mRNA cap analog and method for producing same, method for producing mrna, and kit

Abstract

The mRNA cap analog contains a functional group selected from the following: amino group, carboxylic acid active ester groups, aminooxy group, hydrazino group, carbonyl group, thiol group, haloacetyl groups, Michael acceptor functional groups, alkenyl groups, and 1,3-dipolar functional groups. The mRNA cap analog is also provided with, in the sugar moiety or base moiety of a nucleotide, a linker having a structure that can be cleaved by light or fluoride ion.

Description

mRNA cap analog and its production method, mRNA production method, and kit

[0001] The present invention relates to an mRNA cap analog and a method for producing the same, a method for producing mRNA, and a kit.

[0002] One known method for producing messenger RNA (mRNA) is in vitro transcription. This method involves polymerizing nucleoside triphosphates (NTPs) using template DNA with RNA polymerase. In this reaction, a cap structure (hereinafter referred to as a "5' cap structure") can be introduced at the 5' end of mRNA by adding an mRNA cap analog, such as an ARCA (anti-reverse CAP analog). Patent Document 1 discloses an mRNA cap analog useful for such in vitro transcription.

[0003] Special Publication No. 2009-544754

[0004] In methods for producing mRNA by in vitro transcription, while mRNA having a 5'-cap structure can be produced, mRNA without a 5'-cap structure (i.e., with a triphosphate structure at the 5'-end) is also produced as a by-product. Because mRNA without a 5'-cap structure induces natural immunity and reduces translation efficiency, it is desirable to separate and remove it; however, separation is difficult using purification methods such as chromatography. Therefore, separating the mRNA without a 5'-cap structure produced as a by-product in mRNA production by in vitro transcription can be a useful technique for the production of pharmaceutical active ingredients using mRNA.

[0005] The present invention has been made to solve the above-mentioned problems, and aims to provide an mRNA cap analog that can separate mRNA with a 5' cap structure from mRNA without a 5' cap structure, a method for producing the same, a method for producing mRNA, and a kit.

[0006] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors discovered that introducing a linker having a specific structure into the sugar moiety or base moiety of the nucleotide constituting an mRNA cap analog makes it possible to separate mRNA with a 5' cap structure from mRNA without a 5' cap structure, and thus completed the present invention. That is, the present invention is exemplified as follows.

[0007] [1] An mRNA cap analogue comprising a linker at the sugar moiety or base moiety of a nucleotide, the linker having a structure that is cleavable by light or fluoride ions and that includes a functional group selected from an amino group, a carboxylic acid activated ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group.

[0008] [2] The mRNA cap analog according to [1], wherein the linker has an o-nitrobenzyl ether structure or a silyl ether structure.

[0009] [3] The mRNA cap analog according to [1] or [2], represented by the following general formula (1) or (2):

[0010]

[0011] In the formula, R represents OH, OCH, or the following general formula (3):

[0012] R2 is OH or OCH3, R3 is O, OH, NH2, NHCH3 or N(CH3)2, and when R3 is O, the bond between the C bonded to R3 and R3 is a double bond and the bond between the C bonded to R3 and the N adjacent thereto is a single bond, and when R3 is OH, NH2, NHCH3 or N(CH3)2, the bond between the C bonded to R3 and R3 is a single bond and the bond between the C bonded to R3 and the N adjacent thereto is a double bond, R4 is NH2, NHCH3, N(CH3)2 or H, R5 is a propargyloxy group or an azidoethoxy group, R6 and R7 may be the same or different and are OH or OCH3, a is 3 to 6 and b is 0 to 19, Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosine-1-yl, 5-methylcytosine-1-yl, uracil-1-yl, uracil-5-yl, or 1-methyluracil-5-yl.

[0013] [4] The mRNA cap analog according to [3], wherein R3 is O and R4 is NH2.

[0014] [5] A method for producing an mRNA having a 5' cap structure, comprising: mixing an mRNA cap analog having a linker at the sugar or base moiety of a nucleotide, the linker including functional group A and having a structure cleavable by light or fluoride ions, a nucleoside triphosphate, a template DNA, and an RNA polymerase, and obtaining a preparation containing an mRNA having a 5' cap structure by in vitro transcription; mixing the preparation with an insoluble carrier having functional group B reactive with functional group A, to produce a conjugate of the mRNA and the insoluble carrier; filtering and washing the conjugate; and applying light or fluoride ions to the conjugate to cleave the linker, thereby isolating the mRNA having a 5' cap structure.

[0015] [6] The production method according to [5], wherein the functional groups A and B are a combination of an amino group and a carboxylic acid activated ester, a combination of an aminooxy group or a hydrazino group and a carbonyl group, a combination of a thiol group and a thiol group, a haloacetyl group or a Michael acceptor functional group, or a combination of an alkynyl group and a 1,3-dipole functional group.

[0016] [7] The method according to [5] or [6], wherein the linker has an o-nitrobenzyl ether structure or a silyl ether structure.

[0017] [8] The method according to [5], wherein the mRNA cap analog is represented by the following general formula (1) or (2):

[0018]

[0019] In the formula, R represents OH, OCH, or the following general formula (3):

[0020] R2 is OH or OCH3, R3 is O, OH, NH2, NHCH3 or N(CH3)2, and when R3 is O, the bond between the C bonded to R3 and R3 is a double bond and the bond between the C bonded to R3 and the N adjacent thereto is a single bond, and when R3 is OH, NH2, NHCH3 or N(CH3)2, the bond between the C bonded to R3 and R3 is a single bond and the bond between the C bonded to R3 and the N adjacent thereto is a double bond, R4 is NH2, NHCH3, N(CH3)2 or H, R5 is a propargyloxy group or an azidoethoxy group, R6 and R7 may be the same or different and are OH or OCH3, a is 3 to 6 and b is 0 to 19, Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosine-1-yl, 5-methylcytosine-1-yl, uracil-1-yl, uracil-5-yl, or 1-methyluracil-5-yl.

[0021] [9] The method according to [8], wherein R3 is O and R4 is NH2.

[0022]

[10] The method according to [8] or [9], wherein when R5 is a propargyloxy group, the insoluble carrier is represented by the following formula (4):

[0023] In the formula, Sup is an insoluble carrier structural part.

[0024]

[11] The method according to [8] or [9], wherein when R5 is an azidoethoxy group, the insoluble carrier is represented by the following formula (5), (6), or (7):

[0025] In the formula, Sup is an insoluble carrier structural part.

[0026]

[12] A method for producing an mRNA cap analog, comprising: introducing a linker, which includes a functional group selected from an amino group, a carboxylic acid activated ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group, and which has a structure that can be cleaved by light or a fluoride ion, into the sugar moiety or the base moiety of a first nucleotide; and linking the first nucleotide having the linker introduced therein to a second nucleotide in the presence of magnesium chloride.

[0027]

[13] A kit for use in producing mRNA having a 5'-cap structure, comprising: an mRNA cap analog having a linker at the sugar moiety or the base moiety of a nucleotide, the linker having a structure cleavable by light or fluoride ions and containing functional group A selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group; and an insoluble support having functional group B capable of reacting with functional group A.

[0028]

[14] The kit according to

[13] , wherein the functional groups A and B are a combination of an amino group and a carboxylic acid activated ester, a combination of an aminooxy group or a hydrazino group and a carbonyl group, a combination of a thiol group and a thiol group, a haloacetyl group or a Michael acceptor functional group, or a combination of an alkynyl group and a 1,3-dipole functional group.

[0029]

[15] The kit according to

[13] or

[14] , wherein the linker has an o-nitrobenzyl ether structure or a silyl ether structure.

