of the 5'-untranslated region are sugar-modified nucleotides, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of

[201] to

[208] .

[210] The polynucleotide according to

[209] , further comprising a portion consisting of 1 to 10 sugar-unmodified nucleotides on the 5' side of the 5' end of the 5' untranslated region.

[211] The polynucleotide according to any one of

[201] to

[210] , wherein nucleotides excluding the first to sixth nucleotides from the 5' end of the 5' untranslated region comprise 2'-deoxyribonucleotides and / or spacer modifications.

[212] The 5' untranslated region and / or the 3' untranslated region comprise a spacer modification, preferably the 5' untranslated region and / or the 3' untranslated region comprise a spacer modification, and the spacer modifications are each independently selected from any one of the following structures: [Wherein, Rx is ethynyl, a hydrogen atom, or OH, M is a hydrogen atom or OH, n1 is 1, 2, or 5, and n2 is 1, 2, or 3.] The polynucleotide according to any one of

[201] to

[211] . Note that, as a spacer modification in

[212] , the oxygen atom of the five-membered ring in the leftmost structure may be substituted with NH.

[213] The polynucleotide according to any one of

[201] to

[212] , wherein the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate.

[214] The 5' untranslated region contains a base-modified nucleotide, and the modified base moiety of the base-modified nucleotide has the following structure: [Wherein R is an alkyl group having 1 to 6 carbon atoms.] The polynucleotide according to any one of

[201] to

[213] .

[215] The polynucleotide according to any one of

[201] to

[214] , wherein the translation region comprises at least two codons whose first nucleotide is a sugar-modified nucleotide.

[216] The polynucleotide according to any one of

[201] to

[215] , wherein the translation region comprises four or more codons, and the first nucleotide of all of the codons is a sugar-modified nucleotide. [216-1] The polynucleotide according to any one of

[201] to

[216] , wherein the translation region comprises 4 to 2000 codons (4 to 2000).

[217] The polynucleotide according to any one of

[201] to

[216] , wherein the translation region comprises a codon in which the first nucleotide of all of the codons, excluding the stop codon, is a sugar-modified nucleotide, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: The polynucleotide according to any one of

[201] to [216-1].

[218] The polynucleotide according to any one of

[201] to

[217] , wherein the translation region contains 2000 or less codons. [218-1] The polynucleotide according to any one of

[201] to

[218] , wherein the translation region contains 4 to 2000 codons.

[219] The polynucleotide according to any one of

[201] to [218-1], wherein all nucleotides of the stop codon are sugar-modified nucleotides.

[220] The polynucleotide according to any one of

[201] to [218-1], wherein the polynucleotide comprises the following structure: [In the formula, R 1 and R 2 are each independently H, OH, F, OCH2CH2OCH3 or OCH3, B 1 and B 2 are each independently a base moiety, 1 is O, S or NH, X 2 is O, S, NH or the following structure: X 3 is OH, SH or a salt thereof, provided that X 1 and X 2and not O at the same time].

[221] A pharmaceutical composition comprising the polynucleotide of any one of

[201] to

[220] .

[0013] The present invention further includes the following embodiments. [1A] A polynucleotide according to any one of [1] to

[21] ,

[101] to

[120] , and

[201] to

[220] , or a pharmaceutical composition according to any one of

[22] ,

[121] , and

[221] , for use in treating a disease. [1B] A method for treating a disease, comprising administering to a patient in need thereof a therapeutically effective amount of a polynucleotide according to any one of [1] to

[21] ,

[101] to

[120] , and

[201] to

[220] , or a pharmaceutical composition according to any one of

[22] ,

[121] , and

[221] . [1C] Use of a polynucleotide according to any one of [1] to

[21] ,

[101] to

[120] , and

[201] to

[220] , or a pharmaceutical composition according to any one of

[22] ,

[121] , and

[221] , for treating a disease. [1D] Use of the polynucleotide described in any one of [1] to

[21] ,

[101] to

[120] , and

[201] to

[220] in the manufacture of a medicament for treating a disease. [1E] The polynucleotide described in any one of [1] to

[21] ,

[101] to

[120] , and

[201] to

[220] for use in the manufacture of a medicament for treating a disease. [1F] A kit for use in the treatment of a disease, comprising the polynucleotide described in any one of [1] to

[21] ,

[101] to

[120] , and

[201] to

[220] or the pharmaceutical composition described in any one of

[22] ,

[121] , and

[221] , and instructions for use.

[0014] <Polynucleotide> In one embodiment of the present invention, a polynucleotide comprises a translated region from an initiation codon to a termination codon, a 5'-side untranslated region, and a poly-A tail, and 65% or more of the nucleotides constituting the poly-A tail are sugar-modified nucleotides. In the present invention, the polynucleotide exhibits excellent translatability when 65% or more of the nucleotides constituting the poly-A tail are sugar-modified nucleotides.

[0015] The polynucleotide of this embodiment includes a translated region and a poly-A tail, but preferably includes a 5'-side untranslated region, a translated region, and a poly-A tail arranged from the 5'-side to the 3'-side of the polynucleotide. The translated region and the poly-A tail may be directly linked, or there may be another region or a sequence not included in the poly-A tail between them. The translated region and the poly-A tail being directly linked means that the poly-A tail is linked following the termination codon of the translated region. In this case, the 3'-side untranslated region is the poly-A tail. The poly-A tail is present within the 3'-side untranslated region, and the polynucleotide includes a 5'-side untranslated region, a translated region, and a 3'-side untranslated region. In this case, the poly-A tail is present at the 3' end of the 3'-side untranslated region.

[0016] The polynucleotide of this embodiment is understood to be a polynucleotide having a function equivalent to, for example, mRNA, small open reading frame (smORF), non-canonical open reading frame, long noncoding RNA (lncRNA), or pri-microRNA (pri-miRNA), in that the translated region is translated into a polypeptide (as used herein, "polypeptide" encompasses a protein). The polynucleotide may be a single-stranded polynucleotide, or a circular polynucleotide in which the ends of the polynucleotide are linked to each other.

[0017] The polynucleotide of this embodiment is composed of a plurality of nucleotides linked together, and each nucleotide constituting the polynucleotide typically comprises a sugar moiety, a base moiety, and a phosphate moiety. The sugar moiety is the moiety in the nucleotide that corresponds to the sugar moiety, the base moiety is the moiety in the nucleotide that corresponds to the base moiety, and the phosphate moiety is the moiety in the nucleotide that corresponds to the phosphate moiety. Generally, the base moiety of a nucleotide is selected from adenine (A), guanine (G), cytosine (C), uracil (U), or thymine (T), and the sugar moiety is selected from ribose or 2'-deoxyribose. Preferably, ribose and 2'-deoxyribose are each in the D-form. A nucleotide is composed of a combination of the above-described base moiety and the above-described sugar moiety, and is preferably a ribonucleotide having adenine (A), guanine (G), cytosine (C), or uracil (U) as the base moiety and D-ribose as the sugar moiety.

[0018] The nucleotides constituting the polynucleotide of this embodiment may be ribonucleotides (AUGC), which are unmodified nucleotides, or deoxyribonucleotides (ATGC), which are unmodified nucleotides, or may be modified nucleotides having a structure in at least a portion of the sugar moiety, base moiety, and phosphate moiety that is not derived from unmodified nucleotides.

[0019] Herein, a nucleotide with a modified sugar moiety is referred to as a "sugar-modified nucleotide," a nucleotide with a modified base moiety is referred to as a "base-modified nucleotide," and a nucleotide with a modified phosphate moiety is referred to as a "phosphate-modified nucleotide." Herein, "modification" refers to changing the structure of the sugar moiety, base moiety, or phosphate moiety. The change in structure due to modification is not particularly limited. Examples of modifications include substitution of any site with any substituent. A nucleotide having a modified sugar moiety, modified base moiety, or modified phosphate moiety is referred to as a modified nucleotide, and a nucleotide having no modification in any of the sugar moiety, base moiety, or phosphate moiety is an unmodified nucleotide. A modified nucleotide may have one modified moiety among the modified sugar moiety, modified base moiety, or modified phosphate moiety, or may have any combination of two or three modified moieties.

[0020] The unmodified sugar moiety is a sugar moiety corresponding to ribose or 2'-deoxyribose, and more preferably a sugar moiety corresponding to ribose. That is, in the polynucleotide of this embodiment, nucleotides other than sugar-modified nucleotides preferably contain a sugar moiety corresponding to ribose or 2'-deoxyribose, and more preferably contain a sugar moiety corresponding to ribose.

[0021] (Sugar-modified nucleotide) The sugar-modified nucleotide is not particularly limited as long as the sugar moiety of the nucleotide is modified, but preferably contains a sugar moiety modified at least at the 2'-position. Modification at the 2'-position can improve stability against enzymes. The sugar moiety modified at least at the 2'-position may be a sugar moiety in which the 2'- and 4'-positions are cross-linked.

[0022] Examples of modified sugar moieties include the following: wherein M is R 1 , OR 1 , R 2 OR 1 , OR 2 OR 1 , S.H., S.R. 1 , NH2, NHR 1 , N.R. 12, N3, CN, F, Cl, Br or I, R 1 are each independently alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, more preferably alkyl having 1 to 3 carbon atoms; R 2 is alkylene, preferably alkylene having 1 to 6 carbon atoms. When M is H or OH, the sugar moiety is unmodified. A nucleotide having an unmodified sugar moiety in which M is H is a 2'-deoxyribonucleotide, and a nucleotide having an unmodified sugar moiety in which M is OH is a ribonucleotide.

[0023] In this specification, examples of alkyl having 1 to 6 carbon atoms include linear or branched alkyl having 1 to 6 carbon atoms. Examples of linear alkyl having 1 to 6 carbon atoms include methyl, ethyl, propyl, butyl, pentyl, and hexyl. Examples of branched alkyl having 1 to 6 carbon atoms include isopropyl, isobutyl, sec-butyl, tert-butyl, and pentyl substituted with methyl. Examples of alkyl having 1 to 3 carbon atoms include methyl, ethyl, propyl, and isopropyl.

[0024] As used herein, aryl includes, for example, optionally substituted phenyl and optionally substituted naphthyl.

[0025] In this specification, alkylene having 1 to 6 carbon atoms is a group in which one hydrogen atom bonded to a carbon atom of alkyl having 1 to 6 carbon atoms has been removed.

[0026] As used herein, the term "modified sugar moiety" refers to a modified sugar structure contained in a sugar-modified nucleotide. The modified sugar moiety M can also include 2-(methoxy)ethoxy, 3-aminopropoxy, 2-[(N,N-dimethylamino)oxy]ethoxy, 3-(N,N-dimethylamino)propoxy, 2-[2-(N,N-dimethylamino)ethoxy]ethoxy, 2-(methylamino)-2-oxoethoxy, 2-(N-methylcarbamoyl)ethoxy, and 2-cyanoethoxy.

[0027] Further examples of modified sugar moieties include the sugar moieties of the following nucleic acids: Locked Nucleic Acid (LNA) [Tetrahedron Letters, 38, 8735 (1997) and Tetrahedron, 54, 3607 (1998)]; Ethylene bridged nucleic acid (ENA) [Nucleic Acids Research, 32, e175 (2004)]; Constrained Ethyl (cEt) [The Journal of Organic Chemistry, 75, 1569 (2010)]; Amido-Bridged Nucleic Acid (AmNA) [Chem Bio Chem, 13, 2513 (2012)]; 2'-O,4'-c-Spirocyclopropylene bridged nucleic acid (scpBNA) [Chem. Commun., 51, 9737 (2015)]; ・tricycloDNA (tcDNA) [Nat. Biotechnol., 35, 238 (2017)]; ・Unlocked Nucleic Acid (UNA) [Mol. Ther. Nucleic Acids 2, e103 (2013)]; ・3'-fluorohexitol nucleic acid (FHNA) [Nat. Biotechnol., 35, 238 (2017)]; ・Peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)]; ・Oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)]; ・Peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

[0028] The modified sugar moiety is not particularly limited, but is preferably selected from the following:

[0029] The sugar-modified nucleotide preferably contains a base moiety corresponding to a base selected from the group consisting of adenine (A), guanine (G), cytosine (C), and uracil (U), and preferably contains at least two types of bases. Here, "at least two types of bases" means, for example, that one sugar-modified nucleotide contains a base moiety corresponding to adenine, and another sugar-modified nucleotide contains a base moiety corresponding to guanine.

[0030] The sugar-modified nucleotides may be base-modified and / or phosphate-modified nucleotides (in other words, the sugar-modified nucleotides may further comprise a modified base moiety and / or a modified phosphate moiety). At least one of the sugar-modified nucleotides may comprise a modified base moiety.

[0031] (Base-Modified Nucleotide) The base-modified nucleotide is not particularly limited as long as the base moiety of the nucleotide is modified. Examples of unmodified base moieties include base moieties corresponding to adenine, guanine, cytosine, and uracil. Examples of modified base moieties include base moieties in which the oxygen atom of the unmodified base moiety is replaced with a sulfur atom, base moieties in which the hydrogen atom of the unmodified base moiety is replaced with alkyl having 1 to 6 carbon atoms, halogen, etc., base moieties in which the methyl of the unmodified base moiety is replaced with a hydrogen atom, hydroxymethyl, alkyl having 2 to 6 carbon atoms, etc., and base moieties in which the amino of the unmodified base moiety is replaced with alkyl having 1 to 6 carbon atoms, alkanoyl having 1 to 6 carbon atoms, oxo, hydroxy, etc.

[0032] Specific examples of the modified base moiety of the base-modified nucleotide include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propyladenine, 2-propylguanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-pseudouracil, 4-thiouracil, 8-haloadenine, 8-haloguanine, 8-aminoadenine, 8-aminoguanine, 8-mercaptoadenine, 8-mercaptoguanine, and 8-alkylthioadenine. thioguanine, 8-alkylthioguanine, 8-hydroxyadenine, 8-hydroxyguanine, 5-bromouracil, 5-bromocytosine, 5-trifluoromethyluracil, 5-trifluoromethyluracil, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 3-deazaguanine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, and 1,3,5-triazine.

[0033] The base-modified nucleotide may be a sugar- and / or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further comprise a modified sugar moiety and / or a modified phosphate moiety).

[0034] (Phosphate-modified nucleotide) The phosphate-modified nucleotide is not particularly limited as long as the phosphate moiety (phosphodiester bond) of the nucleotide is modified. Examples of the modified phosphate moiety include a phosphorothioate bond, a phosphorodithioate bond, an alkylphosphonate bond, and a phosphoramidate bond.

[0035] The translation region may contain phosphate-modified nucleotides in which the modified phosphate moiety is an optical isomer (Rp, Sp). Methods for selectively synthesizing optical isomers of phosphorothioate bonds are disclosed, for example, in J. Am. Chem. Soc., 124, 4962 (2002), Nucleic Acids Research, 42, 13546 (2014), and Science 361, 1234 (2018).

[0036] The phosphate-modified nucleotide may be a sugar- and / or base-modified nucleotide (in other words, the phosphate-modified nucleotide may further comprise a modified sugar moiety and / or a modified base moiety).

[0037] <Translated Region> The polynucleotide of this embodiment includes a translated region. The translated region is also called a coding sequence (CDS). The translated region is composed of multiple codons from an initiation codon to a termination codon (or terminator codon), and is a region where a polypeptide is synthesized through translation. A codon is a unit that encodes each amino acid that constitutes a polypeptide, and each unit is composed of three nucleotides. The polynucleotide of this embodiment may include multiple translated regions in one polynucleotide, and in a polynucleotide including multiple translated regions, the translated region portion of a polynucleotide including one translated region may include multiple translated regions.

[0038] Although not limited to the natural codon table, examples of start codons based on the natural codon table include AUG, which encodes methionine. Non-canonical start codons other than AUG include CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA, and AGG. Examples of stop codons include UAA, UAG, and UGA. The type of codons constituting the translation region is not particularly limited and can be selected appropriately depending on the polypeptide of interest.

[0039] The number (n) of codons constituting the translation region is preferably an integer of 2 to 2000, more preferably an integer of 2 to 1500, even more preferably an integer of 2 to 1000, and most preferably an integer of 2 to 500. The lower limit of the numerical range may be changed to 5, 10, 50, 100, 200, etc. When the lower limit is changed, the number of codons (n) constituting the translation region is preferably an integer of 5 to 2000, 10 to 2000, 50 to 2000, 100 to 2000, or 200 to 2000, more preferably an integer of 5 to 1500, 10 to 1500, 50 to 1500, 100 to 1500, or 200 to 1500, even more preferably an integer of 5 to 1000, 10 to 1000, 50 to 1000, 100 to 1000, or 200 to 1000, and most preferably an integer of 5 to 500, 10 to 500, 50 to 500, 100 to 500, or 200 to 500. The number of nucleotides constituting the translation region is three times the number of codons (n).

[0040] Each codon contains a first, second, and third nucleotide. For example, for the start codon (AUG), the first nucleotide is A, the second nucleotide is U, and the third nucleotide is G.

[0041] When a translation region includes n codons, where n is a positive integer of 2 or greater and the n codons each include a first, second, and third nucleotide, it is preferable that the first nucleotide of at least two of the n codons is a sugar-modified nucleotide. In other words, it is preferable that the translation region includes at least two codons whose first nucleotide is a sugar-modified nucleotide, and the at least two codons whose first nucleotide is a sugar-modified nucleotide may be codons at any position in the translation region.

[0042] Since translation activity is maintained even when the sugar moiety of the first nucleotide in multiple codons constituting the translation region is modified, the polynucleotide of this embodiment maintains translation activity even though it has modification sites in the translation region. As used herein, "translation activity" refers to the activity of mRNA being translated to synthesize a polypeptide. The polynucleotide of this embodiment also has excellent stability against enzymes (e.g., nucleases). As long as the translation region maintains translation activity, the polynucleotide of this embodiment will exhibit excellent translation ability if 65% or more of the nucleotides constituting the polyA tail are sugar-modified nucleotides.

[0043] As used herein, the phrase "translation activity is maintained" refers to a polynucleotide in which the sugar moieties of the first nucleotides in multiple codons are modified, and the translation activity is 60% or more compared to an unmodified polynucleotide. The translation activity of the modified polynucleotide is preferably 70% or more, 80% or more, 90% or more, or 100% or more compared to an unmodified polynucleotide.

[0044] In the polynucleotide of this embodiment, at least two of the first nucleotides contained in a codon constituting the translated region may be sugar-modified nucleotides. The position of the codon containing the sugar-modified nucleotide is not particularly limited. The percentage of first nucleotides that are sugar-modified nucleotides is preferably 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. A percentage of 100% means that all first nucleotides are sugar-modified nucleotides. The higher the percentage, the more likely the stability against enzymes is to be excellent. The translated region may be one in which all first nucleotides are sugar-modified nucleotides. Although not particularly limited, when the first nucleotide is a sugar-modified nucleotide, it is preferable that the substituent at the 2'-position of the sugar moiety of the first nucleotide is fluorine.

[0045] In the polynucleotide of this embodiment, at least one of the second nucleotides contained in a codon constituting the translation region may be a sugar-modified nucleotide, but the sugar moiety of the second nucleotide may be unmodified. The proportion of second nucleotides that are sugar-modified nucleotides may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 0%. A proportion of 0% means that none of the second nucleotides are sugar-modified nucleotides. Although not particularly limited, when the second nucleotide is a sugar-modified nucleotide, the substituent at the 2'-position of the sugar moiety of the second nucleotide is preferably fluorine.

[0046] In the polynucleotide of this embodiment, at least one of the third nucleotides contained in a codon constituting the translation region may be a sugar-modified nucleotide. The percentage of sugar-modified third nucleotides may be 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 0%.

[0047] In the polynucleotide of this embodiment, the first, second, and third nucleotides of the stop codon may be sugar-modified nucleotides in order to improve translation activity. All of the first nucleotides and all of the nucleotides of the stop codon may be sugar-modified nucleotides in the translation region.

[0048] In the polynucleotide of this embodiment, the first, second, and third nucleotides of the start codon may be sugar-modified nucleotides in order to improve stability against nucleases. Although not particularly limited, it is preferred that the substituents at the 2'-positions of the sugar moieties of the first, second, and third nucleotides of the start codon are all fluorine.

[0049] In the polynucleotide of this embodiment, the first nucleotide of all of the remaining codons, excluding the stop codon, may be a sugar-modified nucleotide. Although not particularly limited, it is preferred that the substituent at the 2'-position of the sugar moiety of the first nucleotide of all of the codons, excluding the stop codon, is fluorine.

[0050] The translation region may contain a base-modified nucleotide. The position in the translation region where the base-modified nucleotide is present is not particularly limited. The translation region may contain a phosphate-modified nucleotide. The position in the translation region where the phosphate-modified nucleotide is present is not particularly limited, but it is preferable that the phosphate group connecting the first and second nucleotides of the codon is a phosphorothioate bond.