[0030]

[16] The kit according to

[13] , wherein the mRNA cap analog is represented by the following general formula (1) or (2):

[0031]

[0032] In the formula, R represents OH, OCH, or the following general formula (3):

[0033] R2 is OH or OCH3, R3 is O, OH, NH2, NHCH3 or N(CH3)2, and when R3 is O, the bond between the C bonded to R3 and R3 is a double bond and the bond between the C bonded to R3 and the N adjacent thereto is a single bond, and when R3 is OH, NH2, NHCH3 or N(CH3)2, the bond between the C bonded to R3 and R3 is a single bond and the bond between the C bonded to R3 and the N adjacent thereto is a double bond, R4 is NH2, NHCH3, N(CH3)2 or H, R5 is a propargyloxy group or an azidoethoxy group, R6 and R7 may be the same or different and are OH or OCH3, a is 3 to 6 and b is 0 to 19, Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosine-1-yl, 5-methylcytosine-1-yl, uracil-1-yl, uracil-5-yl, or 1-methyluracil-5-yl.

[0034]

[17] The kit according to

[16] , wherein R3 is O and R4 is NH2.

[0035]

[18] The kit according to

[16] or

[17] , wherein when R5 is a propargyloxy group, the insoluble carrier is represented by the following formula (4):

[0036] In the formula, Sup is an insoluble carrier structural part.

[0037]

[19] The kit according to

[16] or

[17] , wherein when R5 is an azidoethoxy group, the insoluble carrier is represented by the following formula (5), (6), or (7):

[0038] In the formula, Sup is an insoluble carrier structural part.

[0039]

[20] The kit according to any one of

[13] to

[19] , further comprising one or more selected from a nucleoside triphosphate, a template DNA, and an RNA polymerase.

[0040] According to the present invention, it is possible to provide an mRNA cap analog capable of separating mRNA having a 5' cap structure from mRNA not having a 5' cap structure, a method for producing the same, a method for producing mRNA, and a kit.

[0041] FIG. 1 is a schematic diagram illustrating the structure of an mRNA cap analog of the present invention. FIG. 2 is a schematic diagram illustrating a process for separating mRNA with a 5' cap structure from mRNA without a 5' cap structure produced by in vitro transcription using an mRNA cap analog of the present invention. FIG. 3 is a HPLC result of an aqueous solution of Compound 15 before and after UV irradiation. FIG. 4 is a HPLC result of a solution of a dimeric oligonucleotide derivative before and after a click reaction. FIG. 5 is a HPLC result of a solution of a conjugate (Compound 32) after UV irradiation. FIG. 6 is a HPLC result of a solution of a conjugate (Compound 33) after UV irradiation. FIG. 7 is a HPLC result of the click reaction of Compound 47 and Compound 60. FIG. 8 is a HPLC result of Compound 61 after UV irradiation. FIG. 9 is a HPLC result of the click reaction of Compound 54 and Compound 60. FIG. 10 is a HPLC result of Compound 63 after UV irradiation. FIG. 11 is a HPLC result of the click reaction of Compound 54 and Compound 72. FIG. 12 is a HPLC result of Compound 73 after UV irradiation.

[0042] The concept of the mRNA cap analog of the present invention will be explained using Figures 1 and 2. Figure 1 is a schematic diagram illustrating the structure of the mRNA cap analog of the present invention. As shown in Figure 1, the mRNA cap analog of the present invention has a nucleotide (represented as "CAP") that provides a 5' cap structure and a functional group A (represented as "A") that can react with functional group B, and they are connected by a structure (represented by a dotted line) that can be cleaved by light or fluoride ions. In this specification, the portion represented by A and the dotted line in Figure 1 is referred to as a "linker."

[0043] Figure 2 is a schematic diagram illustrating the process of separating mRNA with a 5'-cap structure and mRNA without a 5'-cap structure produced by in vitro transcription using the mRNA cap analog of the present invention. As shown in Figure 2, in vitro transcription produces mRNA with a 5'-cap structure (referred to as "Capped RNA") and mRNA without a 5'-cap structure (referred to as "Uncapped RNA"). Because the 5'-capped mRNA has a functional group A in the 5'-cap structure that can react with functional group B (referred to as "B"), it is reacted with an insoluble support having functional group B to produce a conjugate, which can then be separated and removed by filtration and washing. The linker of the separated and recovered conjugate is cleaved (cut) by application of light or fluoride ions, and only the mRNA with a 5'-cap structure is isolated.

[0044] The following is a detailed description of the embodiments of the present invention. The present invention is not limited to the following embodiments, and it should be understood that modifications and improvements made to the following embodiments based on the ordinary knowledge of those skilled in the art without departing from the spirit of the present invention are also within the scope of the present invention.

[0045] <mRNA Cap Analog> The mRNA cap analog according to the present invention includes a functional group A selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group, and includes a linker at the sugar or base moiety of a nucleotide, the linker having a structure that can be cleaved by light or fluoride ions. An mRNA cap analog having such a structure can be bound to an insoluble support having a functional group B that can react with functional group A by various known reactions, thereby enabling separation of mRNA with a 5' cap structure from mRNA without a 5' cap structure.

[0046] As used herein, the term "mRNA cap analog" refers to a capping reagent used to produce mRNA with a 5' cap structure by in vitro transcription. The term "nucleotide" as used herein refers to a compound containing a base and a sugar to which a phosphate group is covalently bound. Nucleotides may be modified with any of a variety of substituents.

[0047] When the functional group A of the mRNA cap analog is an amino group, by using it in combination with an insoluble support in which the functional group B is a carboxylic acid active ester group, it is possible to produce a conjugate having an amide bond (-CONH-) formed by the reaction between the mRNA having a 5' cap structure into which the functional group A has been introduced and the insoluble support having the functional group B. Similarly, when the functional group A of the mRNA cap analog is a carboxylic acid active ester group, by using it in combination with an insoluble support in which the functional group B is an amino group, it is possible to produce a conjugate having an amide bond formed by the reaction between the mRNA having a 5' cap structure into which the functional group A has been introduced and the insoluble support having the functional group B.

[0048] The amino group is not particularly limited, but examples thereof include a monomethylamino group, a monoethylamino group, a mono-n-propylamino group, a monoisopropylamino group, a mono-n-butylamino group, a mono-sec-butylamino group, a mono-tert-butylamino group, a mono-isobutylamino group, a tert-butylisopropylamino group, a mono-n-hexylamino group, a mono-n-octylamino group, a mono-n-decylamino group, a monophenylamino group, a trimethylsilylamino group, a mono-tert-butyldimethylsilylamino group, a pyrrolidinyl group, a piperidinyl group, a carbazolyl group, a dihydroindolyl group, a dihydroisoindolyl group, etc. The amino group may be optionally substituted.

[0049] The carboxylic acid activated ester group is not particularly limited, but examples thereof include carboxylic acid halides, thiophenyl esters, 4-nitrophenyl thioesters, cyanomethyl esters, nitro esters, dinitrophenyl esters, N-hydroxysuccinimide esters, p-nitrophenyl esters, 2,3,5-trichlorophenyl esters, 2,4,6-trichlorophenyl esters, 2,4,5-trichlorophenyl esters, pentachlorophenyl esters, 2,4-dinitrophenyl esters, and N-hydroxyphthalimide esters.

[0050] When the functional group A of the mRNA cap analog is an aminooxy group, by using it in combination with an insoluble support whose functional group B is a carbonyl group, it is possible to produce a conjugate having an oxime bond (-CR=N-; R is H or alkyl) formed by the reaction between an mRNA having a 5' cap structure into which functional group A has been introduced and an insoluble support having functional group B. Similarly, when the functional group A of the mRNA cap analog is a carbonyl group, by using it in combination with an insoluble support whose functional group B is an aminooxy group, it is possible to produce a conjugate having an oxime bond formed by the reaction between an mRNA having a 5' cap structure into which functional group A has been introduced and an insoluble support having functional group B.

[0051] The carbonyl group is not particularly limited, but examples thereof include a methoxycarbonyl group, a tert-butoxycarbonyl group, an ethoxycarbonyl group, an aldehyde group, a ketone group, etc. The carbonyl group may be optionally substituted.

[0052] When the functional group A of the mRNA cap analog is a hydrazino group, by combining it with an insoluble support in which the functional group B is a carbonyl group, a conjugate having a hydrazone bond (-CR=N-NHR-; R is H or alkyl) formed by the reaction between an mRNA having a 5' cap structure to which functional group A has been introduced and an insoluble support having functional group B can be produced. Similarly, when the functional group A of the mRNA cap analog is a carbonyl group, by combining it with an insoluble support in which the functional group B is a hydrazino group, a conjugate having a hydrazone bond formed by the reaction between an mRNA having a 5' cap structure to which functional group A has been introduced and an insoluble support having functional group B can be produced. Examples of hydrazino groups include, but are not limited to, methylhydrazino and ethylhydrazino. The hydrazino group may be optionally substituted.

[0053] When functional group A of the mRNA cap analog is a thiol group, by using it in combination with an insoluble support whose functional group B is a thiol group, a conjugate having a disulfide bond (-SS-) formed by the reaction between mRNA having a 5' cap structure into which functional group A has been introduced and the insoluble support having functional group B can be produced.

[0054] When the functional group A of the mRNA cap analog is a haloacetyl group, by combining it with an insoluble support in which the functional group B is a thiol group, a conjugate having a thioether bond (-S-) formed by the reaction between the mRNA having a 5' cap structure to which the functional group A has been introduced and the insoluble support having the functional group B can be produced. Similarly, when the functional group A of the mRNA cap analog is a thiol group, by combining it with an insoluble support in which the functional group B is a haloacetyl group, a conjugate having a thioether bond formed by the reaction between the mRNA having a 5' cap structure to which the functional group A has been introduced and the insoluble support having the functional group B can be produced. Examples of haloacetyl groups include, but are not limited to, chloroacetyl, bromoacetyl, and iodoacetyl. The haloacetyl group may be optionally substituted.

[0055] When the functional group A of the mRNA cap analog is a Michael acceptor functional group, by combining it with an insoluble support in which the functional group B is a thiol group, a conjugate can be produced that is a Michael adduct formed by the reaction between the mRNA having a 5' cap structure to which the functional group A has been introduced and the insoluble support having the functional group B. Similarly, when the functional group A of the mRNA cap analog is a thiol group, by combining it with an insoluble support in which the functional group B is a Michael acceptor functional group, a conjugate can be produced that is a Michael adduct of the mRNA having a 5' cap structure to which the functional group A has been introduced and the insoluble support having the functional group B. Here, in this specification, the term "Michael acceptor functional group" refers to a functional group that can serve as a Michael acceptor. Examples of the Michael acceptor functional group include, but are not limited to, a maleimide group and an α,β-unsaturated carbonyl group. The Michael acceptor functional group may be optionally substituted.

[0056] When the functional group A of the mRNA cap analog is an alkynyl group, by combining it with an insoluble support in which the functional group B is a 1,3-dipole functional group, it is possible to produce a conjugate having a bond formed by 1,3-dipole cyclization that occurs when an mRNA having a 5'-cap structure to which the functional group A has been introduced reacts with an insoluble support having the functional group B. Similarly, when the functional group A of the mRNA cap analog is a 1,3-dipole functional group, by combining it with an insoluble support in which the functional group B is an alkynyl group, it is possible to produce a conjugate having a bond formed by 1,3-dipole cyclization that occurs when an mRNA having a 5'-cap structure to which the functional group A has been introduced reacts with an insoluble support having the functional group B. The reaction using this combination is called a click reaction. The click reaction is particularly preferred among the various combinations described above because it allows for quantitative reaction under very mild conditions.

[0057] An alkynyl group is a straight or branched hydrocarbon chain containing one or more triple bonds. An alkynyl group can have, for example, 2 to 20 carbon atoms. Typical alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. An alkynyl group may be optionally substituted.

[0058] The 1,3-dipole functional group is not particularly limited, but examples thereof include a nitrone group, an azomethine imine group, an azomethine ylide group, an azimine group, an azoxy group, a nitro group, a carbonyl ylide group, a carbonylimine group, a carbonyl oxide group, a nitrosoimine group, a nitrosoxide group, a nitrile oxide group, a nitrile imine group, a nitrile ylide group, a diazoalkane group, and an azide group. These may be optionally substituted. For example, when the 1,3-dipole functional group is an azide group, a triazole ring can be formed by bonding it to an alkynyl group via a click reaction.

[0059] The linker structure that can be cleaved by light or fluoride ions is not particularly limited, and examples thereof include an o-nitrobenzyl ether structure or a silyl ether structure. An o-nitrobenzyl ether structure can be cleaved (cut) by application of light (typically, irradiation with ultraviolet light having a wavelength of 365 nm). Furthermore, a silyl ether structure can be cleaved (cut) by the action of fluoride ions. Tetrabutylammonium fluoride (TBAF), cesium fluoride (CsF), etc. can be used as a source of fluoride ions.

[0060] The base moiety of the nucleotide to which the linker is connected is not particularly limited and may be adenine (A), guanine (G), cytosine (C) or uracil (U), with guanine (G) being preferred.

[0061] The position of the sugar moiety of the nucleotide to which the linker is attached is not particularly limited, and may be the 2'-carbon or 3'-carbon, but is preferably the 2'-carbon.

[0062] The position of the base moiety of the nucleotide to which the linker is connected is not particularly limited, but in the case of adenine or guanine having a purine skeleton, it is preferably carbon 2 or carbon 6. In addition, in the case of cytosine or uracil having a pyrimidine skeleton, it is preferably nitrogen at position 3, carbon 4, carbon 5 or carbon 6.

[0063] The mRNA cap analogue according to the present invention can be typically represented by the following general formula (1) or (2): mRNA cap analogues having such a structure also have good efficiency in in vitro transcription reactions.

[0064]

[0065]

[0066] In the above formula, R1 is OH, OCH3 or a group represented by the following general formula (3).

[0067]

[0068] In the above formula, R2 is OH or OCH3. In the above formula, R3 is O, OH, NH2, NHCH3, or N(CH3)2. When R3 is O, the bond between the C bonded to R3 and R3 (represented by a dotted line and a solid line) is a double bond, and the bond between the C bonded to R3 and the adjacent N (represented by a dotted line and a solid line) is a single bond. When R3 is OH, NH2, NHCH3, or N(CH3)2, the bond between the C bonded to R3 and R3 (represented by a dotted line and a solid line) is a single bond, and the bond between the C bonded to R3 and the adjacent N (represented by a dotted line and a solid line) is a double bond. R3 is preferably O.

[0069] In the above formula, R4 is NH2, NHCH3, N(CH3)2, or H. R4 is preferably NH2. In the above formula, R5 is a propargyloxy group or an azidoethoxy group. R5 is preferably an azidoethoxy group. In the above formula, R6 and R7 may be the same or different and are OH or OCH3. In the above formula, a is 3 to 6. It is preferable that a is 3. In the above formula, b is 0 to 19. In the above formula, Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosin-1-yl, 5-methylcytosin-1-yl, uracil-1-yl, uracil-5-yl, or 1-methyluracil-5-yl.

[0070] The mRNA cap analogs according to the embodiments of the present invention can be produced according to known methods. An example of a method for producing an mRNA cap analog according to the embodiments of the present invention is described below. The method for producing an mRNA cap analog according to the embodiments of the present invention includes the steps of: introducing a linker, which includes a functional group A selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group, and which has a structure cleavable by light or fluoride ions, into the sugar moiety or base moiety of a first nucleotide; and linking the first nucleotide with the linker introduced therein to a second nucleotide in the presence of magnesium chloride. By employing a production method including these steps, mRNA cap analogs can be produced efficiently.

[0071] The linker can be synthesized by a known method depending on the type of functional group A and the structure that can be cleaved by light or fluoride ions. The conditions for introducing the linker into the sugar moiety or the base moiety of the first nucleotide are not particularly limited and may be appropriately set depending on the type of linker and the first nucleotide. The first nucleotide is not particularly limited, but for example, guanylic acid can be used.

[0072] The conditions for linking the first nucleotide into which a linker has been introduced to the second nucleotide are not particularly limited except that the linking is performed in the presence of magnesium chloride, and may be appropriately set depending on the types of the linker, the first nucleotide, and the second nucleotide. The second nucleotide is not particularly limited, and for example, guanosine diphosphate can be used.