[0051] <5' Untranslated Region> The polynucleotide of this embodiment includes a 5' untranslated region (5'UTR). The 5' untranslated region is located upstream of the translated region (towards the 5' end) and is not translated for polypeptide synthesis. The number of nucleotides constituting the 5' untranslated region is preferably 1 or more and may be 6 or more. The number of nucleotides constituting the 5' untranslated region is preferably 1,000 or less, and may be 500 or less, 250 or less, or 100 or less. The number of nucleotides constituting the 5' untranslated region may be any number within a range selected from the above upper and lower limits, but is preferably an integer between 1 and 1,000, more preferably an integer between 1 and 500, even more preferably an integer between 6 and 250, and particularly preferably an integer between 6 and 100. In the polynucleotide of this embodiment, the 5' untranslated region and the translated region are linked in this order.

[0052] The 5'-untranslated region may contain 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides. The positions of these nucleotides within the 5'-untranslated region are not particularly limited. From the viewpoint of improving translation activity, the first, second and third nucleotides from the 5'-end may be sugar-modified nucleotides, and it is preferable that the first to sixth nucleotides from the 5'-end are all sugar-modified nucleotides. Furthermore, all nucleotides in the 5'-untranslated region may be sugar-modified nucleotides. In sugar-modified nucleotides, the substituent at the 2'-position of the sugar moiety is preferably a methoxyethoxy group (OCH2CHOCH3) or fluorine (F).

[0053] One embodiment of the present invention is a polynucleotide comprising: a translated region from an initiation codon to a termination codon; a 5' untranslated region; and a polyA tail, wherein the nucleotides in the 5' untranslated region are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, or sugar-modified nucleotides. In the polynucleotide of the present invention, the nucleotides in the 5' untranslated region are each independently selected from 2'-deoxyribonucleotides, spacer-modified nucleotides, or sugar-modified nucleotides, thereby exhibiting excellent translation ability.

[0054] When the nucleotides in the 5'-untranslated region are composed of 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides, it is preferred that sugar-modified nucleotides are included.

[0055] The polynucleotide of this embodiment also includes those in which an appropriate non-sugar-modified nucleotide of 1 to 10 bases in length has been added to the original 5'-end.

[0056] (5' Cap Structure) The polynucleotide of this embodiment may further comprise a 5' cap structure at the original 5' end. The 5' cap structure may be present in a form added to the 5' untranslated region. The inclusion of a 5' cap structure tends to improve translation activity.

[0057] The 5' cap structure in this application refers to the following structure in which a triline structure is attached to 7-methylguanylic acid (m7G).

[0058] In addition to the 7-methylguanylic acid (m7G) cap, the 5'-cap structure may also include 5'-cap analogs such as those disclosed in the following papers: ARCA: RNA, Vol. 7, pp. 1486-1495 (2001), Cell Cycle, Vol. 17, No. 13, pp. 1624-1636 (2018); LNA: Journal of American Chemical Society, Vol. 131, No. 18, pp. 6364-6365 (2009); S-Cap: RNA, Vol. 14, pp. 1119-1131 (2008); Nature Reviews Drug Discovery, Vol. 13, pp. 759-780 (2014).

[0059] The 5'-untranslated region may contain a base-modified nucleotide. The position of the base-modified nucleotide in the 5'-untranslated region is not particularly limited. The base-modified nucleotide may be a sugar-modified nucleotide and / or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further contain a modified sugar moiety and / or a modified phosphate moiety).

[0060] Although not particularly limited, from the viewpoint of improving translation activity, the 5'-side untranslated region preferably contains the following modified base moieties. [wherein R is an alkyl group having 1 to 6 carbon atoms] The alkyl group R of the modified base moiety is preferably methyl or ethyl.

[0061] The 5'-untranslated region may contain a phosphate-modified nucleotide. The position of the phosphate-modified nucleotide in the 5'-untranslated region is not particularly limited. The phosphate-modified nucleotide may be a sugar-modified nucleotide and / or a base-modified nucleotide (in other words, the phosphate-modified nucleotide may further contain a modified sugar moiety and / or a modified base moiety).

[0062] The 5' untranslated region may contain a 2'-deoxyribonucleotide or spacer modification. The position of the 2'-deoxyribonucleotide or spacer modification in the 5' untranslated region is not particularly limited, but it is preferred that a 2'-deoxyribonucleotide or spacer modification be contained in any nucleotide position except for the first to sixth nucleotides from the 5' end. In this embodiment, it is preferred that the translated region does not contain a spacer modification.

[0063] (Spacer Modification) The spacer modification contained in the 5'-untranslated region is not particularly limited as long as it does not contain a base portion and has a structure that can be used as a substitute for a nucleotide, and examples thereof include the following structures. [wherein, Rx is alkyl having 1 to 6 carbon atoms, alkenyl having 1 to 6 carbon atoms, alkynyl having 1 to 6 carbon atoms, a hydrogen atom, or OH; 1 , OR 1 , R 2 OR 1 , OR 2 OR 1 , S.H., S.R. 1 , NH2, NHR 1 , N.R. 1 2, N3, a hydrogen atom, OH, CN, F, Cl, Br or I, and X is O, S or NR 1 and R 1 are each independently alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, more preferably alkyl having 1 to 3 carbon atoms; R 2represents alkylene, preferably alkylene having 1 to 6 carbon atoms, and n1 and n2 each represent an integer of 1 to 10. As a spacer modification, the oxygen atom of the 5-membered ring in the leftmost structure may be substituted with NH.

[0064] The structures used as spacer modifications are disclosed in the following papers:・M. Takeshita, CN Chang, F. Johnson, S. Will, and AP Grollman, J. Biol. Chem., 1987, 262, 10171-10179. ・MW Kalnik, CN Chang, AP Grollman, and DJ Patel, Biochemistry, 1988, 27, 924-931. ・IG Shishkina and F. Johnson, Chem. Res Toxicol, 2000, 13, 907-912. ・K. Groebke, and CJ Leumann, Helv Chim Acta, 1990, 73, 608-617. ・T. Kuboyama, M. Nakahara, M. Yoshino, Y. Cui, T. Sako, Y. Wada, T. Imanishi, S. Obika, Y. Watanabe, M. Suzuki, H. Doi, Bioorg. Med. Chem. 2011, 19, 249-255. ・M. Salunkhe, TF Wu, and RL Letsinger, J. Amer. Chem. Soc., 1992, 114, 8768-8772.

[0065] The spacer modification is not particularly limited, but the following structures are preferred. [Wherein, Rx is ethynyl, a hydrogen atom, or OH, M is a hydrogen atom or OH, n1 is 1, 2, or 5, and n2 is 1, 2, or 3.]

[0066] <Poly A Chain> The polynucleotide of this embodiment includes a poly A chain. In one aspect of this embodiment, 65% or more of the nucleotides constituting the poly A chain are sugar-modified nucleotides. The poly A chain is contained in the 3'-untranslated region. In this embodiment, at least one poly A chain is contained in the 3'-untranslated region. The poly A chain is a polyadenylic acid composed of two or more AMPs. In this application, AMP includes nucleotides corresponding to AMP (e.g., sugar-modified nucleotides of AMP, 2'-deoxyribonucleotides of AMP, phosphate-modified nucleotides of AMP, and base-modified nucleotides of AMP). Hereinafter, in this application, AMP or nucleotides corresponding to AMP will be collectively referred to as AMP. The poly A chain may include ribonucleotides other than AMP (e.g., CMP, GMP, UMP, or nucleotides corresponding to each of them) as long as it has a polyadenylic acid structure containing two or more AMPs. When the polyA chain contains ribonucleotides other than AMP, the nucleotide at the 5' end of the polyA chain is understood to be the AMP that serves as the starting point of a sequence of two or more consecutive AMPs. When the polyA chain contains ribonucleotides other than AMP, the proportion of ribonucleotides other than AMP among the nucleotides constituting the polyA chain is 40% or less, 30% or less, 20% or less, or 10% or less, preferably 30% or less, more preferably 20% or less, and even more preferably 10% or less. In the polynucleotide of this embodiment, 65% or more of the nucleotides in the polyA chain are neither ribonucleotides nor 2'-deoxyribonucleotides.

[0067] Examples of poly(A) chains containing ribonucleotides other than AMP are disclosed in, for example, Nature Medicine, Vol. 23, No. 7, pp. 815-817 (2017), Science, Vol. 361, pp. 701-704 (2018), and RNA, Vol. 25, pp. 507-518 (2019).

[0068] As used herein, the term "poly-A chain" also refers to a sequence in which two or more consecutive AMP regions present at two or more locations are linked by an arbitrary linker. Examples of linkers include, but are not limited to, polyethylene glycol, polypeptides, and alkyl chains. For example, International Publication No. 2016 / 011306 discloses a method for linking nucleotides with a specific linker.

[0069] The poly-A chain in one aspect of this embodiment may contain 2'-deoxyribonucleotides, spacer-modified, or sugar-modified nucleotides. The location of these nucleotides within the 3'-untranslated region is not particularly limited. The poly-A chain of this aspect may not contain AMP, but the description of the poly-A chain described in the above aspect is also applicable. The poly-A chain may contain 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% 2'-deoxyribonucleotides, spacer-modified, or sugar-modified nucleotides. The poly-A chain is preferably composed of 2'-deoxyribonucleotides, spacer-modified, or sugar-modified nucleotides. When the nucleotides of the poly-A chain are composed of 2'-deoxyribonucleotides, spacer-modified, or sugar-modified nucleotides, it is preferable that sugar-modified nucleotides are included. When the poly-A chain is composed of 2'-deoxyribonucleotides, spacer-modified, or sugar-modified nucleotides, sugar-modified nucleotides may account for 65% or more of the nucleotides constituting the poly-A chain.

[0070] (3' Untranslated Region) The 3' untranslated region (3'UTR) is located downstream of the translated region (towards the 3' end) and is a region where translation for polypeptide synthesis does not occur. The number of nucleotides constituting the 3' untranslated region is preferably an integer between 2 and 6,000, more preferably an integer between 2 and 3,000, even more preferably an integer between 2 and 1,000, and particularly preferably an integer between 2 and 500. The region other than the polyA tail in the 3' untranslated region may be any nucleotide, and each nucleotide in the region other than the polyA tail in the 3' untranslated region may be either an unmodified nucleotide or a modified nucleotide. The polynucleotide of this embodiment is linked in the order of the translated region and the 3' untranslated region.

[0071] The length of the poly-A tail is preferably 2 to 500 bases, more preferably 2 to 200 bases, even more preferably 2 to 80 bases, even more preferably 2 to 40 bases, even more preferably 3 to 40 bases, even more preferably 5 to 40 bases, even more preferably 10 to 40 bases, and particularly preferably 20 to 40 bases.

[0072] In a polyA chain, 65% or more of the nucleotides constituting the polyA chain are sugar-modified nucleotides. The position of the sugar-modified nucleotides within the polyA chain is not particularly limited. The proportion of sugar-modified nucleotides within the polyA chain is preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. A proportion of 100% means that all nucleotides in the polyA chain are sugar-modified nucleotides.

[0073] When the poly A tail is composed of 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides, it is preferred that sugar-modified nucleotides are included.

[0074] When the polyA chain is composed of 2'-deoxyribonucleotides, spacer-modified nucleotides, and sugar-modified nucleotides, it is preferable that, of the nucleotides constituting the polyA chain, sugar-modified nucleotides account for 50% or more, 2'-deoxyribonucleotides for 30% or less, and spacer-modified nucleotides for 20% or less.

[0075] When the polyA chain is composed of 2'-deoxyribonucleotides and sugar-modified nucleotides, it is preferred that the sugar-modified nucleotides account for 50% or more of the nucleotides constituting the polyA chain, and the 2'-deoxyribonucleotides account for 50% or less.

[0076] When the polyA chain is composed of spacer-modified and sugar-modified nucleotides, it is preferred that sugar-modified nucleotides account for 80% or more of the nucleotides constituting the polyA chain, and spacer-modified nucleotides account for 20% or less.

[0077] From the viewpoint of improving translation activity, the first, second, and third nucleotides from the 3'-end of the 3'-untranslated region may be sugar-modified nucleotides. Although not particularly limited, the substituent at the 2'-position of the sugar moiety of the first, second, and third nucleotides from the 3'-end is preferably a methoxyethoxy group (OCH2CH2OCH3).

[0078] Specific examples of modified sugar moieties of sugar-modified nucleotides are preferably selected independently from any one of the following structures: Preferably, each independently is selected from one of the following structures:

[0079] The polyA strand may contain a base-modified nucleotide. The position of the base-modified nucleotide in the polyA strand is not particularly limited. The base-modified nucleotide may be a sugar-modified nucleotide and / or a phosphate-modified nucleotide (in other words, the base-modified nucleotide may further contain a modified sugar moiety and / or a modified phosphate moiety).

[0080] The 3' untranslated region may preferably contain 2'-deoxyribonucleotides or spacer modifications in the 3' untranslated region other than the polyA tail. Specific examples of spacer modifications include those described above in the section (Spacer Modification) of (5' Untranslated Region). The polynucleotide of this embodiment also includes polynucleotides in which an appropriate non-sugar-modified nucleotide of 1 to 10 bases in length has been added to the original 3' end.

[0081] The polyA tail may contain a phosphate-modified nucleotide. The position of the phosphate-modified nucleotide in the polyA tail is not particularly limited. The phosphate-modified nucleotide may be a sugar-modified nucleotide and / or a base-modified nucleotide (in other words, the phosphate-modified nucleotide may contain a modified sugar moiety and / or a modified base moiety).

[0082] The modified phosphate moiety contained in the poly-A chain is preferably phosphorothioate. The positions of the nucleotides linked with phosphorothioate in the poly-A chain are preferably consecutive from the 3'-end.

[0083] The proportion of nucleotides linked by phosphorothioate among the phosphate bonds in the polyA chain is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more, preferably 50% or more, more preferably 80% or more, and particularly preferably 100%. A proportion of 100% means that all of the nucleotides in the polyA chain are linked by phosphorothioate.

[0084] Since phosphate-modified nucleotides can impart stability to endonucleases, which are a type of nuclease, it is preferable that two or more consecutive phosphate-modified nucleotides are contained at the 5'-end and / or 3'-end of the polynucleotide of the present invention.

[0085] (Linking Part) The polynucleotide of this embodiment may contain the following linking part. [In the formula, R 1 and R 2 are each independently H, OH, F, OCH2CH2OCH3 or OCH3, B 1 and B 2 are each independently a base moiety, 1 is O, S or NH, X 2 is O, S, NH or the following structure: X 3 is OH, SH or a salt thereof (X 3 The OH and SH are respectively O -and S - may be written as), where X 1 and X 2 is not simultaneously O.] The base moiety may be an unmodified base moiety or a modified base moiety.

[0086] The left and right nucleotides in the linking portion are the two nucleotides that constitute the polynucleotide of this embodiment. Even if the linking portion is included, translation activity can be maintained. Nucleotide A on the right side (5'-end) of the linking portion and nucleotide B on the left side (3'-end) of the linking portion, as well as nucleotide C on the 3'-end adjacent to nucleotide B and nucleotide D on the 3'-end adjacent to nucleotide C, may not be modified.

[0087] X of the connecting portion 3 Examples of OH and SH salts include pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include alkali metal salts, alkaline earth metal salts, ammonium salts, organic amine salts, and amino acid salts. Examples of alkali metal salts include sodium salts, lithium salts, and potassium salts. Examples of alkaline earth metal salts include calcium salts and magnesium salts.

[0088] Specific examples of the connecting portion include the following. [In the formula, R 1 , R 2 , B 1 , B 2 , and X 3 is as described above]

[0089] The position of the linking portion is not particularly limited. The linking portion may be present in any of the translated region, the 5'-untranslated region, and the 3'-untranslated region (including the polyA tail), but when a linking portion is present, it is preferably present at least in the translated region.

[0090] The number of linking moieties is not particularly limited and can be appropriately selected depending on the length of the polynucleotide, and may be, for example, 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 or 2.

[0091] In the polynucleotide of this embodiment, the first and second nucleotides in at least one of the codons constituting the translation region may be linked by a phosphorothioate bond. The number of phosphorothioate bonds is not particularly limited and can be selected appropriately depending on the length of the polynucleotide. Examples of the number of phosphorothioate bonds include 1 to 200, 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 or 2.

[0092] From the viewpoint of improving translation activity, the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region may be linked by phosphorothioate. Note that "the first and second nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate" is synonymous with "the first and second nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate." For example, "the first to third nucleotides are linked by phosphorothioate" means that the first and second nucleotides are linked by phosphorothioate, and the second and third nucleotides are linked by phosphorothioate. When the first to third nucleotides are linked by phosphorothioate, the structures on the 5' side of the first nucleotide and the 3' side of the third nucleotide may be any.

[0093] From the viewpoint of improving translation activity, the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the 3' untranslated region may be linked by phosphorothioate. From the viewpoint of improving translation activity, the first and second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the polyA tail may be linked by phosphorothioate. Alternatively, all nucleotides of the polyA tail may be linked by phosphorothioate.

[0094] Another embodiment of the present invention relates to a polynucleotide, wherein the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides.

[0095] Another embodiment of the present invention relates to a polynucleotide, wherein the first, second, and third nucleotides from the 3' end of the polyA tail are sugar-modified nucleotides.

[0096] Another embodiment of the present invention relates to a polynucleotide, wherein the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and the first, second, and third nucleotides from the 3' end of the polyA tail are sugar-modified nucleotides.

[0097] Another embodiment of the present invention relates to a polynucleotide in which the first to third, first to fourth, or first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioate.

[0098] Another embodiment of the present invention relates to a polynucleotide in which the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the polyA tail are linked by phosphorothioates.

[0099] Another embodiment of the present invention relates to a polynucleotide in which the first to third, the first to fourth, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by a phosphorothioate group, and the first to third, the first to fourth, or the first to fifth nucleotides from the 3' end of the polyA tail are linked by a phosphorothioate group.

[0100] Another embodiment of the present invention relates to a polynucleotide in which the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by phosphorothioates.

[0101] Another embodiment of the present invention relates to a polynucleotide in which the first, second, and third nucleotides from the 3' end of the polyA tail are sugar-modified nucleotides, and the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the polyA tail are linked by phosphorothioates.

[0102] Another embodiment of the present invention relates to a polynucleotide in which the first, second, and third nucleotides from the 5' end of the 5' untranslated region are sugar-modified nucleotides, and the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5' end of the 5' untranslated region are linked by a phosphorothioate group; and the first, second, and third nucleotides from the 3' end of the polyA tail are sugar-modified nucleotides, and the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3' end of the polyA tail are linked by a phosphorothioate group.

[0103] In the present invention, exemplary embodiments and preferred embodiments are described in each description of the 5' untranslated region, the translated region, and the polyA tail, but the 5' untranslated region, the translated region, and the polyA tail may be present in any combination of the embodiments, or any combination of preferred embodiments of any one or two of the 5' untranslated region, the translated region, and the polyA tail. Furthermore, regions other than those described as the 5' untranslated region, the translated region, and the polyA tail may also be appropriately combined from the exemplary embodiments and preferred embodiments. In other words, in the present specification, all combinations of exemplary embodiments and preferred embodiments in each description of the 5' untranslated region, the translated region, and the polyA tail are described and exemplified as embodiments in the present specification.

[0104] (Other Sequences) The polynucleotide of this embodiment may further include a Kozak sequence and / or a Ribosome Binding sequence (RBS).

[0105] <Method for Producing Polynucleotides> The polynucleotides of this embodiment can be produced, for example, by chemical synthesis. Specifically, the polynucleotides of this embodiment can be produced by using a known chemical synthesis method to introduce predetermined sugar-modified nucleotides at predetermined positions while extending the polynucleotide chain. Known chemical synthesis methods include, for example, the phosphoramidite method, the phosphorothioate method, the phosphotriester method, and the CEM method (see Nucleic Acids Research, 35, 3287 (2007)). Alternatively, an ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems) can be used.

[0106] More specifically, known chemical synthesis methods include those described in the following literature: Tetrahedron, Vol. 48, No. 12, pp. 2223-2311 (1992); Current Protocols in Nucleic Acids Chemistry, John Wiley & Sons (2000); Protocols for Oligonucleotides and Analogs, Human Press (1993); Chemistry and Biology of Artificial Nucleic Acids, Wiley-VCH (2012); Genome Chemistry: A Scientific Approach Utilizing Artificial Nucleic Acids, Kodansha (2003);・New Trends in Nucleic Acid Chemistry, Kagaku Dojin (2011).

[0107] A phosphoramidite that is not commercially available can be chemically synthesized and used as a raw material to produce the polynucleotide of this embodiment. A method for synthesizing phosphoramidite (f), which is the raw material for base-modified nucleotides, is shown below. [In the synthesis scheme, Ra is a hydrogen atom, F, OCH2CHOCH3, or OCH3, Rb is a protecting group that can be removed with a fluoride ion, such as di-tert-butylsilyl, Rc is an alkyl group having 1 to 6 carbon atoms, and Rd is a protecting group used in solid-phase nucleic acid synthesis, such as a p,p'-dimethoxytrityl group.]

[0108] (Step A) Compound (b) can be produced by reacting compound (a) with, for example, a corresponding silylating agent in a solvent in the presence of a base at a temperature between 0°C and 80°C for 10 minutes to 3 days. Examples of solvents include DMF, DMA, NMP, etc., which can be used alone or in combination. Examples of bases include imidazole, triethylamine, diisopropylethylamine, etc. Examples of silylating agents include di-tert-butylsilyl bis(trifluoromethanesulfonate), etc.

[0109] (Step B) Compound (c) can be produced by reacting compound (b) with the corresponding alkylating agent in a solvent in the presence of a base at a temperature between 0°C and 150°C for 10 minutes to 3 days. The reaction can also be accelerated by the addition of an appropriate additive. Examples of solvents include DMF, pyridine, dichloromethane, THF, ethyl acetate, 1,4-dioxane, NMP, etc., which can be used alone or in combination. Examples of bases include aqueous sodium hydroxide, potassium carbonate, pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, etc. Examples of alkylating agents include methyl iodide, ethyl iodide, methyl bromide, etc. Examples of additives include tetrabutylammonium bromide.

[0110] (Step C) Compound (d) can be produced by reacting compound (c) with a fluorine reagent in a solvent at a temperature between -80°C and 200°C for 10 seconds to 72 hours. A base can also be added at this time. Examples of the fluorine reagent include hydrogen fluoride, triethylamine hydrofluoride, and tetrabutylammonium fluoride (TBAF). Examples of the base include triethylamine and N,N-diisopropylethylamine. Examples of the solvent include dichloromethane, chloroform, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, N,N-dimethylacetamide (DMA), NMP, and dimethyl sulfoxide (DMSO).

[0111] (Step D) Compound (e) can be produced by reacting compound (d) with the corresponding alkylating agent in a solvent in the presence of a base at a temperature between 0°C and 150°C for 10 minutes to 3 days. The reaction can also be accelerated by the use of an appropriate activating agent. Examples of solvents include DMF, pyridine, dichloromethane, THF, ethyl acetate, 1,4-dioxane, and NMP, which can be used alone or in combination. Examples of bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, and 2,6-lutidine. Examples of alkylating agents include trityl chloride and p,p'-dimethoxytrityl chloride. Examples of activating agents include 4-dimethylaminopyridine.

[0112] (Step E) Compound (f) can be produced by reacting compound (e) and compound (g) in a solvent in the presence of a base at a temperature between 0°C and 100°C for 10 seconds to 24 hours. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, NMP, etc., which can be used alone or in combination. Examples of the base include triethylamine, N,N-diisopropylethylamine, pyridine, etc., which can be used alone or in combination.

[0113] The 5'-cap structure can be introduced using known methods (e.g., enzymatic methods and chemical synthesis methods), such as those described in Top. Curr. Chem. (Z) (2017) 375:16 and Beilstein J. Org. Chem. 2017, 13, 2819-2832.

[0114] When the polynucleotide of this embodiment has a long base length, a plurality of polynucleotide units may be linked together. The linking method is not particularly limited, and examples thereof include enzymatic methods and chemical synthesis methods.

[0115] Enzymatic ligation can be achieved, for example, by ligation using a ligase. Examples of ligases include T4 DNA Ligase, T4 RNA Ligase 1, T4 RNA Ligase 2, T4 RNA Ligase 2, truncated T4 RNA Ligase 2, truncated KQ, E. coli DNA Ligase, and Taq DNA Ligase. These can be used alone or in combination. In enzymatic ligation, it is generally preferred that the 3'-terminal nucleotide A of the polynucleotide unit constituting the 5'-end of the polynucleotide (hereinafter referred to as the "5'-end polynucleotide unit"), the 5'-terminal nucleotide B of the polynucleotide unit constituting the 3'-end of the polynucleotide (hereinafter referred to as the "3'-end polynucleotide unit") (nucleotides A and B are adjacent to each other in the linked polynucleotide), the nucleotide C adjacent to nucleotide B, and the nucleotide D adjacent to nucleotide C are unmodified. On the other hand, when T4 RNA ligase 2 described in Molecular Cell, Vol. 16, 211-221, October 22, 2004, or the like is used, the nucleotides A to D may be modified.

[0116] In enzymatic linkage, polydisperse polyethylene glycol (PEG) may be used to promote the linkage reaction by the molecular crowding effect. Examples of polydisperse PEG include PEG4000, PEG6000, PEG8000, and PEG10000, which may be used alone or in combination.

[0117] An example of linkage by chemical synthesis (also referred to as "chemical ligation") is a method in which the 3' end of the 5'-end polynucleotide unit (on the right side below) and the 5' end of the 3'-end polynucleotide unit (on the left side below) are condensed in the presence of a condensing agent, as shown below. [In the formula, R 1 , R 2 , B 1 , B 2 and X 3is as described above, and X 1 is O, S or NH, X 2 is O, S, NH or the following structure:

[0118] Examples of the condensing agent include 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), iodide Examples include 2-chloro-1-methylpyridinium, 1H-imidazole-1-carbonitrile, 1-cyano-1H-benzimidazole, and 1-cyano-1H-benzotriazole.

[0119] The condensation reaction is preferably carried out in the presence of template DNA comprising a nucleotide chain complementary to the 3'-terminal nucleotide chain of the 5'-terminal polynucleotide unit and the 5'-terminal nucleotide chain of the 3'-terminal polynucleotide unit. The template DNA is preferably a nucleotide chain having a length of preferably 2 to 50 bases, more preferably 5 to 40 bases, from the 3'-terminal of the 5'-terminal polynucleotide unit, and a nucleotide chain complementary to the nucleotide chain having a length of preferably 2 to 50 bases, more preferably 5 to 40 bases, from the 5'-terminal of the 3'-terminal polynucleotide unit. Here, "complementary" means that the base sequence identity is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0120] In the condensation reaction, an additive may be added, such as 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP).

[0121] In the condensation reaction, a metal salt may be added, such as zinc chloride(II), zinc bromide(II), zinc acetate(II), nickel chloride(II), or manganese chloride(II).

[0122] The condensation reaction may be carried out in the presence of a buffer solution, such as an acetate buffer solution, a Tris buffer solution, a citrate buffer solution, a phosphate buffer solution, or water.

[0123] The temperature of the condensation reaction is not particularly limited, but may be, for example, room temperature to 200° C. The time period of the condensation reaction is not particularly limited, but may be, for example, 5 minutes to 100 hours.

[0124] Specific examples of the condensation reaction between the 3' end of the 5' end polynucleotide unit (right side below) and the 5' end of the 3' end polynucleotide unit (left side below) include the following. [In the formula, R 1 , R 2 , B 1 , B 2 and X 3 is as described above, and X 4 is a leaving group.

[0125] Specific examples of the leaving group include a chloro group, a bromo group, an iodo group, a methanesulfonyl group, a p-toluenesulfonyl group, and a trifluoromethanesulfonyl group. The leaving group is not particularly limited, but is preferably a chloro group or a bromo group.

[0126] The polynucleotide units may be linked multiple times depending on the length of the desired polynucleotide. The number of times of linkage is not particularly limited, and may be, for example, 1 to 200 times, 1 to 100 times, 1 to 50 times, 1 to 20 times, 1 to 10 times, 1 to 8 times, 1 to 6 times, 1 to 4 times, 1 to 3 times, or 1 or 2 times.

[0127] The methods for producing Compound (M) and Compound (N), which are the 5'-terminal polynucleotide units used for linking, are shown below. [In the formula, B pis a base which may be protected by a protecting group, B is a base, and Polymer is a solid support. 4 is a selectively deprotectable protecting group, such as a tert-butyldimethylsilyl group or a triethylsilyl group, and R 3 is a protecting group used in solid-phase nucleic acid synthesis, for example, p,p'-dimethoxytrityl group, and X a is a nucleic acid sequence, and Y a and Y b are each independently a leaving group, such as a halogen atom, preferably a chlorine atom or a bromine atom. In this specification, a nucleic acid sequence is a partial structure in a nucleic acid that forms a nucleic acid together with a compound to which it is bound. When there are multiple Bs in a molecule, each B may be the same or different.

[0128] (Step 1) Compound (B) can be produced by reacting compound (A) in a solvent at a temperature between 60°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include toluene, xylene, 1,2-dichloroethane, 1,4-dioxane, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), 1,2-dichlorobenzene, water, etc., and these can be used alone or in combination. Compound (A) can be produced, for example, by the method described in J. Am. Chem. Soc. (1999), 121, 5661-5665.

[0129] B in compound (A) p is not particularly limited, but preferably has one of the following structures:

[0130] R 6 is a group constituting a part of a protecting group for a base, and represents, for example, a methyl group, an isopropyl group, an optionally substituted phenyl group, etc. Examples of the substituent in the optionally substituted phenyl group include a methyl group, an isopropyl group, and a tert-butyl group.

[0131] (Step 2) Compound (C) can be produced by reacting compound (B) with, preferably 1 to 100 equivalents of an additive in a solvent in the presence of 1 to 100 equivalents of an oxidizing agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include aprotic solvents such as chloroform and dichloromethane, which can be used alone or in combination. Examples of the oxidizing agent include organic oxidizing agents such as Jones reagent, chromic acid, pyridinium dichromate, ruthenium tetroxide, sodium chlorite, and Dess-Martin reagent, and inorganic oxidizing agents such as pyridinium chlorochromate, which can be used alone or in combination. Examples of the additive include pyridine, triethylamine, and N,N-diisopropylethylamine, which can be used alone or in combination.

[0132] (Step 3) Compound (D) can be produced by reacting compound (C) in the presence of hydroxylamine hydrochloride in a solvent such as pyridine at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days.

[0133] (Step 4) Compound (E) can be produced by reacting compound (D) in a solvent in the presence of 1 to 100,000 equivalents of a deprotecting agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include toluene, xylene, water, etc., which can be used alone or in combination. Examples of the deprotecting agent include trifluoroacetic acid, trichloroacetic acid, acetic acid, hydrochloric acid, etc., which can be used alone or in combination.

[0134] (Step 5) Compound (F) can be produced by reacting compound (E) in a solvent in the presence of a reducing agent at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include trifluoroacetic acid, trichloroacetic acid, acetic acid, hydrochloric acid, toluene, xylene, tetrahydrofuran, methanol, ethanol, 1,4-dioxane, water, etc., and these can be used alone or in combination. Examples of the reducing agent include sodium borohydride, sodium cyanoborohydride, lithium borohydride, sodium triacetoxyborohydride, etc.

[0135] (Step 6) Compound (G) can be produced by reacting compound (F) in a solvent in the presence of a catalyst under a hydrogen atmosphere at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of the solvent include trifluoroacetic acid, acetic acid, dilute hydrochloric acid, methanol, ethanol, isopropanol, water, etc., and these can be used alone or in combination. Examples of the catalyst include palladium carbon, ruthenium carbon, etc. Compound (G) can also be produced, for example, by the method described in WO 2017 / 123669.

[0136] (Step 7) Compound (H) can be produced by reacting compound (G) with, preferably 1 to 1,000 equivalents of base in a solvent in the presence of 1 to 100 equivalents of compound (G') and a base at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include methanol, ethanol, isopropanol, dichloromethane, acetonitrile, toluene, ethyl acetate, tetrahydrofuran (THF), 1,4-dioxane, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), water, etc., which can be used alone or in combination. Examples of bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, 2,6-lutidine, etc., which can be used alone or in combination. Compound (G') is commercially available.

[0137] (Step 8) Compound (I) can be produced by reacting compound (H) with p,p'-dimethoxytrityl chloride in a solvent such as pyridine, optionally in the presence of a co-solvent, at a temperature between 0°C and 100°C for 5 minutes to 100 hours. Examples of the co-solvent include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone, triethylamine, N,N-diisopropylethylamine, water, etc., and these can be used alone or in combination.

[0138] (Step 9) Compound (J) can be produced by reacting compound (I) with 1 to 10 equivalents of an additive in a solvent at a temperature between 0°C and the boiling point of the solvent used for 10 minutes to 10 days. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, NMP, etc., which can be used alone or in combination. Examples of the additive include tetrabutylammonium fluoride, triethylamine trihydrofluoride, etc., which can be used alone or in combination.

[0139] (Step 10) Compound (K) can be produced by reacting compound (J) with succinic anhydride in a solvent in the presence of 1 to 30 equivalents of a base at a temperature between room temperature and 200° C. for 5 minutes to 100 hours. Examples of the solvent include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone, pyridine, and water, and these can be used alone or in combination. Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP), and the like, and these can be used alone or in combination.

[0140] (Step 11) Compound (L) can be produced by reacting compound (K) with a terminally aminated solid support, either without solvent or in a solvent, in the presence of 1 to 30 equivalents of a base, a condensing agent, and, if necessary, 0.01 to 30 equivalents of an additive, at a temperature between room temperature and 200°C for 5 minutes to 100 hours, followed by reaction in an acetic anhydride / pyridine solution at a temperature between room temperature and 200°C for 5 minutes to 100 hours. Examples of the solvent include those exemplified in Step 4. Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP), and the like, which can be used alone or in combination. Examples of condensing agents include 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 2-chloro-1-methylpyridinium iodide. Examples of additives include 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP), etc., which can be used alone or in combination. The solid-phase support is not particularly limited as long as a known aminated solid-phase support is used for solid-phase synthesis, and examples include solid-phase supports such as controlled pore glass (CPG) and polystyrene resin (PS) modified with a long-chain alkylamino group.For example, a commercially available long-chain alkylamine controlled pore glass (LCAA-CPG) can be used.

[0141] (Step 12) Compound (M) can be produced by using compound (L) to elongate the corresponding nucleotide chain using a known oligonucleotide chemical synthesis method, followed by detachment from the solid phase, deprotection of the protecting groups, and purification. Dedetachment from the solid phase and deprotection can be achieved by treating the oligonucleotide after chemical synthesis with a base in a solvent or without a solvent at a temperature between −80° C. and 200° C. for 10 seconds to 72 hours. Examples of bases include ammonia, methylamine, dimethylamine, ethylamine, diethylamine, isopropylamine, diisopropylamine, piperidine, triethylamine, ethylenediamine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), potassium carbonate, etc., which can be used alone or in combination. Examples of solvents include water, methanol, ethanol, THF, etc., which can be used alone or in combination. The oligonucleotide can be purified using a C18 reverse-phase column or an anion exchange column, or preferably a combination of the two methods described above. The purity of the nucleic acid complex after purification is preferably 90% or more, more preferably 95% or more.

[0142] (Step 13) Compound (N) can be produced by reacting compound (M) in a buffer solution in the presence of 1 to 1,000 equivalents of compound (O) at a temperature between room temperature and 100°C for 5 minutes to 100 hours. Examples of buffer solutions include acetate buffer, Tris buffer, citrate buffer, phosphate buffer, water, etc., which can be used alone or in combination. A commercially available product can be used as compound (O).

[0143] The method for producing compound (W), which is the 3'-terminal polynucleotide unit used for linking, is shown below. [In the formula, B p is a base which may be protected by a protecting group, B is a base, and R 7is a protecting group, for example, a tert-butyldimethylsilyl group or a triethylsilyl group; Yc is, for example, a chlorine atom, a bromine atom or a tosylate group; X b is a nucleic acid sequence. When multiple Bs are present in a molecule, each B may be the same or different.]

[0144] (Step 14) Compound (Q) can be produced by reacting compound (P) in a solvent in the presence of an additive and a base at a temperature between 0°C and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, NMP, etc., which can be used alone or in combination. Examples of additives include tosylic anhydride, tosyl chloride, thionyl chloride, oxalyl chloride, etc., which can be used alone or in combination. Examples of bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate, etc., which can be used alone or in combination. A commercially available product can be used as compound (P).

[0145] (Step 15) Compound (R) can be produced by reacting compound (Q) with an azidation agent in a solvent, optionally in the presence of a base, at a temperature between room temperature and the boiling point of the solvent used, for 10 seconds to 3 days. Examples of solvents include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, and NMP, which can be used alone or in combination. Examples of azidation agents include sodium azide. Examples of bases include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, and potassium carbonate, which can be used alone or in combination.

[0146] (Step 16) Compound (S) can be produced by reacting compound (R) in a solvent in the presence of a silylating agent and a base at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, DMA, and NMP, and these can be used alone or in combination. Examples of silylating agents include tert-butyldimethylsilyl chloride, tert-butyldimethylsilyl triflate, and triethylsilyl chloride. Examples of the base include pyridine, triethylamine, N-ethyl-N,N-diisopropylamine, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP), and the like, and these can be used alone or in combination.

[0147] (Step 17) Compound (T) can be produced by adding a reducing agent to compound (S) in a solvent and reacting the mixture at a temperature between room temperature and the boiling point of the solvent used for 10 seconds to 3 days. Examples of solvents include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, N-methylpyrrolidone, triethylamine, N,N-diisopropylethylamine, acetic acid, and water. These solvents can be used alone or in combination. Examples of reducing agents include sodium borohydride, sodium cyanoborohydride, lithium borohydride, sodium triacetoxyborohydride, and palladium on carbon under a hydrogen atmosphere.

[0148] (Step 18) Compound (U) can be produced in the same manner as in Step 7 using compound (T).

[0149] (Step 19) Compound (V) can be produced by reacting compound (U) with compound (AA) in a solvent in the presence of a base at a temperature between 0°C and 100°C for 10 seconds to 24 hours. Examples of the solvent include dichloromethane, acetonitrile, toluene, ethyl acetate, THF, 1,4-dioxane, DMF, NMP, etc., which can be used alone or in combination. Examples of the base include triethylamine, N,N-diisopropylethylamine, pyridine, etc., which can be used alone or in combination. A commercially available product can be used as compound (AA).

[0150] (Step 20) Compound (W) can be produced in the same manner as in Step 12 using compound (V).

[0151] When the polynucleotide of this embodiment is produced by linking multiple polynucleotide units, it may contain some polynucleotide units produced by IVT. The method for linking polynucleotides produced by IVT is not particularly limited, and examples include the enzymatic method and chemical synthesis method described above. Examples of methods for producing polynucleotide units using IVT include a method in which RNA is transcribed from a template DNA having a promoter sequence using RNA polymerase. More specifically, known IVT methods include the methods described in the following literature. RNA, Methods in Moleculer Biology (Methods and Protocols), Vol. 703, Chapter 3 (2011); Cardiac Gene Therapy: Methods in Moleculer Biology (Methods and Protocols), Vol. 1521, Chapter 8 (2016); Journal of Molecular Biology, Vol. 249, pp. 398-408 (1995).

[0152] Template DNA used in IVT includes, for example, those produced by chemical synthesis, those produced by polymerase chain reaction, plasmid DNA, and those produced by linearizing plasmid DNA with restriction enzymes, and these can be used alone or in combination. RNA polymerases include T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, etc., and these can be used alone or in combination. The ribonucleoside triphosphate used during transcription may be modified, and multiple types of ribonucleoside triphosphates can be used in combination. As described in Cardiac Gene Therapy: Methods in Molecular Biology (Methods and Protocols), Vol. 1521, Chapter 8 (2016), a 5' Cap structure can be added using compounds such as m7G(5')ppp(5')G (Trilink, Catalog No. S1404) or Anti Reverse Cap Analog, 3'-O-Me-m7G(5')ppp(5')G (Trilink, Catalog No. N-7003). As described in Journal of Molecular Biology, Vol. 249, pp. 398-408 (1995), inserting a sequence such as the Hepatitis delta virus (HDV) ribozyme into template DNA allows for cleavage of the 5' or 3' end of RNA after transcription.

[0153] One embodiment of the present invention relates to a pharmaceutical composition comprising the polynucleotide. By administering the pharmaceutical composition of this embodiment to a patient with a disease, the polynucleotide is translated, a polypeptide encoded by the polynucleotide is synthesized, and the disease is treated.

[0154] Although not particularly limited, the present invention provides a method for treating diseases characterized by the loss or abnormality of the function or activity of a specific protein by compensating for that function or activity with a polypeptide translated from the polynucleotide. Alternatively, the present invention also provides a therapeutic method for artificially controlling immune responses by expressing a foreign antigen peptide or its analog in vivo using a polypeptide translated from the polynucleotide. Furthermore, by expressing in vivo a specific protein in the body, such as a transcription factor, or a polypeptide not naturally occurring in the body, using a polypeptide translated from the polynucleotide, it is also possible to artificially control or modify the function, differentiation, proliferation, etc. of cells, thereby providing a therapeutic method for restoring the function of tissues or cells in diseases characterized by damaged tissues or cells or reduced or abnormal function or activity.

[0155] Diseases include, but are not limited to, cancer and proliferative diseases, infectious and parasitic diseases, blood and hematopoietic diseases, autoimmune diseases, endocrine, nutritional and metabolic diseases (including congenital metabolic disorders), mental and nervous system diseases, skin and subcutaneous tissue diseases, eye diseases, ear diseases, respiratory system diseases, digestive system diseases, renal, urinary and reproductive system diseases, cardiovascular diseases, cerebrovascular diseases, musculoskeletal and connective tissue diseases, miscarriage, perinatal diseases, congenital malformations, acquired injuries, and poisoning.

[0156] Pharmaceutical compositions may be administered in a prescribed formulation. Examples of such formulations include liquid dosage forms for oral or parenteral administration, such as pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art (e.g., water or other solvents), solubilizers, and emulsifiers (e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof). Oral formulations may also contain adjuvants (e.g., wetting agents, emulsifying and suspending agents), sweeteners, flavorings, and / or perfumes. Formulations for parenteral administration may include solubilizing agents (e.g., Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof).