[0073] <Method for Producing mRNA Having a 5'-Cap Structure> A method for producing mRNA having a 5'-cap structure (hereinafter sometimes abbreviated as "mRNA production method") according to an embodiment of the present invention comprises a step of preparing a preparation containing mRNA having a 5'-cap structure (hereinafter referred to as "Step 1"), a step of producing a conjugate (hereinafter referred to as "Step 2"), a step of filtering and washing the conjugate (hereinafter referred to as "Step 3"), and a step of isolating mRNA having a 5'-cap structure (hereinafter referred to as "Step 4"). The production method including these steps enables separation and removal of mRNA without a 5'-cap structure that is by-produced in mRNA production by in vitro transcription, and allows mRNA having a 5'-cap structure to be isolated, making the method useful for use as a pharmaceutical active ingredient.

[0074] The first step involves mixing an mRNA cap analogue having a nucleotide sugar or base moiety with a linker containing functional group A and cleavable by light or fluoride ions, a nucleoside triphosphate, template DNA, and RNA polymerase, and obtaining a preparation containing mRNA with a 5' cap structure by in vitro transcription. The preparation obtained in this step also includes mRNA without a 5' cap structure. Step 1 can be performed according to existing conditions, except for the use of an mRNA cap analogue with a specific structure. Details of the mRNA cap analogue with a specific structure have already been described above, so further description is omitted.

[0075] The second step is to mix the above-mentioned preparation with an insoluble carrier having a functional group B capable of reacting with functional group A to produce a conjugate between an mRNA having a 5'-cap structure and the insoluble carrier. The conditions for producing the conjugate are not particularly limited and may be set depending on the type of functional group A of the mRNA having a 5'-cap structure and the type of functional group B of the insoluble carrier. The functional groups A and B may be a combination of an amino group and a carboxylic acid activated ester, an aminooxy group or a hydrazino group and a carbonyl group, a thiol group and a thiol group, a haloacetyl group, or a Michael acceptor functional group, or an alkynyl group and a 1,3-dipole functional group. Here, for example, a combination of an amino group and a carboxylic acid activated ester refers to both a combination in which functional group A is an amino group and functional group B is a carboxylic acid activated ester, and a combination in which functional group A is a carboxylic acid activated ester and functional group B is an amino group. The same applies to other combinations. By using such combinations, the various bonds described above can be formed. Among these combinations, the combination of an alkynyl group and a 1,3-dipole functional group, which allows for a click reaction that can be carried out under simple conditions, is preferred.

[0076] The insoluble carrier is not particularly limited as long as it is insoluble in aqueous solution. Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Various commercially available insoluble carriers may also be used. Examples of commercially available carriers include Primer Support (registered trademark) 5G (manufactured by Cytiva) and EAH Sepharose (registered trademark) 4B (manufactured by Cytiva), which are used in oligonucleotide synthesis.

[0077] When an mRNA cap analogue represented by the above general formula (1) or (2) in which R5 is a propargyloxy group is used, the insoluble carrier can be represented by the following formula (4).

[0078]

[0079] In formula (4), Sup is an insoluble carrier structural part. The insoluble carrier structural part is a part constituted by the above-mentioned carrier.

[0080] When an mRNA cap analogue represented by the above general formula (1) or (2) in which R is an azidoethoxy group is used, the insoluble support can be represented by the following formula (5), (6), or (7): With such an insoluble support having a cyclooctyne group, the click reaction can be carried out even in the absence of copper(I) ions, thereby preventing copper(I) ion contamination during mRNA production.

[0081]

[0082] In the formula (5), (6) or (7), Sup is an insoluble carrier structural part. The insoluble carrier structural part is a part constituted by the above-mentioned carrier.

[0083] The third step is filtering and washing the conjugate. The conjugate of the mRNA having a 5'-cap structure and the insoluble carrier is solid, whereas the mRNA without a 5'-cap structure is liquid. Therefore, by filtering and washing the conjugate, the mRNA without a 5'-cap structure can be separated and removed. The filtering and washing methods and conditions are not particularly limited, and can be performed according to known methods.

[0084] The fourth step is to cleave the linker by applying light or fluoride ions to the conjugate and isolate mRNA having a 5'-cap structure. Cleavage of the linker using an acid or alkali may affect mRNA having a 5'-cap structure, but cleavage of the linker using light or fluoride ions reduces the effect on mRNA having a 5'-cap structure. The linker cleavage conditions are not particularly limited and may be appropriately determined depending on the structure of the linker that can be cleaved by light or fluoride ions. For example, if the structure is an o-nitrobenzyl ether structure, the linker can be cleaved (cut) by irradiating the compound with ultraviolet light having a wavelength of 365 nm. Furthermore, if the structure is a silyl ether structure, the linker can be cleaved (cut) by applying a solution containing fluoride ions to the compound.

[0085] <Kit> A kit according to an embodiment of the present invention is used for producing mRNA having a 5'-cap structure. The kit according to an embodiment of the present invention includes an mRNA cap analog having a linker at the sugar or base moiety of a nucleotide, the linker having a structure cleavable by light or fluoride ions and containing functional group A selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group; and an insoluble support having functional group B reactive with functional group A. By providing a kit including such an mRNA cap analog and an insoluble support, it is possible to separate and remove mRNA that does not have a 5'-cap structure, which is a by-product of the production of mRNA having a 5'-cap structure, and to isolate mRNA having a 5'-cap structure. The details of the mRNA cap analog and insoluble support used in the kit according to an embodiment of the present invention have already been described above, and therefore further description thereof will be omitted.

[0086] The kit according to the embodiment of the present invention may further include one or more components selected from nucleoside triphosphates, template DNA, and RNA polymerase, as needed. These components are raw materials used in preparing mRNA having a 5'-cap structure by in vitro transcription. Therefore, a kit including these components facilitates the preparation of mRNA having a 5'-cap structure.

[0087] The present invention will be described below with reference to examples, but is not limited to these examples. In the examples, Me represents a methyl group, Et represents an ethyl group, and Ac represents an acetyl group.

[0088] (1. Model Experiments on Linker Cleavage) To conduct model experiments on linker cleavage, nucleoside derivatives incorporating various linkers were synthesized and cleavage experiments were carried out. 2-nitro-5-hydroxybenzaldehyde was used as the starting material to synthesize three types of linkers, each having a methoxy group, a propargyloxy group, and a 2-azidoethoxy group as the functional group A. Nucleoside derivatives were synthesized by introducing these linkers into the base moiety of guanosine.

[0089] <Synthesis of a linker having a methoxy group as functional group A [2-nitro-5-methoxybenzyl alcohol (compound 3)]> First, 2-nitro-5-methoxybenzyl alcohol (compound 3) was synthesized according to the following scheme.

[0090]

[0091] Compound 1 (200 mg, 1.20 mmol) was dissolved in N,N-dimethylformamide (3 mL), and methyl iodide (149 μL, 2.39 mmol) and potassium carbonate (662 mg, 4.79 mmol) were added, followed by stirring at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate and washed twice with deionized water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (hexane:ethyl acetate=3:1) to obtain compound 2 (157 mg, 867 μmol, 72.4%). 1H-NMR (CDCl3): δ10.49 (s, 1H), 8.17 (d, 1H), 7.34 (d, 1H), 7.16 (dd, 1H), 3.96 (s, 3H)

[0092] Next, compound 3 was synthesized according to the following scheme.

[0093]

[0094] Compound 2 (157 mg, 867 μmol) was dissolved in tetrahydrofuran (2 mL), methanol (4.3 mL) was added, and the mixture was ice-cooled. Next, sodium borohydride (16.4 mg, 433 μmol) was added, and the mixture was stirred under the same conditions for 1 hour. The reaction solution was diluted with ethyl acetate and washed with water. The organic layer was then dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (hexane:ethyl acetate = 3:1) to obtain compound 3 (157 mg, 857 μmol, 98.9%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 7.22 (d, 1H), 6.90 (dd, 1H), 5.00 (d, 2H) 3.92 (s, 3H), 2.57 (t, 1H)

[0095] <Synthesis of linker having propargyloxy group as functional group A [2-nitro-5-propargyloxybenzyl alcohol (compound 5)]> 2-Nitro-5-propargyloxybenzyl alcohol (compound 5) was synthesized according to the following scheme.