[0157] Methods for administering the pharmaceutical composition include, for example, local administration into lymph nodes, local administration into tumors, intramuscular administration, intradermal administration, subcutaneous administration, intratracheal administration, intrathecal administration, intraventricular administration, intraocular administration, intratympanic administration, catheter administration into coronary arteries, catheter administration into hepatic portal vein, catheter administration into myocardium, transurethral catheter administration, and intravenous administration.

[0158] In addition to the polynucleotide, the pharmaceutical composition may contain optional ingredients, such as one or more pharmaceutically acceptable additives selected from solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonicity agents, thickeners, emulsifiers, preservatives, lipids, lipidoid liposomes, lipid nanoparticles, core-shell nanoparticles, polymers, lipoplexes, peptides, proteins, cells, hyaluronidase, and mixtures thereof.

[0159] The present invention will be described in more detail below using examples and reference examples, but the technical scope of the present invention is not limited to these.

[0160] Reagents used in the synthesis of compounds were purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd., Wako Pure Chemical Industries, Ltd., and Kanto Chemical Co., Ltd. and were used without further purification. Anhydrous solvents were prepared by drying over activated molecular sieves 4A for 12 hours, or commercially available anhydrous-grade solvents were used. Reactions were monitored by thin-layer silica gel chromatography (silica gel 70F254 TLC plate - Wako, Wako Pure Chemical Industries, Ltd.). Compounds were purified using flash chromatography silica gel 60N (spherical, neutral, particle size 40-50 μm) (Kanto Chemical Co., Ltd.). NMR was measured using a JEOL ECS 400 MHz (JEOL Ltd.) with deuterated solvents (CDCl3, CD3OD, DMSO-d6) (Kanto Chemical Co., Ltd.). The acquired NMR data was analyzed using JEOL Delta software (JEOL Ltd.), and chemical shift values ​​were corrected using residual signals in deuterated solvents (CDCl3: 7.26, CD3OD: 3.31, DMSO-d6: 2.50) (Organometallics 2010, 29, 2176-2179). 1 H NMR data are reported as chemical shift (δ), integral (H), signal splitting mode, and coupling constant (Hz) (s: Singlet, d: Doublet, t: Triplet, sept.: Septet, m: Multiplet, br.: Broad). High-resolution mass spectrometry was performed using a microTOF-QII ESI (Bruker Daltonics), and accurate mass correction was performed using ESI TUNING MIX (Agilent Technologies) as an internal standard.

[0161] Compound 12, which is a raw material for polynucleotides, was synthesized according to the following scheme.

[0162] Step 1: Synthesis of Compound 4. N-(9-((2R,3S,4S,5R)-3-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy)methyl)-4-hydroxy-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide. Compound 3, obtained by a method described in a literature (J. Am. Chem. Soc., 1999, 121, 5661-5665), was dissolved in 1,2-dichlorobenzene (2.0 mL) and stirred in an oil bath (160°C) for 4 hours. The reaction mixture was allowed to cool to room temperature and then purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 40:1) without concentration to give compound 4 as a white solid (0.31 g, 53% yield). 1 H NMR (400 MHz, CDCl3) δ 12.01 (1H, s), 8.50 (1H, s), 8.07 (1H, s), 5.86 (1H, d, J = 6.0 Hz), 4.47 (1H, s), 4.24 - 4.23 (1H, m), 4.22 - 4.21 (1H, m), 3.93 (1H, dd, J = 11.6, 2.0 Hz), 3.82 (1H, dd, J = 11.6, 2.0 Hz), 2.66 (1H, sept., J = 6.8 Hz), 1.27 (3H, d, J = 6.8 Hz), 1.25 (3H, d, J = 6.8 Hz), 0.93 (9H, s), 0.82 (9H, s), 0.13 (3H, s), 0.12 (3H, s), -0.07 (3H, s), -0.20 (3H, s) 13 C NMR (100 MHz, CDCl3) δ 179.0, 155.7, 148.5, 148.7, 147.8, 136.8, 121.0, 87.4, 85.4, 77.6, 71.8, 63.6, 36.2, 25.9, 25.4, 19.1, 18.7, 18.3, 17.8, -5.3, -5.4, -5.5, -5.6 ESI-HRMS: calcd for C26 H 48 N5O6Si2 582.31[M+H] + , found: 582.31[M+H] +

[0163] Step 2 Synthesis of Compound 5 N-(9-((2R,3S,5S)-3-(tert-butyldimethylsilyloxy)-5-((tert-butyldimethylsilyloxy) methyl)-4-(hydroxyimino)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl) isobutyramide Molecular sieves 3A (powder) (258 mg) was added to a solution of chromic acid (129 mg, 1.29 mmol) in anhydrous dichloromethane (2.0 mL) and cooled on an ice bath. Anhydrous pyridine (207 μL, 1.29 mmol) was added dropwise to this solution with stirring, and the mixture was stirred on an ice bath. After 30 minutes, acetic anhydride (122 μL, 1.29 mmol) was added dropwise and the mixture was stirred on an ice bath. After 30 minutes, a solution of compound 4 (250 mg, 0.43 mmol) in dichloromethane (1.3 mL) was added dropwise and stirred at room temperature for 2 hours. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was diluted with ethyl acetate and filtered through a silica pad (2 cm thick). The filtrate was concentrated under reduced pressure to give a colorless solid. This crude product 4' was used directly in the next reaction. To a solution of crude product 4' (0.43 mmol) in pyridine (4 mL), hydroxylamine hydrochloride (299 mg, 4.30 mmol) was added and stirred at room temperature. After 24 hours, the reaction mixture was concentrated under reduced pressure, and the residue was added with water and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure, and the residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 40:1) to give compound 5 as a white solid (255 mg, 68% yield for 2 steps). 1H NMR (400 MHz, CDCl3) δ 12.14 (1H, s), 9.27 (1H, s), 8.78 (1H, s), 8.11 (1H, s), 5.78 (1H, d, J = 7.6 Hz), 5.09 (1H, s), 4.92 (1H, d, J = 7.2 Hz), 4.14 (1H, d, J = 11.4 Hz), 3.92 (1H, d, J = 11.4 Hz), 2.79 - 2.74 (1H, m), 1.27 - 1.21 (6H, m), 0.91 (9H, s), 0.71 (9H, s), 0.10 (3H, s), 0.07 (3H, s), -0.10 (3H, s), -0.23 (3H, s) 13 C NMR (100 MHz, CDCl3) δ 178.9, 157.8, 155.6, 148.7, 147.8, 136.8, 120.8, 87.5, 86.5, 62.2, 36.3, 25.9, 25.5, 25.2, 19.1, 18.8, 18.3, 18.0, -5.0, -5.5, -5.6, -5.7 ESI-HRMS : calcd for C 26 H 47 N6O6Si2 595.31[M+H] + , found : 595.31[M+H] +

[0164] Step 3 Synthesis of Compound 6 N-(9-((2R,3S,5S)-3-(tert-butyldimethylsilyloxy)-4-(hydroxyimino)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Compound 5 (129 mg, 0.22 mmol) was added with ice-cold 90% aqueous trifluoroacetic acid solution (1.0 mL) and stirred on an ice bath for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the resulting residue was azeotroped three times with toluene and water (1:1, v / v) under reduced pressure. The resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 50:1 to 40:1) to give compound 6 as a white solid (96 mg, 92% yield). 1 H NMR (400 MHz, CD3OD) δ 8.36 (1H, s), 5.87 (1H, d, J = 7.6 Hz), 5.18 (1H, dd, J = 7.6, 2.0 Hz), 5.02 (1H, d, J = 2.0 Hz), 4.11 (1H, dd, J = 12.0, 2.0 Hz), 3.92 (1H, d, J = 12.0, 2.0 Hz), 2.71 (1H, sept., J = 7.2 Hz), 1.21 (6H, d, J = 7.2 Hz), 0.72 (9H, s), 0.00 (3H, s), -0.16 (3H, s) 13 C NMR (100 MHz, CD3OD) δ 181.8, 157.4, 156.8, 151.0, 150.0, 139.8, 121.3, 88.4, 79.7, 76.5, 61.6, 36.9, 25.9, 19.4, 19.2, -4.5, -5.5 ESI-HRMS : calcd forC 20 H 32 N6NaO6Si 503.21[M+Na] + , found: 503.20[M+Na] +

[0165] Step 4 Synthesis of Compound 7 N-(9-((2R,3S,4S,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Sodium borohydride (15 mg, 0.38 mmol) was added to a solution of compound 6 (93 mg, 0.19 mmol) in acetic acid (1.9 mL), and the mixture was stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 20:1) to give compound 7 as a white solid (51 mg, 55% yield). 1 H NMR (400 MHz, CD3OD) δ 8.34 (1H, s), 6.06 (1H, d, J = 6.0 Hz), 4.75 (1H, t, J = 6.4 Hz), 4.27 (1H, d, J = 2.8 Hz), 3.86 (1H, dd, J = 12.4, 2.0 Hz), 3.73 (1H, d, J = 12.4, 2.0 Hz), 3.62 - 3.60 (1H, m), 2.71 (1H, sept., J = 6.8 Hz), 1.21 (6H, d, J = 6.8 Hz), 0.82 (9H, s), -0.02 (3H, s), -0.23 (3H, s) 13 C NMR (100 MHz, CD3OD) δ 181.8, 157.4, 150.8, 149.8, 139.6, 139.4, 121.1, 89.7, 84.5, 77.7, 65.4, 65.2, 36.9, 26.0, -5.2, -5.3 ESI-HRMS: calcd for C 20 H 35 N6O6Si 483.24[M+H] + , found: 483.23[M+H] +

[0166] Step 5 Synthesis of Compound 8 N-(9-((2R,3S,4S,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide 10% palladium-carbon (20 mg) was added to a solution of compound 7 (50 mg, 0.10 mmol) in 90% aqueous acetic acid (1.5 mL), and the mixture was stirred under a hydrogen atmosphere at room temperature for 18 hours. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was diluted with methanol, and the palladium-carbon was removed by filtration through Celite. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 15:1 to 10:1) to give compound 8 as a white solid (41 mg as acetate, 75% yield). 1 H NMR (400 MHz, CD3OD) δ 8.32 (1H, s), 5.99 (1H, s), 4.60 (1H, s), 3.95 - 3.68 (4H, m), 2.73 (1H, br. s), 1.22 (6H, br. s), 0.05 (3H, s), -0.06 (3H, s) ESI-HRMS : calcd for C 20 H 34 N6NaO5Si 489.2258[M+Na] + , found: 489.2231[M+Na] +

[0167] Step 6 Synthesis of Compound 9 N-(9-((2R,3S,4R,5S)-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Ethyl trifluoroacetate (0.76 mL) was added to a solution of compound 8 (40 mg, 0.076 mmol) and triethylamine (45 L, 0.38 mmol) in methanol (0.76 mL) as described in the literature (WO2017 / 123669), and the mixture was stirred at room temperature for 24 hours. After confirming the disappearance of the raw materials by thin layer chromatography, the reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 20:1 to 12:1) to obtain compound 9 as a white solid (12 mg, yield 28%). 1 H NMR (400 MHz, CDCl3) δ 12.26 (1H, s), 10.11 (1H, s), 7.76 (1H, s), 7.26 (1H, d, J = 3.6 Hz), 5.71 (1H, d, J = 3.6 Hz), 4.98 (1H, dd, J = 6.8 Hz), 4.78 (1H, dd, J = 6.8, 3.6 Hz), 4.21 (1H, d, J = 6.8 Hz), 4.03 (1H, dd, J =11.2 Hz), 3.82 (1H, dd, J =11.2 Hz), 2.79 (1H, sept., J = 6.8 Hz), 1.26 (3H, d, J = 6.8 Hz), 1.24 (3H, d, J = 6.8 Hz), 0.85 (9H, s), -0.01 (3H, s), -0.11 (3H, s) 13C NMR (100 MHz, CDCl3) δ 179.8, 158.0, 157.7, 157.3, 156.9, 155.2, 148.3, 147.3, 138.6, 122.0, 120.0, 117.1, 114.2, 111.3, 91.6, 83.7, 74.5, 61.3, 51.0, 36.1, 25.2, 18.9, 17.7, -5.0, -5.4 ESI-HRMS : calcd for C 22 H 33 F3N6NaO6Si 585.21[M+Na] + , found: 585.21[M+Na] +

[0168] Step 7 Synthesis of Compound 10 N-(9-((2R,3S,4R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Dimethoxytrityl chloride (18 mg, 0.053 mmol) was added to a solution of compound 9 (10 mg, 0.017 mmol) in anhydrous pyridine (1 mL), and the mixture was stirred at room temperature for 1.5 hours. Then, dimethoxytrityl chloride (18 mg, 0.053 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After confirming the disappearance of the starting materials by thin-layer chromatography, methanol (1 mL) was added to the reaction mixture, which was then concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed with water and then with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (neutral silica gel, hexane / ethyl acetate = 5:1 to 2:1) to give compound 10 as a white solid (15.2 mg, yield 99%). 1H NMR (400 MHz, CDCl3) δ 11.99 (1H, s), 10.11 (1H, s), 8.07 (1H, s), 7.81 (1H, s), 7.45 (2H, dd, J = 8.2, 2.0 Hz), 7.32 (4H, dd, J = 9.2, 3.6 Hz), 7.24 - 7.29 (3H, m), 7.01 (1H, d, J = 7.2 Hz), 6.76 (4H, J = 9.2, 3.6 Hz), 5.71 (1H, d, J = 4.2 Hz), 5.16 (1H, dd, J = 6.4, 4.2 Hz), 4.20 - 4.17 (1H, m), 3.76 (3H, s), 3.75 (3H, s), 3.56 (1H, dd, J = 11.2, 2.8 Hz), 3.22 (1H, dd, J = 11.2, 2.8 Hz), 1.82 (1H, d, J = 6.8 Hz), 0.97 (3H, d, J = 6.8 Hz), 0.68 (9H, s), 0.79 (3H, d, J = 6.8 Hz), 0.04 (3H, s), -0.06 (3H, s) 13 C NMR (100 MHz, CDCl3) δ 171.2, 158.7, 158.0, 157.6, 157.2, 156.8, 155.4, 147.6, 147.2, 144.8, 139.2, 135.9, 135.4, 130.0, 127.9, 127.1, 122.6, 120.0, 117.0, 114.1, 111.2, 90.1, 86.3, 81.7, 73.4, 62.4, 60.4, 55.2, 51.4, 36.1, 25.4, 18.4, 17.8, -5.0, -5.3 ESI-HRMS : calcd for C 43 H 52 F3N6O8Si 865.36[M+H] + , found : 865.35[M+H] +

[0169] Step 8 Synthesis of Compound 11 N-(9-((2R,3S,4S,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxy-4-(2,2,2-trifluoroacetamido)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide To a solution of compound 10 (14 mg, 0.016 mmol) in tetrahydrofuran (1 mL), tetrabutylammonium fluoride (1 M tetrahydrofuran solution, 19 μL, 0.019 mmol) was added and stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (neutral silica gel, dichloromethane / methanol = 30:1 to 15:1) to give compound 11 as a white solid (10.8 mg, 83% yield). 1 H NMR (400 MHz, CDCl3) δ 12.12 (1H, br. s), 8.76 (1H, br. s), 7.74 (1H, s), 7.81 (1H, s), 7.68 (1H, d, J = 5.4 Hz), 7.48 (2H, d, J = 7.6 Hz), 7.37 (2H, d, J = 9.2 Hz), 7.34 (2H, d, J = 9.2 Hz), 7.25 - 7.21 (2H, m), 7.17 (1H, t, J = 7.2 Hz), 6.81 (2H, d, J = 9.2 Hz), 6.78 (2H, d, J = 9.2 Hz), 5.80 (1H, d, J = 4.0 Hz), 5.35 (1H, br. s), 5.08 (1H, dd, J = 12.4, 6.4 Hz), 4.30 - 4.29 (1H, m), 3.76 (3H, s), 3.74 (3H, s), 3.57 - 3.53 (1H, m), 3.29 - 3.26 (1H, m), 1.84 - 1.57 (1H, m), 0.94 (3H, d, J = 6.8 Hz), 0.68 (3H, d, J = 6.8 Hz) 13C NMR (100 MHz, CDCl3) δ 179.4, 158.6, 158.4, 158.0, 157.6, 157.3, 147.8, 147.2, 144.8, 139.4, 136.3, 135.7, 130.1, 129.9, 128.1, 128.0, 127.0, 121.0, 120.0, 117.1, 114.2, 111.3, 91.2, 86.1, 82.5, 71.5, 62.7, 55.1, 51.2, 35.9, 18.5, 18.2ESI-HRMS : calcd for C 37 H 38 F3N6O8 751.27[M+H] + , found: 751.27[M+H] +

[0170] Step 9: Synthesis of Compound 12. To a solution of compound 11 (0.90 g, 1.20 mmol) and triethylamine (0.42 mL, 3.0 mmol) in acetonitrile (12 mL), succinic anhydride (0.24 g, 2.40 mmol) and dimethylaminopyridine (29 mg, 0.24 mmol) were added and stirred at room temperature for 1 hour. After confirming the disappearance of the starting materials by thin-layer chromatography, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed twice with saturated aqueous sodium bicarbonate and then with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was azeotropically evaporated with a dichloromethane / methanol solution (1:1, v / v) to yield a white foamy solid (1.11 g, 97% as the triethylamine salt). This compound 12 was used directly in the next reaction.

[0171] Compound 12 can also be synthesized from the starting material 13 shown below to obtain intermediate 6.

[0172] Step 10 Synthesis of Compound 14 N-(9-((2R,3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-4-(hydroxyimino)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide Under an argon atmosphere, compound 13 (ChemGenes, 5.0 g, 6.5 mmol) was dissolved in anhydrous dichloromethane (50 mL) and stirred while cooling in an ice bath. While cooling the reaction mixture, sodium bicarbonate (8.2 g, 97.3 mmol) and nor-AZADO (36 mg, 0.260 mmol) were added, and iodobenzene diacetate (3.14 g, 9.73 mmol) was added in portions, carefully monitoring the internal temperature. The mixture was then stirred for 21 hours and 10 minutes while warming to room temperature. After confirming the disappearance of the starting materials, isopropyl alcohol (7.5 mL) was added to the reaction mixture and stirred for 4 hours (to quench excess oxidant). The reaction mixture was poured into ice water, and chloroform was added to separate the layers. The aqueous layer was extracted again with chloroform. The combined organic layers were washed once with water and once with saturated brine, then dehydrated over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated to obtain the crude product (9.01 g, including a compound in which some of the DMTr groups had been deprotected) as an orange solid. Under an argon atmosphere, the crude product (9.01 g) was dissolved in anhydrous pyridine (40 mL) and stirred while cooling in an ice bath. While the reaction mixture was cooled, hydroxylamine hydrochloride (4.06 g, 58.7 mmol) was added, and the mixture was stirred for 17 hours and 25 minutes while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was transferred to a recovery flask while washing with chloroform (containing 1% triethylamine) and concentrated. The residue was added to saturated aqueous sodium bicarbonate and stirred for 15 minutes, then extracted twice with chloroform. The organic layers were combined, washed once with saturated brine, and dehydrated over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated to give compound 14 (4.13 g, diastereomeric mixture, 81% yield for two steps) as an orange foam. 1H NMR (400MHz, CDCl3) δ: 12.04 (1H, d, J = 23.3 Hz), 9.23 (1H, s), 8.49 (1H, s), 7.89 (1H, s), 7.79 (1H, s), 7.66-7.58 (2H, m), 7.49-7.39 (4H, m), 7.31-7.14 (5H, m), 6.81-6.76 (2H, m), 6.73-6.68 (2H, m), 5.92 (1H, dd, J = 8.0, 1.6 Hz), 5.83 (1H, d, J = 3.7 Hz), 5.64 (1H, d, J = 8.2 Hz), 5.54 (1H, dd. d, J = 6.9 Hz), 0.86-0.76 (9H, m), 0.47 (2H, m), 0.11 (1H, s), 0.02--0.02 (3H, m), -0.07 (2H, s)[diastereomer mixture] ESI-HRMS : calcd for C 41 H 50 N6O8Si 781.97[MH] - , found: 781.84[MH] -

[0173] Step 11: Synthesis of Compound 6 from Compound 14 Compound 14 (3.80 g) obtained in Step 10 was used to obtain Compound 6 (2.12 g, 4.41 mmol, 91% yield) in the same manner as in Step 3. Detailed information on Compound 6 is as described in Step 3.