[0096]

[0097] Compound 1 (3.00 g, 18.0 mmol) was dissolved in N,N-dimethylformamide (45.0 mL), and propargyl bromide (1.40 mL, 18.0 mmol) and potassium carbonate (5.0 g, 36 mmol) were added. The mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with ethyl acetate and washed twice with deionized water. The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain crude compound 4. Crude compound 4 was then dissolved in tetrahydrofuran (18.0 mL), and methanol (90 mL) was added. The mixture was then ice-cooled. Sodium borohydride (0.34 g, 9.0 mmol) was then added, and the mixture was stirred under the same conditions for 20 minutes. The reaction mixture was concentrated, diluted with ethyl acetate, and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated, and the concentrate was crystallized from hexane-ethyl acetate. The crystals were collected by filtration, washed, and dried under vacuum to obtain compound 5 (3.32 g, 16.0 mmol, 89.0%). 1 H-NMR (CDCl3): δ8.20 (d, 1H), 7.33 (d, 1H), 7.00 (dd, 1H), 5.01 (d, 1H), 4.82 (d, 1H), 2.59 (t, 1H), 2.54 (t, 1H)

[0098] <Synthesis of a linker having a 2-azidoethoxy group as functional group A [(5-(2-azidoethoxy)-2-nitrophenyl)methanol (compound 12)]> First, methyl 2-(3-formyl-4-phenoxy)acetate (compound 6) was synthesized according to the following scheme.

[0099]

[0100] Compound 1 (10.0 g, 59.8 mmol) was dissolved in N,N-dimethylformamide (120 mL), and then potassium carbonate (13.7 g, 120 mmol) and methyl bromoacetate (8.27 mL, 89.8 mmol) were added sequentially, followed by stirring for 1 hour. After the reaction was complete, the mixture was diluted with ethyl acetate, and the organic layer was washed twice with water (120 mL). The aqueous layer was extracted with ethyl acetate (120 mL), and the organic layers were combined. Subsequently, the organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. Hexane was added to the residue. The resulting solid was filtered, washed with hexane, and dried to obtain compound 6 (12.9 g, 90.1%). 1H-NMR (CDCl3): δ10.45 (s, 1H), 8.16 (d, 1H), 7.29 (d, 1H), 7.19 (dd, 1H), 4.79 (s, 2H), 3.82 (s, 3H)

[0101] Next, compound 7 was synthesized according to the following scheme.

[0102]

[0103] Compound 6 (12.7 g, 53.1 mmol) was dissolved in a mixed solvent of tetrahydrofuran and methanol (1:1, 106 mL), and then sodium borohydride (703 mg, 18.6 mmol) was added under ice cooling and stirred for 1 hour. After completion of the reaction, the reaction mixture was quenched with acetone (5 mL) and then concentrated. The concentrate was dissolved in ethyl acetate, and the organic layer was washed with saturated aqueous ammonium chloride. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, and hexane was added to the residue. The resulting solid was filtered, washed with hexane, and dried to obtain compound 7 (11.31 g, 88.3%). 1 H-NMR (DMSO-d6): δ8.12 (d, 1H), 7.33 (d, 1H), 7.04 (dd, 1H), 5.61 (t, 1H), 4.99 (s, 2H), 4.84 (d, 2H), 3.72 (s, 3H)

[0104] Next, compound 8 was synthesized according to the following scheme.

[0105]

[0106] Compound 7 (11.1 g, 46.2 mmol) was dissolved in acetonitrile (92 mL), and then imidazole (7.55 g, 111 mmol) and tert-butylchlorodimethylsilane (7.66 g, 50.8 mmol) were added sequentially, followed by stirring for 1 hour. After completion of the reaction, the reaction mixture was quenched with methanol (5 mL) and then concentrated. The concentrate was dissolved in ethyl acetate, and the organic layer was washed with water. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 3 / 17) to obtain compound 8 (15.4 g, 93.9%). 1H-NMR (CDCl3): δ8.18 (d, 1H), 7.41 (t, 1H), 6.88 (dd, 1H), 5.11 (s, 2H), 4.75 (s, 2H), 3.81 (s, 3H), 0.98 (s, 9H), 0.15 (s, 6H)

[0107] Next, 2-(3-(((tert-butyldimethylsilyl)oxy)methyl)-4-nitrophenoxy)ethan-1-ol (compound 9) was synthesized according to the following scheme.

[0108]

[0109] Compound 8 (15.2 g, 42.8 mmol) was dissolved in tetrahydrofuran (86 mL), and then a 4 mol / L tetrahydrofuran solution of lithium borohydride (10.7 mL, 42.8 mmol) was added under ice-cooling, followed by stirring at room temperature for 2 hours. After completion of the reaction, the reaction was quenched with acetone (10 mL) under ice-cooling, and the reaction solution was concentrated. The concentrate was dissolved in ethyl acetate, and the organic layer was washed with a saturated aqueous ammonium chloride solution. Subsequently, the organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / n-hexane = 1 / 4 → 1 / 1) to obtain compound 9 (13.1 g, 93.6%). 1 H-NMR (CDCl3): δ8.18 (d, 1H), 7.46 (t, 1H), 6.88 (dd, 1H), 5.12 (s, 2H), 4.19 (t, 2H), 4.03 (td, 2H), 2.04-1.98 (m, 1H), 0.98 (s, 9H), 0.16 (s, 6H)

[0110] Next, 2-(3-formyl-4-nitrophenoxy)ethyl methanesulfonate (compound 10) was synthesized according to the following scheme.

[0111]

[0112] Compound 9 (3.00 g, 9.16 mmol) was dissolved in pyridine (18 mL), and then N,N,N',N'-tetramethylethylenediamine (0.34 mL, 2.29 mmol) and methanesulfonyl chloride (0.851 mL, 11.0 mmol) were added sequentially under ice cooling, followed by stirring for 30 minutes. After completion of the reaction, the mixture was quenched with saturated aqueous sodium bicarbonate solution and then extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and the solvent was evaporated under reduced pressure, followed by azeotropy with toluene twice. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1 / 4 → 1 / 1) to obtain compound 10 (3.48 g, 93.7%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 7.44 (t, 1H), 6.87 (dd, 1H), 5.11 (s, 2H), 4. 62-4.60 (m, 2H), 4.35-4.33 (m, 2H), 3.10 (s, 3H), 0.98 (s, 9H), 0.15 (s, 6H)

[0113] Next, compound 11 was synthesized according to the following scheme.

[0114]

[0115] Compound 10 (3.35 g, 8.26 mmol) was dissolved in N,N-dimethylformamide (17 mL), and then sodium azide (806 mg, 12.4 mmol) was added and stirred at 80°C for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, water (30 mL) was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water, dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1 / 19 → 1 / 9) to obtain compound 11 (2.82 g, 96.9%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 7.46 (t, 1H), 6.88 (dd, 1H), 5.12 (s, 2H), 4.24 (t, 2H), 3.66 (t, 2H), 0.98 (s, 9H), 0.16 (s, 6H)

[0116] Next, (5-(2-azidoethoxy)-2-nitrophenyl)methanol (compound 12) was synthesized according to the following scheme.

[0117]

[0118] Compound 11 (1.51 g, 4.28 mmol) was dissolved in tetrahydrofuran (21.4 mL), and a 1.0 mol / L solution of tetrabutylammonium fluoride in tetrahydrofuran (8.56 mL, 8.56 mmol) was added, followed by stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (Biotage® Sfar Silica HC D, 25 g, 10-25 wt% AcOEt in hexane) to obtain compound 12 (0.68 g, 2.85 mmol, 66.7%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 7.27 (d, 1H), 6.93 (dd, 1H), 5.01 (d, 2H), 4.26 (t, 2H), 3.67 (t, 2H), 2.54 (t, 1H)

[0119] <Nucleoside derivative [O 6 Synthesis of 2-nitro-5-methoxybenzylguanosine (Compound 15)

[0120] First, according to the following scheme, 6 3',5'-tri-O-tert-butyldimethylsilylguanosine (compound 14) was synthesized.

[0121] Compound 13 (6.05 g, 6.78 mmol) and compound 3 (1.86 g, 10.2 mmol) were dissolved in 1,2-dimethoxyethane (67.8 mL), molecular sieves 4A (3.00 g) were added, and the mixture was stirred at room temperature for 1 hour. 1,4-diazabicyclo[2.2.2]octane (1.52 g, 13.6 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 1,8-diazabicyclo[5.4.0]undec-7-ene (2.03 mL, 13.6 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The molecular sieves were removed by filtration, and the filtrate was diluted with ethyl acetate and washed with 1 mol / L hydrochloric acid and saturated sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was then purified by medium-pressure silica gel column chromatography (Biotage (registered trademark) Sfar Silica HC D, 25 g, 10 to 20 wt% AcOEt in Hexane) to obtain Compound 14 (4.20 g, 5.31 mmol, 78.3%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 8.01 (s, 1H), 7.30 (d, 1H), 6.87 (dd, 1H), 6.02 (d , 1H), 5.97 (d, 1H), 5.91 (d, 1H), 4.88 (s, 2H), 4.53 (t, 1H), 4.30 (t, 1H), 4.10 ( dd, 1H), 3.99 (dd, 1H), 3.84 (s, 3H), 3.78 (dd, 1H), 0.95 (s, 9H), 0.93 (s, 9H), 0 .14 ​​(s, 3H), 0.13 (s, 3H), 0.11 (s, 3H), 0.10 (s, 3H), -0.02 (s, 1H), -0.16 (s, 1H)

[0122] Next, according to the following scheme, 6 1-(2-nitro-5-methoxybenzyl)guanosine (compound 15) was synthesized.

[0123]

[0124] Compound 14 (4.20 g, 5.31 mmol) was dissolved in methanol (53.1 mL), and ammonium fluoride (3.03 g, 53.1 mmol) was added. The mixture was heated and stirred at 50° C. for 11 hours. After cooling, the precipitated solid was collected by filtration and washed with methanol (3.26 g). The resulting solid was crystallized from deionized water to give compound 15 (2.28 g, 5.08 mmol, 95.8%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 8.01 (s, 1H), 7.30 (d, 1H), 6.87 (dd, 1H), 6.02 (d , 1H), 5.97 (d, 1H), 5.91 (d, 1H), 4.88 (s, 2H), 4.53 (t, 1H), 4.30 (t, 1H), 4.10 ( dd, 1H), 3.99 (dd, 1H), 3.84 (s, 3H), 3.78 (dd, 1H), 0.95 (s, 9H), 0.93 (s, 9H), 0 .14 ​​(s, 3H), 0.13 (s, 3H), 0.11 (s, 3H), 0.10 (s, 3H), -0.02 (s, 1H), -0.16 (s, 1H)

[0125] <Nucleoside derivative [O 6 Synthesis of 2-nitro-5-propargyloxybenzyl)guanosine (Compound 17)> First, according to the following scheme, 6 -(2-nitro-5-propargyloxybenzyl)-2',3',5'-tri-O-tert-butyldimethylsilylguanosine (Compound 16) was synthesized.

[0126]

[0127] Compound 13 (2.5 g, 2.8 mmol) and compound 5 (0.70 g, 3.4 mmol) were dissolved in 1,2-dimethoxyethane (28 mL), molecular sieves 4A (5.0 g) was added, and the mixture was stirred at room temperature for 1 hour. 1,4-diazabicyclo[2.2.2]octane (0.63 mg, 5.6 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 1,8-diazabicyclo[5.4.0]undec-7-ene (0.84 mL, 5.6 mmol) was then added, and the mixture was stirred at room temperature for 12 hours. The molecular sieves were removed by filtration, and the filtrate was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was then purified by medium-pressure silica gel column chromatography (Biotage (registered trademark) Sfar Silica HC D, 25 g, 20 w / w% AcOEt in Hexane) to obtain Compound 16 (1.60 g, 1.96 mmol, 70%). 1 H-NMR (CDCl3): δ8.20 (d, 1H), 8.02 (s, 1H), 7.35 (d, 1H), 6.97 (dd, 1H), 6.03 (d, 1H), 5.97 (d, 1H), 5.91 (d, 1H), 4.94 (s, 2H), 4.71 (d, 1H), 4.52 (t, 1H), 4.29 (t, 1H), 4.10 ( dd, 1H), 3.98 (dd, 1H), 3.78 (dd, 1H), 2.46 (t, 1H), 0.96 (s, 9H), 0.92 (s. 9H), 0.81 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H), 0.10 (s, 3H), -0.02 (s, 3H), -0.15 (s, 3H)

[0128] Next, according to the following scheme, 6 -(2-nitro-5-propargyloxybenzyl)guanosine (compound 17) was synthesized.

[0129]

[0130] Compound 16 (1.58 g, 1.94 mmol) was dissolved in methanol (19.4 mL), and ammonium fluoride (1.11 g, 19.4 mmol) was added. The mixture was heated and stirred at 50° C. for 12 hours. After cooling, the precipitated solid was collected by filtration and washed with methanol (1.07 g). The resulting solid was crystallized from deionized water to give compound 17 (0.90 g, 1.9 mmol, 98%). 1 H-NMR (DMSO-d6): δ8.27 (d, 1H), 8.16 (s, 1H), 7.27 (d, 1H), 7.21 (dd, 1H), 6.52 (s, 2H), 5.92 (d, 1H), 5.88 (d, 1H), 5.81 (d, 1H), 5.43 (d, 1H), 5.14 (d, 1H), 5.09 (t, 1H), 4.96 (d, 2H), 4.50 (dd, 1H), 4.12 (dd, 1H), 3.90 (dd, 1H), 3.64 (dt, 1H), 3.60 (t, 1H), 3.54 (dt, 1H)

[0131] <(5-(2-azidoethoxy)-2-nitrophenyl)methanol (10)-introduced nucleoside derivative [O 6 Synthesis of 5-(2-nitro-5-(2-azidoethoxy)benzyl)guanosine (Compound 19)> First, according to the following scheme, 6 -(2-nitro-5-(2-azidoethoxy)benzyl)-2′,3′,5′-tri-O-tert-butyldimethylsilylguanosine (compound 18) was synthesized.

[0132]

[0133] Compound 13 (2.56 g, 2.87 mmol) and compound 12 (873 mg, 5.74 mmol) were dissolved in 1,2-dimethoxyethane (39.2 mL), molecular sieves 4A (5.12 g) was added, and the mixture was stirred at room temperature for 1 hour. 1,4-diazabicyclo[2.2.2]octane (644 mg, 5.74 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 1,8-diazabicyclo[5.4.0]undec-7-ene (0.858 mL, 5.74 mmol) was added, and the mixture was stirred at room temperature for 12 hours. The molecular sieves were removed by filtration, and the filtrate was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (Biotage (registered trademark) Sfar Silica HC D, 25 g, 20 w / w% AcOEt in Hexane) to obtain Compound 18 (1.74 g, 2.06 mmol, 71.7%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 8.01 (s, 1H), 7.33 (d, 1H), 6.91 (dd, 1H), 6.04 (d, 1H), 5.96 (d, 1H), 5.92 (d, 1H), 5.03 (s, 2H), 4.52 (t, 1H), 4.29 (t, 1H), 4.18 (t, 2H), 4.11 ( dd, 1H), 3.98 (dd, 1H), 3.78 (dd, 1H), 3.61 (t, 2H), 0.96 (s, 9H), 0.92 (s, 9H), 0.80 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H), 0.11 (s, 3H), 0.10 (s, 3H), -0.03 (s, 3H), -0.17 (s, 3H)

[0134] Next, according to the following scheme, 6 19) was synthesized.