[0174] Synthesis of Compound 15 A solution of compound 12 (380 mg, 0.50 mmol) in N,N-dimethylformamide (2.5 mL) was added to Native amino lcaa CPG (1000A, ChemGenes) (84 μmol / g, 1.20 g, 0.10 mmol), followed by a solution of HOBt (136 mg, 1.01 mmol) and EDC-HCl (193 mg, 1.01 mmol) in DMF (2.5 mL) and the mixture was shaken at room temperature. After 20 h, the reaction mixture was discarded, and the solid support was washed with N,N-dimethylformamide (5 mL, 4 times) and then with dichloromethane (5 mL, 4 times). Unreacted amino groups on the solid support were capped with 10% acetic anhydride / pyridine solution (5 mL) at room temperature for 16 h with shaking. The reaction mixture was discarded, and the solid support was washed with pyridine (5 mL, once) and then with dichloromethane (5 mL, four times). This resulted in compound 12 supported on the solid support (1.20 g). The amount of compound 12 supported on the solid support was calculated using the following method. A specified amount of the resulting solid support was taken, and the color development of the 4,4'-dimethoxytrityl cation produced by the addition of a deblocking reagent (3 w / v% trichloroacetic acid / dichloromethane solution) was measured by UV-visible spectrophotometry (quartz cell, cell length: 10 mm). The amount of compound 12 supported on the solid support was calculated using the Lambert-Beer equation from the absorbance at 504 nm and the molar extinction coefficient of the 4,4'-dimethoxytrityl cation (wavelength 504 nm: 76,000). Specifically, the obtained solid support (2.0 mg) was weighed into a 2 mL volumetric flask, deblocking reagent was added to make the total volume 2 mL, and the mixture was mixed by inversion to prepare the measurement sample. After a blank measurement was performed using a 3 w / v% trichloroacetic acid / dichloromethane solution, measurements were performed using the measurement sample. The absorbance at 504 nm was 0.377, which indicated a loading amount of 24.8 μmol / g.

[0175] Compound 24 was synthesized according to the following scheme.

[0176] Step 12 Synthesis of Compound 17 N-(9-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide Commercially available N6-Benzyladenosine (Compound 16) (100 g, 269 mmol, 1.0 eq.), acetone (2.70 L), and dimethoxypropane (166 mL, 1.35 mol, 5.0 eq.) were sequentially added to a 10 L four-neck flask under an argon atmosphere. Concentrated sulfuric acid (1.44 mL, 26.9 mmol, 0.10 eq.) was added to the reaction solution, and the mixture was stirred at room temperature for 15 hours. After confirming that the starting material remained, concentrated sulfuric acid (1.44 mL, 26.9 mmol, 0.10 eq.) was added and the mixture was stirred for 24 hours. After confirming that the starting material remained, concentrated sulfuric acid (1.44 mL, 26.9 mmol, 0.10 eq.) was added and the mixture was stirred for 1 hour and 30 minutes. After that, concentrated sulfuric acid (2.87 mL, 53.8 mmol, 0.20 eq.) was added and the mixture was stirred for 4 hours. After confirming the progress of the reaction by LC / MS, the reaction mixture was cooled in an ice bath, and saturated aqueous sodium bicarbonate (400 mL) was added dropwise over 5 minutes to maintain the internal temperature at 3-5°C until the solution became neutral. The reaction mixture was concentrated under reduced pressure, and distilled water (2.0 L) was added to the residue. The solution was extracted three times with chloroform (1.0 L), and the organic layer was dehydrated over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain compound 17 (222 g). The obtained compound 17 was used in the next step without further purification.

[0177] Step 13 Synthesis of Compound 18 ((3aR,4R,6R,6aR)-6-(6-benzamido-9H-purin-9-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl methanesulfonate Under an argon atmosphere, compound 17 (222 g) obtained in Step 12 and pyridine (520 mL) were placed in a 2 L four-neck flask, and the reaction solution was cooled in an ice bath. Methanesulfonyl chloride (25.0 mL, 321 mmol, 1.2 eq.) was added dropwise over 15 minutes so that the internal temperature became 4°C to 9°C, and the mixture was stirred for 2 hours. After confirming the progress of the reaction by LC / MS, distilled water (500 mL) was added to the reaction mixture, and the solution was extracted three times with ethyl acetate (1.0 L). The organic layer was washed sequentially with 1N hydrochloric acid (1.0 L x 1, 500 mL x 2), saturated aqueous sodium bicarbonate (500 mL x 2), and saturated brine (500 mL x 2), and then dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the resulting residue was azeotroped with toluene to yield compound 18 (150 g, 17.6 wt % toluene content). The resulting compound 18 was used in the next step without further purification.

[0178] Step 14 Synthesis of Compound 19 N-(9-((3aR,4R,6R,6aR)-6-(azidomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide Compound 18 (150 g) obtained in Step 13 and dehydrated DMF (1.26 L) were placed in a 3 L four-neck flask under an argon atmosphere. Sodium azide (82.8 g, 1.26 mol, 5.0 eq.) was added to the reaction mixture, and the mixture was heated to 60°C over 30 minutes and stirred at 60°C for 3 hours and 30 minutes. After confirming the progress of the reaction by LC / MS, the reaction mixture was gradually cooled to room temperature, and distilled water (1.0 L) and ethyl acetate (600 mL) were added. Distilled water (3.0 L) was added to the resulting solution, and the aqueous layer was extracted six times with ethyl acetate (500 mL). The organic layer was washed twice with distilled water (800 mL) and twice with saturated brine (800 mL), and then dehydrated over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (SiO2 700 g, ethyl acetate) to give compound 19 (55.7 g, 128 mmol, 48% yield (3 steps from compound 16)).

[0179] Step 15 Synthesis of Compound 20 N-(9-((3aR,4R,6R,6aR)-2,2-dimethyl-6-((2,2,2-trifluoroacetamido)methyl) tetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-yl)benzamide Compound 19 (55.7 g, 128 mmol, 1.0 eq.) obtained in Step 14 and methanol (1.28 L) were placed in a 3 L four-neck flask under an argon atmosphere. 10% Pd / C (76.8 g, 21.2 mmol, 0.17 eq.) was added to the reaction solution, the atmosphere was purged with hydrogen, and the mixture was stirred at room temperature for 16 hours. After confirming the progress of the reaction by LC / MS, the atmosphere was purged with argon gas and the reaction solution was filtered through Celite. The filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in methanol (985 mL) and transferred to a 3 L four-neck flask. The solution was cooled in an ice bath, and 1-(trifluoroacetyl)imidazole (17.0 mL, 149 mmol, 1.2 eq.) was added dropwise over 15 minutes to maintain the internal temperature at 2-4°C. The mixture was stirred at 4°C for 2 hours. After confirming the progress of the reaction by LC / MS, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 800 g, heptane / ethyl acetate = 1:4) to give compound 20 (21.4 g, 42.2 mmol, 33% yield).

[0180] Step 16 Synthesis of Compound 21 N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-((2,2,2-trifluoroacetamido)methyl) tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Compound 20 (10.0 g, 19.8 mmol, 10 eq.) obtained in Step 15 and distilled water (50.0 mL) were added to a 1 L recovery flask, and the solution was cooled in an ice bath. Under ice cooling, trifluoroacetic acid (50.0 mL, 640 mmol, 32.4 eq.) was added dropwise over 5 minutes, and the reaction mixture was warmed to room temperature and stirred for 4 hours and 30 minutes. After confirming the progress of the reaction by LC / MS, the reaction mixture was concentrated under reduced pressure, and the residue was azeotroped with toluene. Isopropyl ether was added to the resulting residue to precipitate a solid, which was collected by filtration. The resulting solid was dried under reduced pressure at room temperature to obtain Compound 21 (8.86 g, 19.0 mmol, yield 96%).

[0181] Step 17 Synthesis of Compound 22 N-(9-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-4-hydroxy-5-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Compound 21 (15.6 g, 33.6 mmol, 1.0 eq.) obtained in Step 16 and dehydrated DMF (111 mL) were placed in a 500 mL recovery flask under an argon atmosphere, and the solution was cooled in an ice bath. Under ice cooling, imidazole (9.16 g, 134 mmol, 4.0 eq.) and t-butyldimethylsilyl chloride (15.2 g, 101 mmol, 3.0 eq.) were added so that the internal temperature was less than 6°C, and the mixture was stirred at the same temperature for 30 minutes. After confirming the progress of the reaction by LC / MS, ice water was added to the reaction mixture. The aqueous layer was extracted three times with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (SiO2 800 g, chloroform / 2-butanone = 100:0 to 85:15) to give a mixture of compound 22 and compound 23 (10.9 g). The resulting mixture of compound 22 and compound 23 was used in the next step without further purification.

[0182] Step 18 Synthesis of Compound 24 (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-((2,2,2-trifluoroacetamido)methyl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Under an argon atmosphere, a mixture of Compound 22 obtained in Step 17 and Compound 23 (1.19 g, 2.05 mmol, Compound 22:Compound 23=9:1) and dehydrated dichloromethane (6.83 mL) were placed in a 200 mL recovery flask, and the solution was cooled in an ice bath. A dichloromethane mixture of diisopropylethylamine (0.537 mL, 3.07 mmol, 1.5 eq.) and 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (0.857 mL, 3.07 mmol, 1.5 eq.) was added dropwise under ice cooling. The reaction mixture was then warmed to room temperature and stirred for 2 h. After confirming the disappearance of the starting materials by TLC, distilled water was added to the reaction mixture, which was then extracted twice with chloroform (50 mL). The organic layer was dehydrated over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure. The residue was purified multiple times by silica gel column chromatography (heptane / ethyl acetate = 50:50 to 30:70, containing 0.5% triethylamine) to give the desired compound 24 (608 mg, 0.779 mmol, 38% yield) as a pale yellow amorphous substance. 1H NMR (400 MHz, CDCl3) δ: 9.82 (1H, d, J = 8.8 Hz), 9.07 (1H, s), 8.83-8.80 (1H, m), 8.06-8.01 (3H, m), 7.66-7.52 (3H, m), 5.86 (1H, d, J = 7.8 Hz), 4.88 (1H, dd, J = 7.8, 5.2 Hz), 4.50 (1H, br s), 4.39-4.29 (1H, m), 4.21-3.88 (3H, m), 3.74-3.63 (1H, m), 3.48-3.35 (1H, m), 2.73-2.66 (2H, m), 1.28-1.23 (12H, m), 1.08-1.04 (1H, m), 0.72-0.68 (9H, m), -0.17 (3H, s), -0.45 (3H, s). 31 P NMR(CDCl3) δ: 149.95

[0183] Compound 6a, which is a starting material for polynucleotides, was synthesized according to the following scheme.

[0184] Step 1 Synthesis of Compound 2a N-(9-((3aR,5R,6R,6aS)-2,2-di-tert-butyl-6-methoxytetrahydrofuro[2,3-d][1,3,2]dioxasilol-5-yl)-9H-purin-6-yl)benzamide Di-t-butylsilylbis(trifluoromethanesulfonate) (68.6 g, 156 mmol) was slowly added to a solution of commercially available compound 1a (30.0 g, 78.0 mmol) in DMF (300 mL) under ice-cooling. After stirring for 1 hour under ice-cooling, the reaction mixture was added to a saturated aqueous solution of sodium bicarbonate and extracted twice with a mixed solvent of heptane / ethyl acetate. The organic layer was washed twice with water and once with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 7 / 3 → 3 / 7) to obtain compound 2a (38.7 g, 73.7 mmol) as a colorless solid (95% yield). ESI-MS: calculated value: 524.23 [MH] -, Actual measurement: 524.5 [MH] - 1 H-NMR (CDCl3, 400 MHz) δ: 9.32 (s, 1H), 8.76 (s, 1H), 8.05 (s, 1H), 8.03 (t, J = 6.6 Hz, 2H), 7.60 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.8 Hz, 2H), 6.01 (s, 1H), 4.66 (dd, J = 9.6, 5.0 Hz, 1H), 4.48 (dd, J = 8.9, 4.8 Hz, 1H), 4.31 (d, J = 4.6 Hz, 1H), 4.20 (ddd, J = 10.1, 5.0, 5.0 Hz, 1H), 4.03 (t, J = 9.8 Hz, 1H), 3.70 (s, 3H), 1.10 (s, 9H), 1.06 (s, 9H).

[0185] Step 2 Synthesis of Compound 3a N-(9-((3aR,5R,6R,6aS)-2,2-di-tert-butyl-6-methoxytetrahydrofuro[2,3-d][1,3,2]dioxasilol-5-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 2a (10.0 g, 19.0 mmol) was dissolved in dichloromethane (50 mL), and tetrabutylammonium bromide (9.20 g, 28.5 mmol) and 1 M aqueous sodium hydroxide solution (50 mL) were added. Methyl iodide (4.76 mL, 76.0 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 1 hour and 10 minutes. After confirming the disappearance of the raw materials, the reaction mixture was quenched by adding it to ice-cooled water / chloroform = 1 / 1. The organic layer was washed twice with water, then dehydrated over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10 → 50 / 50) to give compound 3a (6.25 g, 11.6 mmol) as a colorless amorphous solid (61% yield). ESI-MS: calculated value: 540.26 [M+H] + , Measured value: 540.4 [M+H]+ 1 H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.94 (s, 1H), 7.49-7.46 (m, 2H), 7.34-7.29 (m, 1H), 7.21 (t, J = 7.6 Hz, 2H), 5.94 (s, 1H), 4.61 (dd, J = 9.6, 5.0 Hz, 1H), 4.46 (dd, J = 9.2, 5.0 Hz, 1H), 4.22 (d, J = 4.6 Hz, 1H), 4.17 (ddd, J = 10.0, 5.2, 5.0 Hz, 1H), 4.00 (dd, J = 10.5, 9.2 Hz, 1H), 3.79 (s, 3H), 3.67 (s, 3H), 1.08 (s, 9H), 1.05 (s, 9H).

[0186] Step 3 Synthesis of Compound 4a N-(9-((2R,3R,4S,5S)-4,5-dihydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 3a (6.25 g, 11.6 mmol) was dissolved in tetrahydrofuran (63 mL) and cooled in an ice bath. Triethylamine (8.07 mL, 57.9 mmol) and triethylamine trihydrofluoride (1.89 mL, 11.6 mmol) were added, and the mixture was stirred for 1 hour and 5 minutes while cooling in an ice bath. After confirming the disappearance of the raw materials, the reaction was quenched by adding triethylamine (10 mL, 76.0 mmol). The mixture was diluted with chloroform and then concentrated. The concentrated residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 90 / 10) to give compound 4a (4.25 g, 10.6 mmol) as a colorless amorphous solid. (Yield quant.) ESI-MS: Calculated value: 400.16 [M+H] + , Measured value: 400.3 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.97 (s, 1H), 7.50-7.48 (m, 2H), 7.35-7.31 (m, 1H), 7.22 (t, J = 7.8 Hz, 2H), 5.87 (d, J = 7.3 Hz, 1H), 5.86 (dd, J = 11.4, 2.3 Hz, 1H), 4.63 (dd, J = 7.3, 4.6 Hz, 1H), 4.57-4.56 (m, 1H), 4.36-4.34 (m, 1H), 3.99-3.94 (m, 1H), 3.81 (s, 3H), 3.77 (td, J = 12.3, 1.7 Hz, 1H), 3.31 (s, 3H), 2.77 (d, J = 1.4 Hz, 1H).

[0187] Step 4 Synthesis of Compound 5a N-(9-((2R,3R,4S,5S)-5-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 4a (4.25 g, 10.6 mmol) was dissolved in pyridine (43 mL) and stirred in an ice bath. 4,4'-Dimethoxytrityl chloride (5.41 g, 20.0 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours and 25 minutes. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give compound 5a (5.35 g, 7.62 mmol) as a colorless amorphous solid (71% yield). ESI-MS: calculated value: 702.29 [M+H] + , Measured value: 702.6 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.50 (s, 1H), 8.14 (s, 1H), 7.45-7.40 (m, 4H), 7.33-7.22 (m, 8H), 7.16 (t, J = 7.6 Hz, 2H), 6.81 (dd, J = 8.9, 1.1 Hz, 4H), 6.15 (d, J = 3.7 Hz, 1H), 4.48 (dd, J = 11.9, 5.0 Hz, 1H), 4.35 (dd, J = 5.3, 3.9 Hz, 1H), 4.21-4.19 (m, 1H), 3.80 (s, 3H), 3.79 (s, 6H), 3.53 (s, 3H), 3.50 (dd, J = 10.8, 3.0 Hz, 1H), 3.40 (dd, J = 10.8, 4.4 Hz, 1H), 2.66 (d, J = 6.4 Hz, 1H).

[0188] Step 5: Synthesis of amidite 6a (2S,3S,4R,5R)-2-(bis(4-methoxyphenyl)(phenyl)methoxy)-4-methoxy-5-(6-(N-methylbenzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5a (5.30 g, 7.55 mmol) was dissolved in dichloromethane (48 mL), and diisopropylethylamine (2.64 mL, 15.1 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (2.68 g, 11.3 mmol) dissolved in dichloromethane (5 mL) was added dropwise over 5 minutes. The mixture was then stirred for 1 hour and 10 minutes while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Ethyl acetate was added for extraction. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the drying agent was filtered off. The filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to obtain amidite 6a (6.22 g, 6.90 mmol) as a colorless amorphous solid (yield 91%). ESI-MS: Calculated value: 902.40 [M+H] + , Measured value: 902.5 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.47 (s, 0.35H), 8.47 (s, 0.65H), 8.14 (s, 0.35H), 8.09 (s, 0.65H), 7.44-7.39 (m, 4H), 7.33-7.21 (m, 8H), 7.16-7.12 (m, 2H), 6.93-6.78 (m, 4H), 6.12 (d, J = 5.5, 0.65H), 6.10 (d, J = 5.0, 0.35H), 4.66-4.53 (m, 2H), 4.41-4.38 (m, 0.35H), 4.34-4.32 (m, 0.65H), 3.97-3.78 (m, 10H), 3.70-3.44 (m, 7H), 3.36-3.30 (m, 1H), 2.64 (t, J = 6.2 Hz, 1.3H), 2.38 (t, J = 6.4 Hz, 0.70H), 1.22-1.17 (m, 8H), 1.06 (d, J = 6.9 Hz, 4H). 31 P-NMR(CDCl3, 162 MHz) δ: 150.70, 150.94.

[0189] Compound 6b, which is a starting material for polynucleotides, was synthesized according to the following scheme.

[0190] Step 1 Synthesis of Compound 2b N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)benzamide Di-t-butylsilylbis(trifluoromethanesulfonate) (70.8 g, 161 mmol) was slowly added to a solution of commercially available compound 1b (30.0 g, 80.4 mmol) in DMF (300 mL) under ice-cooling. After stirring for 1 hour under ice-cooling, the reaction mixture was added to saturated aqueous sodium bicarbonate and extracted twice with a heptane / ethyl acetate mixed solvent. The organic layer was washed twice with water and once with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the concentrated residue was purified by slurry with heptane / ethyl acetate = 9 / 1 to obtain compound 2b (38.7 g, 75.4 mmol) as a colorless solid (yield 94%). ESI-MS: calculated value: 514.23 [M+H] + , Measured value: 514.5 [M+H] + 1 H-NMR (DMSO-d6, 400 MHz) δ: 11.27 (s, 1H), 8.74 (s, 1H), 8.65 (s, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 7.5 Hz, 2H), 6.45 (d, J = 23 Hz, 1H), 5.71 (dd, J = 54.5, 4.1 Hz, 1H), 5.03 (m, 1H), 4.44 (q, J = 3.7 Hz, 1H), 4.09 (m, 2H), 1.11 (s, 9H), 1.02 (s, 9H).

[0191] Step 2 Synthesis of Compound 3b N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 2b (10.0 g, 19.5 mmol) was dissolved in dichloromethane (50 mL), and tetrabutylammonium bromide (9.41 g, 29.2 mmol) and 1 M aqueous sodium hydroxide solution (50 mL) were added. Methyl iodide (1.83 mL, 29.2 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 1 hour. After confirming the disappearance of the raw materials, the reaction mixture was quenched by adding it to ice-cooled water / chloroform = 1 / 1. The organic layer was washed twice with water, then dehydrated over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10 → 50 / 50) to give compound 3b (6.86 g, 12.8 mmol) as a colorless amorphous solid (65% yield). ESI-MS: Calculated value: 528.24 [M+H] + , Measured value: 538.6 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.54 (s, 1H), 7.94 (s, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 7.21 (t, J = 7.6 Hz, 2H), 6.10 (d, J = 22.0 Hz, 1H), 5.46 (dd, J = 54.5, 4.1 Hz, 1H), 4.86 (ddd, J = 27.2, 9.8, 4.1 Hz, 1H), 4.47 (dd, J = 9.2, 5.0 Hz, 1H), 4.14 (m, 1H), 4.03 (t, J = 9.8 Hz, 1H), 3.78 (s, 3H), 1.11 (s, 9H), 1.05 (s, 9H).

[0192] Step 3 Synthesis of Compound 4b N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 3b (6.67 g, 12.6 mmol) was dissolved in tetrahydrofuran (66 mL) and cooled in an ice bath. Triethylamine (8.81 ml, 63.2 mmol) and triethylamine trihydrofluoride (2.05 ml, 12.6 mmol) were added, and the mixture was stirred for 1 hour and 5 minutes while cooling in an ice bath. After confirming the disappearance of the raw materials, the reaction was quenched by adding triethylamine (10.6 ml, 76.0 mmol). The mixture was diluted with chloroform and then concentrated. The concentrated residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 90 / 10) to give compound 4b (4.98 g, 12.9 mmol) as a colorless amorphous solid. (Yield quant.) ESI-MS: Calculated value: 388.14 [M+H] + , Measured value: 388.4 [M+H] + 1 H-NMR (DMSO-d6, 400 MHz) δ: 8.70 (s, 1H), 8.58 (s, 1H), 7.30 (m, 5H), 6.31 (dd J = 16.9, 2.3 Hz, 1H), 5.75 (d, J = 6.4 Hz, 1H), 5.41 (m, 1H), 5.15 (t, J = 5.3 Hz, 1H), 4.46 (m, 1H), 3.98 (m, 1H), 3.75 (dq, J = 12.4, 2.6 Hz, 1H), 3.67 (s, 3H), 3.61 - 3.56 (m, 1H).