[0135]

[0136] Compound 18 (1.70 g, 2.01 mmol) was dissolved in methanol (20.1 mL), and ammonium fluoride (1.15 g, 20.1 mmol) was added. The mixture was heated and stirred at 50° C. for 12 hours. After cooling, the precipitated solid was collected by filtration and washed with methanol (1.07 g). The resulting solid was crystallized from deionized water to give compound 19 (0.91 g, 1.8 mmol, 90%). 1 H-NMR (DMSO-d6): δ8.26 (d, 1H), 8.16 (s, 1H), 7.26 (d, 1H), 7.19 (dd, 1H), 6.53 (s, 2H), 5.92 (d, 1H), 5.88 (d, 1H), 5.81 (d, 1H), 5.42 ( d, 1H), 5.14 (d, 1H), 5.09 (t, 1H), 4.49 (dd, 1H), 4.30 (t, 2H), 4.12 (dd, 1H), 3.90 (dd, 1H), 3.71 (t, 2H), 3.64 (dt, 1H), 3.55 (ddd, 1H)

[0137] <Cleavage test of linker-introduced nucleoside derivative> A linker cleavage test was carried out by irradiating compound 15 with ultraviolet light having a wavelength of 365 nm. Specifically, an ultraviolet lamp was placed in an aqueous solution of compound 15, and ultraviolet light having a wavelength of 365 nm was irradiated at room temperature for 2 hours to investigate whether the following photocleavage reaction proceeded.

[0138]

[0139] The photocleavage reaction was evaluated by analyzing an aqueous solution of Compound 15 by HPLC before and after UV irradiation, and the results are shown in Figure 3.

[0140] As shown in Figure 3, after UV irradiation, the peak of compound 15 disappeared and a new peak appeared. A spike test was performed on the component of this peak, and analysis based on the data obtained from the PDA detector revealed that it was guanosine. Therefore, it was confirmed that the above-mentioned photocleavage reaction had progressed. Although no cleavage test was performed on compounds 17 and 19, since they have the same photocleavage structure as compound 15, it is assumed that the photocleavage reaction will proceed in the same way.

[0141] (2. Model Experiments on Binding Between Linkers and Insoluble Carriers and Linker Cleavage) In order to conduct model experiments on binding between linkers and insoluble carriers and linker cleavage, oligonucleotide derivatives incorporating specific linkers were synthesized and bound to insoluble carriers, followed by cleavage tests. As oligonucleotide derivatives, a dimer (compound 30) and a 21-mer (compound 31) having the following sequences were synthesized. Sequence of dimer (compound 30): 5'-QT-3' Sequence of 21-mer (compound 31): 5'-QTT TTT TTT TTT TTT TTT TTT-3' Q represents guanosine (compound 26) incorporating a linker that is cleaved by light.

[0142] <Linker-introduced guanosine amidite [O 6 -(2-nitro-5-propargyloxybenzyl)-N 2 Synthesis of N,N-phenoxyacetyl-5'-O-dimethoxytrityl-2'-O-methylguanosine-3'-(2-cyanoethyl)-N,N-diisopropylphosphoramidite (Compound 26) according to the following scheme: 6 -(2-nitro-5-propargyloxybenzyl)-N 2 -Phenoxyacetyl-5'-O-dimethoxytrityl-2'-O-methylguanosine 3'-amidite (Compound 26) was synthesized.

[0143]

[0144] Compound 20 (Bioorganic & Medicinal Chemistry (2013), 21(22), 6886-6892) (6.14 g, 9.38 mmol) was dissolved in methanol (93.8 mL) and tetrahydrofuran (30 mL), and 28 wt% aqueous ammonia (50 mL) was added and stirred at room temperature for 16 hours. 28 wt% aqueous ammonia (50 mL) was then added, and the mixture was stirred at room temperature for 25 hours, and the reaction solution was concentrated. The concentrate was azeotroped three times with N,N-dimethylformamide, and the residue was dissolved in N,N-dimethylformamide (18.8 mL). tert-Butylchlorodimethylsilane (2.12 g, 14.1 mmol) and imidazole (1.92 g, 28.1 mmol) were added, and the mixture was stirred at room temperature for 21 hours. The reaction solution was diluted with ethyl acetate and washed with water, and the precipitated solid was collected by filtration and washed. The organic layer of the filtrate was concentrated, and the precipitated solid was collected by filtration and washed. The resulting solids were combined and dried in vacuo to give Compound 21 (5.98 g, 8.38 mmol, 89.3%).

[0145] Next, compound 21 (5.93 g, 8.31 mmol) was dissolved in dichloromethane (55.4 mL), and 2,4,6-triisopropylbenzenesulfonyl chloride (4.03 g, 13.3 mmol), triethylamine (1.85 mL, 13.3 mmol), and 4-dimethylaminopyridine (101 mg, 0.831 mmol) were added, followed by stirring at room temperature for 48 hours. The reaction solution was diluted with chloroform and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate and concentrated, and the concentrate was purified by medium-pressure silica gel column chromatography (Biotage® Sfar Silica HC D, 25 g, 20 wt% AcOEt in hexane) to obtain compound 22 (7.52 g, 7.67 mmol, 92.4%).

[0146] Next, compound 22 (6.00 g, 6.12 mmol) and compound 5 (1.52 g, 7.34 mmol) were dissolved in 1,2-dimethoxyethane (61 mL), molecular sieves 4A (12.0 g) was added, and the mixture was stirred at room temperature for 1 hour. 1,4-diazabicyclo[2.2.2]octane (1.37 mg, 12.2 mmol) was added, and the mixture was stirred at room temperature for 1 hour. 1,8-diazabicyclo[5.4.0]undec-7-ene (1.83 mL, 12.2 mmol) was added, and the mixture was stirred at room temperature for 15 hours. The molecular sieves were removed by filtration, and the filtrate was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated, and the concentrate was purified by medium-pressure silica gel column chromatography (Biotage (registered trademark) Sfar Silica HC D, 50 g, 20 to 35 wt% AcOEt in Hexane) to obtain Compound 23 (4.09 g, 4.53 mmol, 74.0%).

[0147] Next, compound 23 (2.0 g, 2.2 mmol) was dissolved in acetonitrile (11 mL) and pyridine (4.4 mL), and phenoxyacetyl chloride (0.37 mL, 2.7 mmol) was added under ice cooling and stirred under the same conditions for 1 hour. The reaction mixture was returned to room temperature and stirred for an additional 24 hours, after which deionized water (0.5 mL) was added and the mixture was concentrated. Ethyl acetate was added to the concentrate, and the mixture was washed with saturated aqueous sodium bicarbonate. The organic layer was then dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (Biotage® Sfar Silica HC D, 25 g, 25-50 wt% AcOEt in hexane) to obtain compound 24 (1.84 g, 1.77 mmol, 80.0%).

[0148] Next, compound 24 (1.80 g, 1.74 mmol) was dissolved in tetrahydrofuran (8.68 mL), and a 1.0 mol / L solution of tetrabutylammonium fluoride in tetrahydrofuran (2.60 mL, 2.60 mmol) was added. The mixture was stirred at room temperature for 30 minutes. After concentrating the reaction mixture, ethyl acetate was added to the concentrate, washed with saturated brine, and the organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (Biotage® Sfar Silica HC1, 25 g, 1 wt% MeOH in CHCl3; fractions containing by-products were repurified under the same conditions) to obtain compound 25 (1.31 g, 1.42 mmol, 81.8%).