[0193] Step 4 Synthesis of Compound 5b N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-methylbenzamide Compound 4b (4.93 g, 12.7 mmol) was dissolved in pyridine (49 mL) and stirred in an ice bath. 4,4'-Dimethoxytrityl chloride (6.47 g, 29.2 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour and 20 minutes. After confirming the disappearance of the raw materials, the reaction mixture was quenched by adding it to ice-cold aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give compound 5b (8.34 g, 12.1 mmol) as a colorless amorphous solid (yield 95%). ESI-MS: calculated value: 690.27 [M+H] + , Measured value: 690.7 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.51 (s, 1H), 8.10 (s, 1H), 7.43 (dd, J = 8.2, 1.4 Hz, 2H), 7.37 (dd, 8.2, 1.4 Hz, 2H), 7.28-7.20 (m, 8H), 7.12 (t, J = 7.5 Hz, 2H), 6.79 (d, J = 8.7 Hz), 6.23 (dd, J = 17.1, 2.5 Hz, 1H), 5.58 (dq, J = 52.9, 2.3 Hz, 1H), 4.78 (m, 1H), 4.19 (m, 1H), 3.78 (s, 6H), 3.47 (ddd, J = 57.9, 10.6, 3.5 Hz, 2H), 2.44 (dd, J = 7.5, 2.5 Hz, 1H).

[0194] Step 5: Synthesis of amidite 6b (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro-5-(6-(N-methylbenzamido)-9H-purin-9-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5b (10.0 g, 14.6 mmol) was dissolved in dichloromethane (80 mL), and diisopropylethylamine (5.08 mL, 29.1 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (4.18 g, 21.8 mmol) dissolved in dichloromethane (15 mL) was added dropwise over 5 minutes. The mixture was then stirred for 1 hour while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Ethyl acetate was added for extraction. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give amidite 6b (12.1 g, 13.6 mmol) as a colorless amorphous solid (yield 93%). ESI-MS: Calculated value: 890.38 [M+H] + , Measured value: 890.8 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.53 (s, 0.49H), 8.50 (s, 0.51H), 8.14 (s, 1H), 7.43-7.39 (m, 2H), 7.37-7.33 (m, 2H), 7.27-7.20 (m, 8H), 7.09-7.04 (m, 2H), 6.77 (t, J = 9.1 Hz, 4H), 6.28-6.19 (m, 1H), 5.74 (dq, J = 18.5, 2.2 Hz, 0.50H), 5.61 (dq, J = 19.2, 2.3 Hz, 0.50H), 5.10-5.00 (m, 0.47H), 4.94-4.85 (m, 0.53H), 4.31 (m, 1H), 3.97-3.82 (m, 1H), 3.79 (s, 3H), 3.79 (s, 3H), 3.63-3.53 (m, 4H), 3.31-3.27 (m, 1H), 2.59 (t, J = 6.2 Hz, 1H), 2.41 (t, J = 6.4 Hz, 1H), 1.20-1.15 (m, 9H), 1.04 (d, J = 6.4 Hz, 3H). 31 P-NMR(CDCl3, 162 MHz) δ: 151.97, 151.92, 151.19, 151.11.

[0195] Compound 6c, which is a starting material for polynucleotides, was synthesized according to the following scheme.

[0196] Step 1 Synthesis of Compound 3c N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-methoxytetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 2a (11.7 g, 22.3 mmol) was dissolved in dichloromethane (58.5 mL), and tetrabutylammonium bromide (10.8 g, 33.4 mmol) and 1 M aqueous sodium hydroxide solution (58.5 mL) were added. Ethyl iodide (10.8 mL, 134 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 2 hours. After confirming the disappearance of the raw materials, the reaction mixture was quenched by adding it to ice-cold water / chloroform = 1 / 1. The organic layer was washed twice with water and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by slurry with toluene, and the filtrate was concentrated again. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10 → 70 / 30) to obtain compound 3c (6.14 g, 11.1 mmol) as a colorless amorphous solid (49% yield). ESI-MS: Calculated value: 554.28 [M+H] + , Measured value: 554.6 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.91 (s, 1H), 7.47-7.45 (m, 2H), 7.31-7.27 (m, 1H), 7.19 (t, J = 7.5 Hz, 2H), 5.94 (s, 1H), 4.61 (dd, J = 9.6, 4.6 Hz, 1H), 4.46 (dd, J = 9.1, 5.0 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 4.22 (d, J = 4.6 Hz, 1H), 4.16 (ddd, J = 10.1, 4.9, 4.8 Hz, 1H), 4.00 (dd, J = 10.5, 9.6 Hz, 1H), 3.67 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H), 1.09 (s, 9H), 1.05 (s, 9H).

[0197] Step 2 Synthesis of Compound 4c N-ethyl-N-(9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide Compound 3c (6.14 g, 11.1 mmol) was dissolved in tetrahydrofuran (61.4 mL) and cooled in an ice bath. Triethylamine (7.73 mL, 55.4 mmol) and triethylamine trihydrofluoride (1.81 mL, 11.1 mmol) were added, and the mixture was stirred for 2 hours while cooling in an ice bath. After confirming the disappearance of the raw materials, the reaction was quenched by adding triethylamine (10 mL, 76.0 mmol). The mixture was diluted with chloroform and then concentrated. The concentrated residue was purified by silica gel column chromatography (chloroform / methanol = 100 / 0 → 90 / 10) to give compound 4c (4.60 g, 11.1 mmol) as a colorless amorphous solid. (Yield quant.) ESI-MS: Calculated value: 414.18 [M+H] + , Measured value: 414.3 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.56 (s, 1H), 7.92 (s, 1H), 7.50-7.46 (m, 2H), 7.32-7.28 (m, 1H), 7.20 (t, J = 7.5 Hz, 2H), 5.89 (dd, J = 11.6, 2.1 Hz, 1H), 5.85 (d, J = 7.3 Hz, 1H), 4.62 (dd, J = 7.3, 4.6 Hz, 1H), 4.57-4.56 (m, 1H), 4.45-4.39 (m, 2H), 4.36-4.34 (m, 1H), 3.96 (dt, J = 12.9, 1.9, 1H), 3.80-3.73 (m, 1H), 3.29 (s, 3H), 2.70 (d, J = 1.4 Hz, 1H), 1.37 (t, J = 7.1 Hz, 3H).

[0198] Step 3 Synthesis of Compound 5c N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 4c (4.58 g, 11.1 mmol) was dissolved in pyridine (46 mL) and stirred in an ice bath. 4,4'-Dimethoxytrityl chloride (5.63 g, 16.6 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 2 hours. After confirming the disappearance of the raw materials, the reaction mixture was quenched by adding it to ice-cold aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to give compound 5c (7.55 g, 10.6 mmol) as a colorless amorphous solid (95% yield). ESI-MS: calculated value: 716.31 [M+H] + , Measured value: 716.2 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.51 (s, 1H), 8.11 (s, 1H), 7.43-7.40 (m, 4H), 7.32-7.22 (m, 8H), 7.13 (t, J = 7.5 Hz, 2H), 6.81 (dd, J = 9.1, 1.4 Hz, 4H), 6.14 (d, J = 3.7 Hz, 1H), 4.47 (dd, J = 5.7, 5.6 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H), 4.34 (dd, J = 8.4, 4.1 Hz, 1H), 4.21-4.17 (m, 1H), 3.79 (s, 6H), 3.53 (s, 3H), 3.50 (dd, J = 10.5, 3.2 Hz, 1H), 3.39 (dd, J = 10.7, 4.3 Hz, 1H), 2.64 (d, J = 6.4 Hz, 1H), 1.34 (t, J = 7.1 Hz, 3H).

[0199] Step 4: Synthesis of amidite 6c (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-ethylbenzamido)-9H-purin-9-yl)-4-methoxytetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5c (8.83 g, 12.3 mmol) was dissolved in dichloromethane (74 mL), and diisopropylethylamine (4.31 mL, 24.7 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (4.40 g, 18.6 mmol) dissolved in anhydrous dichloromethane (14 mL) was added dropwise over 9 minutes. The mixture was then stirred for 1 hour while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Ethyl acetate was added for extraction. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 70 / 30 → 50 / 50) to obtain amidite 6c (10.4 g, 11.3 mmol) as a colorless amorphous substance (yield 92%). ESI-MS: Calculated value: 916.42 [M+H] + , Measured value: 917.3 [M+H] + 1H-NMR (CDCl3, 400 MHz) δ: 8.49 (s, 0.37H), 8.48 (s, 0.63H), 8.11 (s, 0.34H), 8.05 (s, 0.66H), 7.43-7.38 (m, 4H), 7.32-7.21 (m, 8H), 7.11 (t, J = 7.8 Hz, 2H), 6.80 (m, 4H), 6.10 (m, 1H), 4.64-4.52 (m, 2H), 4.43-4.32 (m, 3H), 3.94-3.83 (m, 1H), 3.79-3.78 (m, 6H), 3.67 -3.46 (m, 4H), 3.35-3.29 (m, 1H), 2.64 (t, J = 6.4 Hz, 1.3H), 2.37 (t, J = 6.4 Hz, 0.70H), 1.33 (t, J = 7.1 Hz, 3H), 1.18 (m, 8H), 1.06 (d, J = 6.9 Hz, 4H). 31 P-NMR(CDCl3, 162 MHz) δ: 151.67, 150.92.

[0200] Compound 6d, which is a starting material for polynucleotides, was synthesized according to the following scheme.

[0201] Step 1 Synthesis of Compound 3d N-(9-((4aR,6R,7R,7aS)-2,2-di-tert-butyl-7-fluorotetrahydro-4H-furo[3,2-d][1,3,2]dioxasilin-6-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 2b (1.00 g, 1.95 mmol) was dissolved in dichloromethane (5.0 mL), and tetrabutylammonium bromide (0.942 g, 2.92 mmol) and 1 M aqueous sodium hydroxide solution (5.0 mL) were added. Methyl iodide (0.942 mL, 11.7 mmol) was slowly added dropwise. The mixture was then stirred at room temperature for 2 hours. After confirming the disappearance of the raw materials, the reaction mixture was quenched by adding it to ice-cooled water / chloroform = 1 / 1. The organic layer was washed twice with water, then dehydrated over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate = 80 / 20 → 70 / 30) to give compound 3d (629 mg, 1.16 mmol) as a colorless amorphous solid (60% yield). ESI-MS: Calculated value: 542.26 [M+H] + , Measured value: 542.6 [M+H] + 1 H-NMR (CDCl3, 400 MHz) δ: 8.55 (s, 1H), 7.91 (s, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.30 (t, J = 7.1 Hz, 1H), 7.19 (t, J = 7.8 hz, 2H), 6.09 (d, J = 22.4 Hz, 1H), 5.45 (dd, J = 54.1, 3.9 Hz, 1H), 4.86 (ddd, J = 27.2, 9.8, 4.1 Hz, 1H), 4.48 (dd, J = 9.1, 5.0, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.04 (t, J = 9.8 Hz, 1H), 1.34 (t, J = 7.1 Hz, 3H), 1.11 (s, 9H), 1.05 (s, 9H).

[0202] Process 2: Synthesis of N-ethyl-N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide. Compound 4c is the same process as Compound 4d. ESI-MS: Calculated value: 401.40 [M+H] + , measured value: 402.1 [M+H] +

[0203] Process 3 Synthesis of compound 5d N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)-N-ethylbenzamide Compound 5c is the same engineering as compound 5d. ESI-MS: Calculated: 738.25 [M+Cl] - , measured value: 738.7 [M+Cl] - 1 H-NMR (CDCl3, 400 MHz) δ: 8.52 (s, 1H), 8.07 (s, 1H), 7.43-7.41 (m, 2H), 7.38-7.35 (m, 2H), 7.30-7.20 (m, 8H), 7.10 (t, J = 7.8 Hz, 2H), 6.79 (d, J = 8.2 Hz, 4H), 6.22 (dd, J = 17.4, 2.3 Hz, 1H), 5.58 (ddd, J = 53.0, 2.4, 1.2 Hz, 1H), 4.93-4.74 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 4.19-4.16 (m, 1H), 3.78 (s, 6H), 3.54 (dd, J = 11.0, 3.2 Hz, 1H), 3.40 (dd, J = 10.5, 3.1 Hz, 1H), 2.23 (dd, J = 6.9, 2.3 Hz, 1H), 1.33 (t, J = 7.1 Hz, 3H).

[0204] Step 4: Synthesis of amidite 6d (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(6-(N-ethylbenzamido)-9H-purin-9-yl)-4-fluorotetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite Compound 5d (1.93 g, 2.74 mmol) was dissolved in dichloromethane (16 mL), and diisopropylethylamine (0.958 mL, 5.48 mmol) was added. The mixture was cooled in an ice bath. 2-Cyanoethyldiisopropylchlorophosphoramidite (970 mg, 4.11 mmol) dissolved in dehydrated dichloromethane (3.8 mL) was added dropwise. The mixture was then stirred for 1 hour while warming to room temperature. After confirming the disappearance of the starting materials, the reaction mixture was quenched by adding it to ice-cold saturated aqueous sodium bicarbonate. Ethyl acetate was added for extraction. The organic layer was washed with saturated brine and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated. The concentrated residue was purified by silica gel column chromatography (heptane / ethyl acetate (containing 1% triethylamine) = 90 / 10 → 60 / 40) to obtain amidite 6d (2.29 g, 2.53 mmol) as a colorless amorphous solid (yield 92%). ESI-MS: calculated value: 938.36 [M+Cl] - , Measured value: 938.7 [M+Cl] - 1H-NMR (CDCl3, 400 MHz) δ: 8.53 (s, 0.5H), 8.51 (s, 0.5H), 8.11 (s, 1H), 7.41-7.33 (m, 4H), 7.27-7.16 (m, 8H), 7.03 (t, J = 7.8 Hz, 2H), 6.79-6.75 (m, 4H), 6.27-6.18 (m, 1H), 5.78-5.58 (m, 1H), 5.10-4.85 (m, 1H), 4.42-4.38 (m, 2H), 4.31-4.30 (m, 1H), 3.96-3.72 (m, 7H), 3.68-3.51 (m, 4H), 3.29-3.27 (m, 1H), 2.59 (t, J = 6.2 Hz, 1H), 2.40 (t, J = 6.4 Hz, 1H), 1.33-1.31 (m, 3H), 1.19-1.15 (m, 9H), 1.04 (d, J = 6.9 Hz, 3H). 31 P-NMR(CDCl3, 162 MHz) δ: 151.94, 151.89, 151.20, 151.11.

[0205] RNA oligonucleotides were synthesized using 2'-TOM (triisopropylsilyloxymethyl)-protected β-cyanoethyl phosphoramidites (DMT-2'-O-TOM-rA(Ac), DMT-2'-O-TOM-rG(Ac), DMT-2'-O-TOM-rC(Ac), and DMT-2'-O-TOM-rU) (Glen Research and ChemGenes, respectively). DNA oligonucleotides were synthesized using β-cyanoethyl phosphoramidites (DMT-dA(Bz), DMT-dG(iBu), DMT-dC(Ac), and DMT-T). Each phosphoramidite monomer was prepared in 0.05 mol / L acetonitrile and synthesized using 0.2 μmol or 0.8 μmol of solid support on a DNA / RNA solid-phase synthesizer (NTS M-2-MX, Nippon Techno Service Co., Ltd.). DNA oligonucleotides were synthesized using CPG 1000A (dA-CPG, dG-CPG, Ac-dC-CPG, dT-CPG) (Glen Research) as the solid support, with a condensation time of 2 minutes. RNA with a phosphate group at the 5' end (5'-monophosphate RNA) was synthesized using Universal UnyLinker Support 2000A (ChemGenes) as the solid support, with a condensation time of 15 minutes for the first base and 3 minutes for each subsequent base. Phosphorylation of the hydroxyl group at the 5' end was performed using a chemical phosphorylation reagent (0.05 mol / L acetonitrile solution) (Glen Research or ChemGenes). Solid-phase synthesis of RNA oligonucleotides with a 3'-aminoguanosine monomer at the 3' end was performed using compound 15. The condensation time for the first base was 15 minutes, with 3 minutes for each subsequent base.

[0206] The following reagents were used in the solid-phase synthesis system. The dimethoxytrityl group at the 5'-terminus hydroxyl group was removed using a commercially available deblocking reagent (Deblocking Solution-1, 3 w / v% trichloroacetic acid / dichloromethane solution) (Wako Pure Chemical Industries, Ltd.) and allowed to react for 10 seconds. A commercially available activator solution (Activator Solution 3) (Wako Pure Chemical Industries, Ltd.) was used as the phosphoramidite activator. Unreacted 5'-terminus hydroxyl groups were capped using commercially available capping solutions (Cap A Solution-2 and Cap B Solution-2) (Wako Pure Chemical Industries, Ltd.) and allowed to react for 10 seconds. The oxidizing agent used to produce the phosphate ester was a solution containing pyridine, THF, water, and iodine (Oxidizer, 0.01 M iodine, 29.2% water, 6.3% pyridine, 64.5% acetonitrile, Honeywell), and allowed to react for 10 seconds. After solid-phase synthesis, the dimethoxytrityl group at the 5'-terminus of the RNA oligonucleotide was deprotected on the solid support. The synthesized DNA and RNA oligonucleotides were deprotected using standard methods (concentrated aqueous ammonia, 55°C, 12 hours). DNA oligonucleotides were purified using a cartridge column (MicroPure II Column, LGC Biosearch Technology) according to the manufacturer's protocol. The solution obtained by deprotection was concentrated to dryness using a centrifugal evaporator. The TOM protecting group at the 2'-hydroxyl group of the RNA oligonucleotide was then removed using 1 mL of tetrabutylammonium fluoride (1 M tetrahydrofuran solution) (50°C for 10 minutes, followed by 12 hours at room temperature, or 50°C for 10 minutes, followed by 6 hours at 35°C). Tris-HCl buffer (1 M, pH 7.4) (1 mL) was added to the solution, mixed, and the tetrahydrofuran was removed by concentration using a centrifugal evaporator. The resulting solution was treated using a gel filtration column (NAP-25, GE Healthcare) equilibrated with ultrapure water according to the manufacturer's protocol.The resulting fraction containing the RNA oligonucleotide was concentrated using a centrifugal evaporator and then purified using denaturing polyacrylamide gel (hereinafter referred to as dPAGE).

[0207] (Purification of RNA fragments using dPAGE) A gel was prepared by adding an aqueous solution of ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED) as a polymerization agent to an acrylamide gel solution (containing 7 M urea as a denaturant) and allowing it to solidify (room temperature for 6–12 hours). The RNA sample was mixed with gel loading buffer (80% formamide, TBE), heated to 90°C for 3 minutes, and then loaded onto the gel. After electrophoresis, RNA bands were detected by UV light (254 nm) and excised from the gel with a razor blade. The excised gel fragments were finely crushed and extracted with ultrapure water (shaking at room temperature for 6–12 hours). The RNA extract was desalted and concentrated using an Amicon Ultra 10K (Millipore) and ethanol precipitated with 0.3 M sodium acetate (pH 5.2) / 70% ethanol to obtain an RNA pellet. The RNA pellet was rinsed with 80% ethanol and air-dried at room temperature for 1 hour. The resulting RNA pellet was dissolved in ultrapure water and diluted to the appropriate concentration. The absorbance at 260 nm was measured using UV-visible spectrophotometry (NanoDrop, Thermo Scientific). The concentration was determined from the molar extinction coefficient of each RNA sequence (the following molar extinction coefficients for each base were used: A = 15300, G = 11800, C = 7400, T = 9300, U = 9900). The structure of the purified oligonucleotides was determined by mass spectrometry using MALDI-TOF MS (Ultraflex III, Bruker Daltonics) (matrix: 3-hydroxypicolinic acid) or by denaturing polyacrylamide gel electrophoresis.

[0208] (Analysis of Chemical Ligation Reactions Using dPAGE) For analysis of chemical ligation reactions, the reaction solution was appropriately diluted with ultrapure water and used as a sample. The diluted sample was mixed with gel loading buffer (80% formamide / TBE), heated at 90°C for 3 minutes, and then loaded onto a gel. After electrophoresis, the RNA bands were detected by gel staining (room temperature, 15 minutes) using SYBR® Green II Nucleic Acid Stain (Lonza) diluted 10,000-fold with ultrapure water (equipment used: ChemiDoc, BIORAD). The yield of the chemical ligation reaction was calculated by comparing the band intensity of the RNA ligation product with the isolated and purified ligation product as a reference material by dPAGE.

[0209] (Purification of chemical ligation products using dPAGE) The RNA ligation products obtained by the chemical ligation reaction were recovered as RNA pellets from the reaction solution by ethanol precipitation (0.3 M sodium acetate (pH 5.2) / 70% ethanol), and then purified by dPAGE.

[0210] In Tables 1 to 27-5 below, each nucleotide N (uppercase) represents RNA, each nucleotide n (lowercase) represents DNA, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-F modified RNA, N(L) represents LNA, and N(MOE) represents 2'-O-methoxyethyl modified RNA. Am6 indicates that the base moiety is N6-methyladenine. DNA may also be written as dN. p indicates that the 3' or 5' end is phosphorylated. ^ indicates that the phosphate group connecting the sugar moieties is phosphorothioate. The underlined "AUG" represents the start codon, and the underlined "UGA" or "TGA" represents the stop codon.

[0211] Table 1 below shows the sequence information of the compounds (polynucleotides) used in Examples 1 and 2.

[0212] Table 1:

[0213] Example 1 (Enzymatic Ligation) Three batches of ultrapure aqueous solutions (200 μL, final nucleic acid concentration: 50 μM) containing RNA fragment E1-1 (10 nmol), 5'-phosphate RNA fragment E1-2 (10 nmol), and template DNA1 (10 nmol) obtained by solid phase synthesis were prepared. 100 μL of T4 RNA Ligase 2 Reaction Buffer (10X) (New England BioLabs) and 440 μL of ultrapure water were added to each prepared solution, and the mixture was heated at 90°C for 5 minutes and then returned to room temperature over 30 minutes. A 60% aqueous PEG6000 solution was added to each solution to a final concentration of 15%. 10 μL of T4 RNA Ligase 2 (New England BioLabs) (10 units / μL) was added to each solution, mixed, and then placed on a temperature-controlled heat block (37 The reaction mixture was incubated at 37°C for 16 hours. An equal volume of chloroform was added to the reaction mixture, mixed by vortexing, and centrifuged. The upper layer was separated and subjected to alcohol precipitation (0.3 M sodium acetate aqueous solution (pH 5.2) / 70% ethanol) to obtain an RNA pellet. The RNA obtained from each batch was pooled and purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product E1 (9.7 nmol, 32% yield).

[0214] Example 2 (Ligation by Chemical Reaction) A nucleic acid mixture containing 10 nmol of the 3'-phosphate RNA fragment E2-1 obtained by solid-phase synthesis, 10 nmol of the RNA fragment E2-2 obtained as sequence 7, and 20 nmol of template DNA1 was mixed with 1M aqueous sodium chloride and ultrapure water to prepare a 100 mM aqueous sodium chloride solution (180 μL). The prepared solution was heated at 90°C for 5 minutes and then allowed to return to room temperature over 30 minutes. A 100 mM aqueous zinc(II) chloride solution was added to this solution to a final concentration of 5 mM. A 100 mM 1H-imidazole-1-carbonitrile (Apollo Scientific) / DMSO solution was added to this solution to a final concentration of 5 mM, mixed, and then placed on a temperature-controlled heat block (30°C, 20 hours). The reaction mixture was purified using NAP-10 Columns (Cytiva) after adding 50 μL of 2 M triethylamine acetate (TEAA). The fraction with an absorption wavelength of A260 was concentrated by centrifugation or lyophilized. The crude RNA obtained by this purification step was pooled and purified on a 7.5% denaturing polyacrylamide gel to obtain the RNA ligation product E2 (2.5 nmol, 25% yield).

[0215] Example 3 In the following Tables 2-1 to 2-60, rSpacer, dSpacer, Pyrrolidine, Ethynyl-dSpacer, C3, C2 and Spacer9 are spacer modifications in place of the sugar moiety of the nucleotide and have the structures shown below.

[0216] Tables 2-1 to 2-60 below show the sequence information and synthesis method of the compounds (polynucleotides) used in Example 3. Tables 3-1 to 3-14 below show the yield (%) and MS (measured values) of the compounds (polynucleotides) of Example 3. The MS (measured values) were measured using an Agilent Technologies LC (1260 Infinity II) / MSD XT (G6135B). Tables 2-1 to 2-60:

[0217]

[0218] Table 3-1 to Table 3-14:

[0219]

[0220] The sequence information of the compounds (polynucleotides) used in Example 4 is shown in Tables 4-1 to 4-3 below.

[0221] Table 4-1 to Table 4-3:

[0222]

[0223]

[0224] Example 4 (Enzymatic Ligation of Three Fragments) RNA fragments E217-1, E217-2, and E217-3 obtained by solid-phase synthesis, template DNA2, and template DNA3 were simultaneously used, with the remainder being the same as in Example 1, to obtain the RNA ligation product E217 (8.9 nmol, yield 45%). RNA fragments E218-1, E218-2, and E218-3 obtained by solid-phase synthesis, template DNA2, and template DNA3 were simultaneously used, with the remainder being the same as in Example 1, to obtain the RNA ligation product E218 (2.6 nmol, yield 13%). RNA fragments E219-1, E219-2, and E219-3 obtained by solid-phase synthesis, template DNA2, and template DNA3 were simultaneously used, with the remainder being the same as in Example 1, to obtain the RNA ligation product E219 (1.4 nmol, yield 7%).

[0225] Test Example 1 (Translation reaction test using HeLa cell line lysate as an mRNA sample) For each of the mRNAs listed in Tables 5-1 to 5-25 below, the translation activity in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). First, each mRNA was diluted with THE RNA storage solution (Thermo Fisher Scientific, catalog number AM7001) to a final concentration of 0.3 μM, and 1 μL of each was dispensed into a 96-well PCR plate (AS ONE). Next, a master mix was prepared by mixing 5.0 μL of Hela Lysate per reaction, 1.0 μL of Accessory Proteins per reaction, 2.0 μL of Reaction Mix per reaction, 0.2 μL of RNase Inhibitor Murine (New England BioLabs, catalog number M0314) per reaction, and 0.8 μL of purified water per reaction. 9 μL of this mixture was dispensed into the PCR plate containing the mRNA samples. After addition and mixing, the plate was left to stand at 37°C for 45 minutes, allowing the translation reaction to proceed. The translation products in the reaction solution after the translation reaction were detected by the sandwich ELISA method described below. First, 6*His, His-Tag antibody (Proteintech, catalog no. 66005-1-Ig) was diluted to 3 μg / mL in 0.1M carbonate buffer (pH 9.4) and dispensed at 50 μL per well into a 96-well ELISA plate (Nunc). The plate was then left to stand overnight at 4°C to prepare an antibody-immobilized plate. The plate was then washed with Tris-Buffered Saline with Tween 20 (Santa Cruz, catalog no. sc-24953) diluted 1x with purified water (hereafter referred to as the washing solution). Then, 200 μL of a washing solution (hereafter referred to as the blocking solution) prepared by diluting bovine serum albumin (Wako Pure Chemical Industries, catalog no. 017-22231) to a final concentration of 3% was dispensed per well and left to stand at room temperature for 1 hour.After washing the plate with washing solution, 50 μL of the translation reaction solution diluted with blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. The following translation product polypeptide preparations (Cosmobio) were also diluted to various concentrations in blocking solution and dispensed into the plate. After washing the plate with washing solution, 50 μL of Monoclonal Anti-FLAG M2-Peroxidase (HRP) Ab produced in mouse (Sigma, catalog antibody A8592-1MG) diluted 1:10,000 in blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing the plate with washing solution, 50 μL of 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific, catalog number 34028) was dispensed into each well and allowed to stand at room temperature for several minutes. The reaction was then stopped by dispensing 50 μL of 0.5 M sulfuric acid (Wako Pure Chemical Industries, Ltd.) per well, and the absorbance was measured using a spectrophotometer (Bio-Rad) at a measurement wavelength of 450 nm and a reference wavelength of 570 nm. The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and the relative amount of translation product when the amount of unglycosylated compound E3 was set to 1, are shown in Table 5 below. Translation product polypeptide preparation: NH2-MDYKDDDDKIIDYKDDDDKGGDYKDDDDKHHHHHH-COOH (SEQ ID NO: 430).

[0226] Table 5-1 to Table 5-25:

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] As is clear from the test results shown in Tables 5-1 to 5-25 above, after each sugar-modified mRNA was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cell.

[0253] Test Example 2 (In vitro translation reaction test of mRNA samples using HeLa cell line) The in vitro translation activity of each mRNA listed in Tables 6-1 to 6-9 was evaluated using the HeLa cell line. First, HeLa cells suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum were seeded onto a 96-well culture plate for adherent cells at 10,000 cells / 100 μL per well and cultured overnight at 37°C and 5% CO2. After overnight cell culture, the culture supernatant was removed from the cells, and 40 μL of RPMI medium containing 10% fetal bovine serum was added per well. Each mRNA was then diluted with Optimem (Thermo Fisher Scientific, catalog number: LMRNA008) at a final concentration of 0.3% to achieve final mRNA concentrations of 3 nM, 10 nM, and 30 nM, and mixed. 10 μL of the mixture was added per well to each culture plate, and the plates were cultured at 37°C and 5% CO2 for 5 hours. After 5 hours of culture, the culture supernatant was removed from the cells and washed once with ice-cold D-PBS(-) (Nacalai Tesque). Then, 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts, Nacalai Tesque) was added per well, and the cells were lysed by vigorous shaking for 30 seconds. The translation product in the resulting cell lysate was analyzed using the same method as the sandwich ELISA described in Test Example 1. The translation product concentration (nM) in each translation reaction solution was quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation. The results are shown in Table 6.

[0254] Table 6-1 to Table 6-9:

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264] As is clear from the test results shown in Tables 6-1 to 6-9 above, after addition of each sugar-modified mRNA to HeLa cells, it produced the polypeptide encoded by the gene sequence, and the translation amount was superior to that of mRNA without sugar modification.

[0265] Test Example 3 (In vitro translation reaction test using HeLa cell line with mRNA samples) The sustainability of in vitro translation activity of each mRNA listed in Tables 7-1 to 7-4 was evaluated using HeLa cell line. Cell culture and mRNA introduction were performed in the same manner as in Test Example 2, except that the final concentration of each mRNA was adjusted to 30 nM. After 4 hours of culture with each mRNA, the culture supernatant was removed from the cells, and 50 μL of RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum was added per well. The cells were then cultured at 37°C and 5% CO2. Five, eight, and 24 hours after the addition of each mRNA, the culture supernatant was removed from the cultured cells, and the cells were lysed in the same manner as in Test Example 2. The translation products in the resulting cell lysates were analyzed using the same sandwich ELISA method as in Test Example 1. The translation product concentrations (nM) in each translation reaction solution, quantified using a calibration curve constructed based on the absorbance of a polypeptide preparation, are listed in Table 7.

[0266] Table 7-1 to Table 7-4:

[0267]

[0268]

[0269]

[0270]

[0271] As is clear from the test results shown in Tables 7-1 to 7-4 above, after addition of each sugar-modified mRNA to HeLa cells, it produced the polypeptide encoded by the gene sequence, and the translation amount was superior to that of mRNA without sugar modification.

[0272] Test Example 4 (In vitro translation reaction test of mRNA samples using HeLa cell line) The translation activity of each mRNA listed in Tables 8-1 to 8-8 below was evaluated using HeLa cell line. First, each mRNA was diluted to 19 μM with THE RNA Storage Solution (Thermo Fisher Scientific, catalog number AM7000). The Hela cell line was suspended in Opti-MEM I Reduced Serum Media (ThermoFisher Scientific, Catalog No. 31985070) containing a final concentration of 1% bovine serum albumin (Wako Pure Chemical Industries, Catalog No. 017-22231), centrifuged at 90 x g for 10 minutes at room temperature, and the supernatant was carefully removed. The cells were then suspended at 200,000 cells / 19 μL in a mixture of 1% SE Cell Line Nucleofector Solution and Supplement 1 included in the SE Cell Line 96-well Nucleofector Kit (Lonza, Catalog No. V4SC-1096). The prepared mRNA solution and Hela cell suspension were mixed at a volume ratio of 1:19, and then lysed in the Nucleofector. TMElectroporation was performed using a 96-well Shuttle system (Lonza) under pulse conditions of FF-150. Ten minutes after electroporation, cells were suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum and seeded into a 96-well culture plate for adherent cells at 50,000 cells / 145 μL per well. Cultures were incubated at 37°C and 5% CO2. After 3, 8, and 24 hours of incubation, the culture supernatant was removed and the cells were washed once with ice-cold D-PBS(-) (Nacalai Tesque). Then, 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts, Nacalai Tesque) was added per well and vigorously shaken for 30 seconds to lyse the cells. The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 1. As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in Table 8 below.

[0273] Table 8-1 to Table 8-8:

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] As is clear from the test results shown in Tables 8-1 to 8-8 above, sugar-modified mRNA produced the polypeptide encoded by the gene sequence after electroporation into HeLa cells, and its activity was superior to that of mRNA without sugar modification. In particular, the test results shown in Table 8-5 revealed that E26, E27, and E28, in which 65% or more of the nucleotides constituting the polyA tail were sugar-modified, showed superior translation activity to E29, in which 50% of the nucleotides constituting the polyA tail were sugar-modified.

[0283] Test Example 5 (In vitro translation reaction test of mRNA samples using human aortic smooth muscle cells) For each mRNA listed in Tables 9-1 to 9-3, the in vitro translation activity was evaluated using human aortic smooth muscle cells (Human Aortic Smooth Muscle Cells, Lonza, CC-2571; hereafter, sometimes referred to as hAoSMCs). First, hAoSMCs were cultured in SmGM-2 BulletKit medium (Lonza, CC-3182) according to the manufacturer's instructions. The hAoSMCs were suspended in SmGM-2 BulletKit medium and seeded into a 96-well culture plate at 10,000 cells / 100 μL per well. The cells were then cultured overnight at 37°C in 5% CO2. After overnight cell culture, the culture supernatant was removed from the cells, and 40 μL of SmGM-2 BulletKit medium was added per well. Each mRNA was diluted with Optimem (Thermo Fisher Scientific, catalog number: LMRNA008) at a final concentration of 0.3% to achieve final mRNA concentrations of 3 nM, 10 nM, and 30 nM. The mixture was then mixed with Optimem (Thermo Fisher Scientific, catalog number: 31985-070). 10 μL of the mixture was added per well to each culture plate, and the plates were cultured at 37°C and 5% CO2 for 5 hours. After 5 hours of culture, the culture supernatant was removed from the cells and washed once with ice-cold D-PBS(-) (Nacalai Tesque). Then, 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts, Nacalai Tesque) was added per well, and the cells were lysed by vigorous shaking for 30 seconds. The translation product in the resulting cell lysate was analyzed using the same method as the sandwich ELISA described in Test Example 1. The translation product concentration (nM) in each translation reaction solution was quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation. The results are shown in Table 9.

[0284] Table 9-1 to Table 9-3:

[0285]

[0286]

[0287]

[0288] As is clear from the test results shown in Tables 9-1 to 9-3 above, after addition of each sugar-modified mRNA to hAoSMC, it produced the polypeptide encoded by the gene sequence, and the translation amount was superior to that of mRNA without sugar modification.

[0289] Test Example 6 (In vitro translation reaction test of mRNA samples using human aortic smooth muscle cells) The translation activity of each of the mRNAs listed in Tables 10-1 to 10-5 below was evaluated using human aortic smooth muscle cells. First, each mRNA was diluted to 19 μM with THE RNA Storage Solution (Thermo Fisher Scientific, catalog number AM7000). hAoSMCs were suspended in Opti-MEM I Reduced Serum Media (ThermoFisher Scientific, Catalog No. 31985070) containing a final concentration of 1% bovine serum albumin (Wako Pure Chemical Industries, Catalog No. 017-22231), centrifuged at 90 x g for 10 minutes at room temperature, and the supernatant was carefully removed. The cells were then suspended at 100,000 cells / 19 μL in a mixture of P1 Primary Cell Nucleofector Solution and Supplement 1 included in the P1 Primary Cell 96-well Nucleofector Kit (Lonza, Catalog No. V4SP-1096). The prepared mRNA solution and hAoSMC suspension were mixed at a volume ratio of 1:19, and then lysed in the Nucleofector. TMElectroporation was performed using a 96-well Shuttle system (Lonza) under pulse conditions FF-130. Ten minutes after electroporation, cells were suspended in SmGM-2 BulletKit medium (Lonza, CC-3182) and seeded into a 96-well culture plate for adherent cells at 20,000 cells / 145 μL per well. Cultures were incubated at 37°C and 5% CO2. After 4, 8, and 24 hours of incubation, the culture supernatant was removed and the cells were washed once with ice-cold D-PBS(-) (Nacalai Tesque). 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts, Nacalai Tesque) was added per well and vigorously shaken for 30 seconds to lyse the cells. The translation products in the obtained cell lysates were analyzed by the same sandwich ELISA method as described in Test Example 1. As a result of the measurement, the translation product concentrations (nM) in each translation reaction solution were quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and are shown in Table 10 below.

[0290] Tables 10-1 to 10-5:

[0291]

[0292]

[0293]

[0294]

[0295]

[0296] As is clear from the test results shown in Tables 10-1 to 10-5 above, mRNA with sugar modifications produced the polypeptide encoded by the gene sequence after electroporation into hAoSMC, and its activity was superior to that of mRNA without sugar modifications in the translation region.

[0297] Test Example 7 (Serum Stability Test of mRNA Samples) Nucleic acid stability in serum was evaluated for each mRNA listed in Table 11 below using commercially available mouse serum (Kohjin Bio, catalog number 12081001). First, mouse serum was diluted 50-fold with UltraPure DNase / RNase-Free Distilled Water (DW) (Invitrogen, catalog number 10977-015) to prepare a diluted serum solution. Each mRNA was diluted to 5 μM with THE RNA storage solution (Thermo Fisher Scientific, catalog number AM7001). For the enzyme-unreacted (0 min) run, a separate 96-well PCR plate was charged with 10.5 μL of a mixture of 8 μL of diluted serum solution, 2.5 μL of 6 U / μL Ribonuclease Inhibitor (Takara Bio, catalog number 2311B), and 2 μL of 5 μM mRNA, and stored at -30°C. For the enzymatic reaction, 8 μL of diluted serum solution and 2 μL of 5 μM mRNA were added to a separate 96-well PCR plate and mixed thoroughly. After incubation at 37°C for the specified time (15, 30, or 60 min), 2.5 μL of 6 U / μL RNase inhibitor was added and the plate was stored at -30°C until assay. The amount of residual mRNA in the reaction solution after the enzymatic reaction was detected using the RT-qPCR method described below. Standard curves were prepared for each mRNA tested, and a dilution series was created using THE RNA storage solution, with 11 concentrations starting from 4 μM and diluted 4-fold. 2.5 μL of the standard curve and post-enzymatic reaction samples were diluted 1071-fold with DW supplemented with ribonuclease inhibitor to a final concentration of 0.2 U / mL. Five μL of this diluted sample and 1 μL of 2 μM RT primer (Sigma-Aldrich) were used to prepare reverse-transcribed cDNA using the TaqMan Micro RNA RT kit (Thermo Fisher Scientific, catalog number 4366597). The reaction was carried out at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min.qPCR was performed by mixing 5 μL of cDNA, 10 μL of TaqMan Gene Expression Master Mix, 0.28 μL of Fw primer (Sigma-Aldrich), 0.33 μL of Rv primer (Sigma-Aldrich), 0.38 μL of TaqMan MGB Probe (Thermo Fisher Scientific, catalog number 4316033), and 4.01 μL of distilled water. The instrument used was a Quantstudio12K Flex (Applied Biosystems). The DNA sequences of the primers and TaqMan MGB Probe used are as follows. Based on the C values ​​of the standard samples, the concentration of each mRNA in each sample was quantified using a calibration curve. The relative remaining amount compared to the unreacted amount (0 minutes) is shown in Table 11. RT primer: 5'-TCAGTGGTGGTGGTGGTGGTGTTTG-3' (sequence number 431) Fw primer: 5'-ATCTTGTCGTCGTCGTCCTT-3' (sequence number 432) Rv primer: 5'-GAATACAAGCTACTTGTTCTTTT-3' (sequence number 433) Taqman MGB Probe: 5'-CAGCCACCATG-3' (sequence number 434).

[0298] Table 11:

[0299]

[0300] As is clear from the test results shown in Table 11 above, sugar-modified mRNA had improved resistance to degradation in serum compared to non-sugar-modified mRNA.

[0301] Test Example 8 (Translation Reaction Test Using HeLa Cell Line Lysate as an mRNA Sample) For each mRNA listed in Tables 12-1 to 12-7 below, the translation activity in a human cell line was evaluated using a 1-Step Human Coupled IVT Kit (Thermo Fisher Scientific, catalog number 88882). The translation reaction was performed in the same manner as in Test Example 1, with the mRNA at a final concentration of 1 μM. The translation product in the reaction solution after the translation reaction was analyzed by the sandwich ELISA method described in Test Example 1, except that the following peptide (Cosmobio) was used as the translation product polypeptide preparation. The translation product concentration (nM) in each translation reaction solution, quantified using a calibration curve prepared based on the absorbance of the polypeptide preparation, and the relative amount of translation product when the amount of unglycosylated E30 was set to 1, are shown in Table 12 below. Translation product polypeptide preparation: NH2-MDYKDDDDKGGHHHHHH-COOH (SEQ ID NO: 435)

[0302] Tables 12-1 to 12-7:

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] As is clear from the test results shown in Tables 12-1 to 12-7 above, after each mRNA was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cells.

[0311] Example 5 (Synthesis of mRNA that translates VEGF) Sequence information of materials (polynucleotides) used in the synthesis of mRNA that is translated into VEGF protein is shown below.

[0312] Table 13:

[0313]

[0314] mRNA (VEGF-1, VEGF-2, VEGF-3) was obtained by the following series of procedures. (Step 1: Preparation of linearized plasmid DNA and preparation of RNA fragments by in vitro transfection) Plasmid DNA was prepared by inserting the artificially synthesized gene sequence GN shown in Table 13 into the EcoRV and XbaI sites of the commercially available pUC19 vector (GeneWiz Co., Ltd.). Plasmid DNA was linearized using the restriction enzyme XbaI. The final concentrations of the reaction solution were 20 ng / μL plasmid DNA, 0.01% BSA, 0.15 U / μL XbaI (Takara 1093A), and 1x the provided buffer. After incubation at 37°C for 2 hours, the mixture was extracted with phenol / chloroform and precipitated with isopropanol to obtain crude linearized plasmids. Transcription reactions were performed using the resulting template DNA and T7 RNA polymerase. The final concentrations of the reaction solution were as follows: Template DNA 10 ng / μL, DTT 5mM, ATP 2mM, CTP 2mM, UTP 2mM, GMP 2mM, GTP 0.5mM, Murine RNase inhibitor 0.2 U / μL (NEB, M0314), T7 RNA polymerase 2.5 U / μL (Takara, 2540A), 1x provided buffer. After 2 hours of incubation at 37°C, DNase (final concentration 0.1 U / μL) (Takara, 2270A) was added and incubated at the same temperature for 30 minutes. Crude transcription products were obtained by phenol-chloroform extraction, Amicon 10K treatment (Merck Millipore), and isopropanol precipitation. After denaturing polyacrylamide gel electrophoresis, the corresponding bands were excised, extracted with MQ water, purified with Amicon, and isopropanol precipitation to obtain purified RNA. Subsequently, the terminal triphosphate was converted to the monophosphate by treatment with RNA 5' pyrophosphohydrolase (RppH). The final concentrations of the reaction solution were as follows: RNA 0.1 μg / μL, RppH 0.1 U / μL, Murine RNase inhibitor 1 U / μL (NEB, M0314), and 1x NEBuffer 2 (NEB, B7992S).After incubation at 37°C for 30 minutes, phenol-chloroform extraction and isopropanol precipitation were carried out to obtain a crude product of polynucleotide, which was the desired 3'-terminal polynucleotide fragment.

[0315] (Step 2: Preparation of RNA ligation products by RNA ligation) The 5'-end polynucleotide fragments (N1, N2, and N3) shown in Table 13, obtained by standard chemical synthesis, the 3'-end polynucleotide fragment obtained by in vitro transcription in Step 1, and template DNA-4 were used to perform a ligation reaction using RNA ligase 2. The final concentrations were as follows: 5'-end RNA 2 μM, 3'-end RNA 1 μM, template DNA 4 μM, PEG8000 10%, T4 RNA ligase 2 1 U / μL (NEB, M0239), 1x provided buffer, and Murine RNase inhibitor 1 U / μL (NEB, M0314). The mixture before the addition of enzyme and PEG was heated at 90°C for 3 minutes, then gradually cooled to room temperature. The enzyme and PEG were added, and the mixture was incubated at 45°C for 1 hour. Crude transcription products were obtained by phenol-chloroform extraction, Amicon 10K treatment (Merck Millipore), and isopropanol precipitation. After electrophoresis on a denaturing polyacrylamide gel, the corresponding bands were excised, extracted with MQ water, purified with Amicon, and precipitated with isopropanol to obtain purified mRNA.

[0316] Test Example 9 (Translation Reaction of mRNA Sample) The mRNA sequence information obtained in Example 5 above is shown in Tables 14-1 and 14-2.

[0317] Table 14-1 to Table 14-2:

[0318]

[0319]

[0320] The in vitro translation activity of each mRNA was evaluated using HeLa cells. First, HeLa cells were suspended in RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum and seeded onto a 96-well culture plate at 10,000 cells / 100 μL per well. They were then cultured overnight at 37°C in 5% CO2. After overnight culture, the culture supernatant was removed from the cells, and 40 μL of RPMI medium containing 10% fetal bovine serum was added per well. Each compound was diluted with Optimem (Thermo Fisher Scientific, catalog number LMRNA008) at a final concentration of 0.3% to give final concentrations of 0.3, 1, 3, and 10 nM. The mixture was then added to each well at 10 μL per well and cultured at 37°C under 5% CO2 for 24 hours. After 24 hours of culture, the culture supernatant was collected, and VEGF protein levels in the culture supernatant were measured using a Human VEGE Quantikine ELISA (R&D, catalog number DVE00) according to the kit instructions. The VEGF protein concentration (ng / mL) in each culture supernatant quantified as a result of the measurement is shown in Table 15 below.

[0321] Table 15:

[0322]

[0323] As is clear from the evaluation results shown in Table 15 above, after addition of sugar-modified mRNA to HeLa cells, the VEGF protein encoded by the gene sequence was produced with higher efficiency than that of mRNA without sugar modification.

[0324] Example 6 (Synthesis of mRNA by IVT) Sequence information of materials (polynucleotides) used in mRNA synthesis by IVT is shown below.

[0325] Table 16:

[0326]

[0327] mRNA (IVT-1) was obtained by the following series of procedures. (Step 1: Preparation of Linearized DNA) Plasmid DNA was prepared by inserting the artificial synthetic gene sequence GO shown in Table 16 into the BamHI and PstI sites of commercially available pUC57 vector. PCR reactions were performed using the plasmid DNA as follows. Specifically, plasmid DNA (final concentration 250 ng / μL), primers P1 and P2 (final concentrations of 250 nM each), and 200 μL of Primestar MAX (Takara Bio Inc., catalog number R045B) were mixed, and the resulting mixture was adjusted to 400 μL with nuclease-free water. The mixture was heated at 98°C for 30 seconds in a thermal cycler, followed by 30 cycles of heating at 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 5 seconds. The mixture was then heated at 72°C for 5 minutes and cooled to 4°C. To 400 μL of the resulting PCR product, 10 μL of Dpn I (New England BioLab, catalog number R0176L), 50 μL of CutSmart Buffer (New England BioLab), and 40 μL of nuclease-free water were added, mixed, and allowed to stand at 37°C for 30 minutes. The resulting reaction solution was electrophoresed on a 3.0% agarose-TAE gel, and the relevant band was excised. The PCR product was purified using NucleoSpin Gel and PCR Clean-up Midi (MACHEREY-NAGEL, catalog number 740986.20), followed by phenol-chloroform extraction and ethanol precipitation to obtain PCR DNA.

[0328] (Step 2: Preparation of RNA fragments by in vitro transcription) The resulting PCR DNA was used for transcription. The final concentrations of the reaction solution were as follows: PCR DNA 4 ng / μL, ATP, CTP, UTP, and GTP 9 mM each, and 10% T7 Enzyme and 10% T7 Reaction Buffer included with the MEGAScript T7 Transcription Kit (Invitrogen, catalog number AMB13345). The reaction volume was 400 μL and incubated at 37°C for 6 hours. Next, Trubo DNase included with the MEGAScript T7 Transcription Kit was added at 1 / 20 vol of the reaction volume, mixed, and shaken at 37°C for 15 minutes. The RNA fragments were crudely purified by phenol-chloroform extraction and ethanol precipitation to obtain the RNA fragments. The resulting RNA fragments were capped using the Vaccinia Capping System (New England Biolabs, catalog number MB2080S) and the ScriptCap 2'-O-Methyltransferase Kit (CELLSCRIPT, catalog number C-SCMT0625) as follows. The final concentrations of the reaction mixture were as follows: 500 ng / μL RNA fragment, 10% Capping Buffer, 0.5 mM GTP, 0.2 mM SAM, 0.5 U / μL Vaccinia capping enzyme, 1 U / μL RNase inhibitor, and 2.5 U / μL 2'-O-Methyltransferase. The reaction mixture was adjusted to 2000 μL with nuclease-free water and incubated at 37°C for 1 hour. The capped RNA fragments were purified by phenol-chloroform extraction and ethanol precipitation.Subsequently, 105 μL of the resulting capped RNA fragments were mixed with 7 μL of Antarctic Phosphatase (New England Biolabs, Catalog No. M0289S), 14 μL of 10x Antarctic Phosphatase Buffer, and 14 μL of nuclease-free water, and the mixture was incubated at 37°C for 1 hour to carry out an alkaline phosphatase reaction. The reaction sample was purified as described in Example 1 (purification of RNA fragments using dPAGE), yielding 3.04 nmol of capped mRNA IVT-1. The sequence of the resulting IVT-1 is shown in Table 17.

[0329] In each nucleotide in Table 17, N (uppercase) represents RNA, N(M) represents 2'-O-methyl modified RNA, and m7Gppp represents the following structural formula.

[0330]

[0331] Test Example 10 (Translation reaction test using HeLa cell line lysate as an mRNA sample) For each of the mRNAs listed in Tables 18-1 to 18-10 below, the translation activity in a human cell line was evaluated by the same method as in Test Example 1. The translation product concentration (nM) in the translation reaction solution to which 0.3 μM of each mRNA was added is shown in Table 18 below.

[0332] Table 18-1 to Table 18-10:

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343] As is clear from the test results shown in Tables 18-1 to 18-10 above, after each sugar-modified mRNA was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cell.

[0344] Test Example 11 (In vitro translation reaction test of mRNA samples using HeLa cell line) For each of the mRNAs listed in Tables 19-1 to 19-13 below, the translation activity was evaluated in vitro using the human HeLa cell line by the same method as in Test Example 2. The translation product concentrations (nM) in the cell lysates obtained from the cells 5 hours after the addition of 3 to 30 nM of each mRNA are shown in Table 19 below.

[0345] Table 19-1 to Table 19-13:

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359] As is clear from the test results shown in Tables 19-1 to 19-13 above, after addition of each sugar-modified mRNA to HeLa cells, it produced the polypeptide encoded by the gene sequence, and the translation amount was superior to that of the mRNA without sugar modification.

[0360] Test Example 12 (In vitro translation reaction test of mRNA samples using HeLa cell line) For each of the mRNAs listed in Tables 20-1 to 20-8 below, the sustainability of in vitro translation activity was evaluated using HeLa cell line by the same method as in Test Example 3. The translation product concentrations (nM) in the cell lysates obtained from cells to which 30 nM of each mRNA had been added are shown in Table 20 below.

[0361] Table 20-1 to Table 20-8:

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] As is clear from the test results shown in Tables 20-1 to 20-8 above, after addition of each sugar-modified mRNA to HeLa cells, it produced the polypeptide encoded by the gene sequence, and the translation amount was superior to that of mRNA without sugar modification.

[0371] Test Example 13 (In vitro translation reaction test of mRNA samples using HeLa cell line) For each of the mRNAs listed in Tables 21-1 to 21-4 below, the in vitro translation activity was evaluated using HeLa cell line by the same method as in Test Example 4. The translation product concentrations (nM) in the cell lysates obtained from cells to which each mRNA was added are shown in Table 21 below.

[0372] Table 21-1 to Table 21-4:

[0373]

[0374]

[0375]

[0376]

[0377] As is clear from the test results shown in Tables 21-1 to 21-4 above, mRNA with sugar modifications produced the polypeptide encoded by the gene sequence after electroporation into HeLa cells, and its activity was superior to that of mRNA without sugar modifications in the translation region.

[0378] Test Example 14 (Translation reaction test using HeLa cell line lysate as an mRNA sample) For each of the mRNAs listed in Tables 22-1 and 22-2 below, the translation activity in a human cell line was evaluated by the same method as in Test Example 8. The translation product concentration (nM) in the translation reaction solution to which 1 μM of each mRNA was added is shown in Table 22 below.

[0379] Table 22-1 to Table 22-2:

[0380]

[0381]

[0382] As is clear from the test results shown in Tables 22-1 and 22-2 above, after each mRNA was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cells.

[0383] Test Example 15 (Intracellular nucleic acid stability test using HeLa cell line with mRNA sample) The intracellular nucleic acid stability of each mRNA listed in Table 23 below was evaluated using the HeLa cell line. Cell culture and mRNA introduction were performed in the same manner as in Test Example 3, with each mRNA prepared to a final concentration of 30 nM. After 4 hours of culture following the addition of each mRNA, the culture supernatant was removed from the cells, and 50 μL of RPMI medium (Nacalai Tesque) containing 10% fetal bovine serum was added per well. Culture was continued at 37°C and 5% CO2. 4 hours, 8 hours, and 24 hours after the addition of the mRNA, the cells were lysed as follows. Specifically, after removing the culture supernatant from the cells, the cells were washed once with ice-cold D-PBS(-) (Nacalai Tesque). Then, 20 μL of iScript RT-qPCR Sample Preparation Reagent (Bio-Rad, 1708898) containing 2% protease inhibitor cocktail (for animal cell extracts, Nacalai Tesque) was added per well and vigorously shaken for 30 seconds to lyse the cells. The amount of residual mRNA in the resulting cell lysates was detected using the RT-qPCR method described below. First, a DW containing ribonuclease inhibitor at a final concentration of 0.2 U / mL was prepared as sample dilution solution. A standard curve was prepared for each mRNA tested. A dilution series was created by diluting cell lysates prepared from nucleic acid-free cells with a 10-fold dilution of sample dilution solution to obtain 11 concentrations, starting from 1 μM and followed by 4-fold dilutions. Each cell lysate to be measured was diluted 10-fold with sample dilution solution. These standard curves and the cell lysates to be measured were diluted 1071-fold with DW containing ribonuclease inhibitor at a final concentration of 0.2 U / mL. The subsequent reverse transcription reaction and RT-qPCR reaction were carried out in the same manner as in Test Example 7. As a result of the measurement, the concentration of each mRNA in each sample was quantified using the standard curve based on the C value of the standard sample, and the results are shown in Table 23 below.

[0384] Table 23:

[0385]

[0386] As is clear from the test results shown in Table 23 above, sugar-modified mRNA had improved resistance to degradation in cells compared to mRNA prepared by the IVT method.

[0387] Test Example 16 (Translation reaction test using HeLa cell line lysate as an mRNA sample) For each mRNA listed in Table 24 below, the translation activity in a human cell line was evaluated by the same method as in Test Example 1. The translation product concentration (nM) in the translation reaction solution to which 0.3 μM of each mRNA was added is shown in Table 24 below.

[0388]

[0389] As is clear from the test results shown in Table 24 above, after each sugar-modified mRNA was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cell.

[0390] Test Example 17 (In vitro translation reaction test of mRNA samples using HeLa cell line) For each mRNA listed in Table 25 below, the translation activity in vitro was evaluated using a human cell line, HeLa cell line, by the same method as in Test Example 2. The translation product concentrations (nM) in the cell lysates obtained from the cells 5 hours after the addition of 3 to 30 nM of each mRNA are shown in Table 25 below.

[0391]

[0392] E222, which has a sugar-modified polyA tail length of 5, E223, which has a sugar-modified polyA tail length of 10, E65, E224, and E225, which have a sugar-modified polyA tail length of 20, and E59, which has a sugar-modified polyA tail length of 40, each showed superior translation ability compared to mRNAs with unmodified polyA tails of the same length.

[0393] Test Example 18 (In vitro translation reaction test of mRNA sample using HeLa cell line) For each mRNA listed in Table 26 below, the sustainability of in vitro translation activity was evaluated using HeLa cell line by the same method as in Test Example 3. The translation product concentrations (nM) in cell lysates obtained from cells to which 30 nM of each mRNA had been added are shown in Table 26 below.

[0394]

[0395] As is clear from the test results shown in Table 26 above, after addition of each sugar-modified mRNA to HeLa cells, it produced the polypeptide encoded by the gene sequence, and the translation amount was superior to that of the mRNA without sugar modification, depending on the poly(A) tail length.

[0396] Test Example 19 (Translation reaction test using HeLa cell line lysate as an mRNA sample) For each of the mRNAs listed in Tables 27-1 to 27-5 below, the translation activity in a human cell line was evaluated by the same method as in Test Example 8. The translation product concentration (nM) in the translation reaction solution to which 1 μM mRNA was added, and the relative amount of translation product when the amount of unglycosylated mRNA, E226 or E30, was set to 1, are shown in Tables 27-1 to 27-5 below.

[0397] Table 27-1 to Table 27-5:

[0398]

[0399]

[0400]

[0401]

[0402]

[0403] As is clear from the test results shown in Tables 27-1 to 27-5 above, after each mRNA was added to HeLa cell lysate, the polypeptide encoded by the gene sequence was produced by the translation system of the eukaryotic cell, and the translation amount was superior to that of mRNA without sugar modification.

Claims

1. A polynucleotide comprising a translation region from a start codon to a stop codon, a 5'-untranslated region, and a poly-A chain, wherein 65% or more of the nucleotides constituting the poly-A chain are sugar-modified nucleotides.

2. The polynucleotide according to claim 1, wherein all of the nucleotides constituting the poly-A chain are sugar-modified nucleotides.

3. The polynucleotide according to claim 1, wherein the modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures: 【Chemical 1】 The polynucleotide according to claim 1.

4. The polynucleotide according to claim 1, wherein the modified sugar moieties of the sugar-modified nucleotides are each independently selected from any one of the following structures: 【Chemical 2】 The polynucleotide according to claim 1.

5. The polynucleotide according to claim 1, wherein the poly-A chain contains at least one phosphate-modified nucleotide.

6. The polynucleotide according to claim 1, wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 3'-end of the poly-A chain are linked by phosphorothioate.

7. The polynucleotide according to claim 1, wherein all of the nucleotides constituting the poly-A chain are linked by phosphorothioate.

8. The polynucleotide according to claim 1, wherein the poly-A chain is 2 to 40 bases in length.

9. The polynucleotide according to claim 1, wherein the nucleotides of the 5'-untranslated region are each independently selected from 2'-deoxyribonucleotides, spacer-modified or sugar-modified nucleotides.

10. The polynucleotide according to claim 1, wherein the first to sixth nucleotides from the 5'-end of the 5'-untranslated region are sugar-modified nucleotides, and the modified sugar moiety of the sugar-modified nucleotide has the following structure: 【Chemical Formula 3】 The polynucleotide according to claim 1.

11. The polynucleotide according to claim 10, further comprising a portion consisting of 1 to 10 sugar-unmodified nucleotides on the 5'-side of the 5'-end of the 5'-untranslated region.

12. The polynucleotide according to claim 1, wherein the nucleotides excluding the first to sixth nucleotides from the 5'-end of the 5'-untranslated region contain 2'-deoxyribonucleotides and / or spacer-modification.

13. The polynucleotide according to claim 1, wherein the 5'-untranslated region contains spacer-modification, and the spacer-modification is each independently selected from any one of the following structures: 【Chemical Formula 4】 [wherein, Rx is ethynyl, a hydrogen atom or OH, M is a hydrogen atom or OH, n1 is 1, 2 or 5, n2 is 1, 2 or 3.] The polynucleotide according to claim 9.

14. The 5'-untranslated region contains a spacer modification, and the spacer modification is independently selected from any one of the following structures: 【Chemical Formula 5】 [wherein, Rx is ethynyl, a hydrogen atom or OH, M is a hydrogen atom or OH, n1 is 1, 2 or 5, n2 is 1, 2 or 3.] The polynucleotide according to claim 12.

15. The polynucleotide according to claim 1, wherein the first to second nucleotides, the first to third nucleotides, the first to fourth nucleotides, or the first to fifth nucleotides from the 5'-end of the 5'-untranslated region are linked by phosphorothioate.

16. The 5'-untranslated region contains a base-modified nucleotide, and the modified base portion of the base-modified nucleotide has the following structure: 【Chemical Formula 6】 [wherein R is an alkyl group having 1 to 6 carbon atoms.] The polynucleotide according to claim 1.

17. The polynucleotide according to claim 1, wherein the translation region contains at least two codons in which the first nucleotide is a sugar-modified nucleotide.

18. The polynucleotide according to claim 1, wherein the translation region contains 4 or more codons, and the first nucleotide of all codons is a sugar-modified nucleotide.

19. The polynucleotide according to claim 1, wherein the first nucleotide of all codons except the stop codon in the translation region is a sugar-modified nucleotide, and the modified sugar portion of the sugar-modified nucleotide has the following structure: 【Chemical Formula 7】 The polynucleotide according to claim 1.

20. The polynucleotide according to claim 1, wherein the translation region contains 2000 or fewer codons.

21. The polynucleotide according to claim 1, wherein all nucleotides of the stop codon are sugar-modified nucleotides.

22. Containing the following structure: [Chemical Formula 8] [wherein, R １ and R ２ are each independently H, OH, F, OCH ２ CH ２ OCH ３ or OCH ３ and B １ and B ２ are each independently a base moiety, X １ is O, S or NH, X ２ is O, S, NH or has the following structure, 【Chemical Formula 9】 X ３ is OH, SH or a salt thereof, However, X １ and X ２ are not O at the same time The polynucleotide according to claim 1.

23. A pharmaceutical composition comprising the polynucleotide according to any one of claims 1 to 22.

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