[0149] Next, compound 25 (1.03 g, 1.12 mmol) was dissolved in acetonitrile (5.58 mL), and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (0.53 mL, 1.67 mmol) and 1H-tetrazole (117 mg, 1.67 mmol) were added, followed by stirring at room temperature for 3 hours. The reaction solution was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrate was purified by medium-pressure silica gel column chromatography (Yamazen Silica Gel 2 L, 3.0 × 20.0 cm, 55 g, 25-33-50 wt% AcOEt in hexane). The fractions containing the target product were concentrated, and the concentrate was dissolved in dichloromethane (3 mL) and added dropwise to hexane (500 mL). The resulting precipitate was collected by filtration, washed (hexane), and then dried in vacuo to obtain Compound 26 (0.89 g, 0.79 mmol, 71.0%). HRMS (ESI) m / z: C 59 H 62 N8O 13 P - [M-H] - Theoretical value for ions: 1121.4179; Measured value: 1121.4127

[0150] <Synthesis of Insoluble Carrier Having Azide Group (Compound 29)> An insoluble carrier having an azide group (Compound 29) was synthesized according to the following scheme.

[0151]

[0152] Compound 27 (100 mg, 0.429 mmol) and triethylamine (0.0717 mL, 0.515 mmol) were dissolved in acetonitrile (1.95 mL), and di(N-succinimidyl)carbonate (132 mg, 0.515 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The resulting crude compound 28 was azeotroped three times with acetonitrile, and the residue was dissolved in acetonitrile (10 mL). Triethylamine (0.0717 mL, 0.515 mmol) and Cytiva Primer support® 5G amino (1.23 g, 0.45 mmol / g) were added, and the mixture was stirred at room temperature for 17 hours. After confirming the absence of raw materials in the supernatant by TLC, the insoluble matter was collected by filtration. The mixture was then washed with acetonitrile and dried under vacuum (1.29 g). To the resulting support, Cap A [1-methylimidazole-acetonitrile (2:8)] (5 mL), Cap B1 [acetic anhydride-acetonitrile (4:6)] (2.5 mL), and Cap B2 [2,6-lutidine-acetonitrile (4:6)] (2.5 mL) were added and stirred at room temperature for 18 hours. The insoluble material was collected by filtration, washed with acetonitrile, and dried under vacuum to obtain insoluble support (compound 29) (1.28 g, 0.335 mmol / g assuming all azide linkers were bound).

[0153] <Synthesis of Oligonucleotide Derivatives> Using the amidite of the compound (compound 26) synthesized above, dT amidite, and a solid phase support, two types of oligonucleotide derivatives (a dimer (compound 30) and a 21-mer (compound 31)) were synthesized using an automatic nucleic acid synthesizer by the phosphoramidite method. At this time, the amidite and dT amidite of compound 26 were diluted with acetonitrile to a concentration of 0.15 mol / L. Other synthesis conditions were as follows. Carrier: Primer support (registered trademark) 5G T350 Equipment: Akta oligopilot 10 Synthesis scale: 50 μmol Amidite: Q amidite 23 (0.15 mol / L acetonitrile solution, 3 equivalents), dT (0.15 mol / L acetonitrile solution, 1.8 equivalents) Recycling time: 12 minutes Next, the solid phase carrier obtained by the above synthesis was transferred to a sealed glass tube, 28 wt% aqueous ammonia (10 mL) was added, and the mixture was heated and stirred at 60°C for 18 hours. After cooling, the solid phase carrier was removed by filtration and washed with 50 wt% ethanol (100 mL). The filtrate and washings were combined and concentrated, and then the volume was adjusted to 50 mL with deionized water.

[0154] <Binding (Click Reaction) of Oligonucleotide Derivatives (Compounds 30 and 31) with Insoluble Support (Compound 29)> A click reaction between a dimeric oligonucleotide derivative (Compound 30) and an insoluble support (Compound 29) was carried out according to the following scheme.

[0155]

[0156] A dimeric oligonucleotide derivative (compound 30) (3.37 μmol), copper(I) iodide (12.8 mg in 50 w / w% MeCN, 3.37 mL), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (71.5 mg), and sodium ascorbate (66.8 mg) were mixed, and then an insoluble carrier (compound 29) (100 mg, 33.5 μmol) was added and stirred at room temperature for 2 hours to carry out the click reaction. The insoluble carrier (reactant) was then collected by filtration, washed with MeCN, and vacuum dried (104 mg). The resulting insoluble carrier was suspended in acetonitrile (1 mL) and 100 mmol / L aqueous disodium ethylenediaminetetraacetate solution (3 mL) and stirred at room temperature for 16 hours. The insoluble carrier was filtered, washed with deionized water and then with acetonitrile, and dried in vacuo to obtain a conjugate of the oligonucleotide derivative and the insoluble carrier (compound 32).

[0157] The solution (supernatant) after the click reaction was analyzed by HPLC and compared with the HPLC results for the solution before the click reaction (aqueous solution of the dimeric oligonucleotide derivative). The results are shown in Figure 4. As shown in Figure 4, the peak of the dimeric oligonucleotide derivative disappeared in the solution after the click reaction, suggesting that the reaction between the dimeric oligonucleotide derivative (compound 30) and the insoluble carrier (compound 29) had progressed.

[0158] Similarly, a click reaction was carried out between a 21-mer oligonucleotide derivative (compound 31) and an insoluble support (compound 29) according to the following scheme.

[0159]

[0160] A 21-mer oligonucleotide derivative (compound 31) (1.12 mL), deionized water (2.24 mL), copper(I) iodide (12.8 mg in 50 w / w% MeCN, 3.37 mL), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (71.5 mg), and sodium ascorbate (66.8 mg) were mixed, and then the insoluble carrier (29) (100 mg, 33.5 μmol) was added and stirred at room temperature for 6 hours to carry out the click reaction. The insoluble carrier (reactant) was then collected by filtration, washed with MeCN, and vacuum dried. The resulting insoluble carrier was suspended in acetonitrile (1 mL) and 100 mmol / L aqueous disodium ethylenediaminetetraacetate solution (3 mL) and stirred at room temperature for 16 hours. The insoluble carrier was filtered, washed with deionized water and then with acetonitrile, and dried in vacuo to obtain a conjugate of the oligonucleotide derivative and the insoluble carrier (compound 33).

[0161] Cleavage test of conjugates of oligonucleotide derivatives and insoluble carriers (compounds 32 and 33) The conjugates of oligonucleotide derivatives and insoluble carriers (compound 32) were suspended in acetonitrile (3 mL) and deionized water (7 mL), and irradiated with ultraviolet light at a wavelength of 365 nm (room temperature, 3 hours). The supernatant was analyzed by HPLC over time to investigate whether the following photocleavage reaction occurred. The HPLC results are shown in Figure 5.

[0162]

[0163] As shown in FIG. 5, a new peak appeared in HPLC after UV irradiation, and analysis by LC / MS confirmed that this was Compound 34.

[0164] Similarly, a conjugate of an oligonucleotide derivative and an insoluble carrier (compound 33) was suspended in acetonitrile (3 mL) and deionized water (7 mL), irradiated with 365 nm ultraviolet light (room temperature, 3 hours), and the supernatant was analyzed by HPLC over time to investigate whether the following photocleavage reaction was occurring. The HPLC results are shown in Figure 6.

[0165]

[0166] As shown in FIG. 6, a new peak appeared in HPLC after UV irradiation, and analysis by LC / MS confirmed that this was Compound 35.

[0167] (3. Experiments using mRNA cap analogues) The model experiments 1 and 2 above showed that after binding the oligonucleotide derivative to an insoluble carrier, the linker could be cleaved by ultraviolet light irradiation. Therefore, several mRNA cap analogues were actually synthesized and various experiments were carried out.

[0168] <Synthesis of mRNA Cap Analogue (Compound 47) 1> First, Compound 36 was synthesized according to the following scheme.

[0169]

[0170] Compound 12 (10.7 g, 44.9 mmol) was dissolved in acetic acid (103 mL), and then dimethyl sulfoxide (64 mL) and acetic anhydride (85 mL) were added sequentially under ice cooling, followed by stirring at room temperature for 42 hours. After completion of the reaction, the reaction solution was added dropwise to ice-cooled 10 mmol / L aqueous sodium hydroxide solution (400 mL). After completion of the addition, the mixture was stirred at room temperature for 1.5 hours. Subsequently, the mixture was extracted with ethyl acetate, and the organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1 / 19 → 1 / 4) to obtain compound 36 (9.58 g, 71.5%). 1 H-NMR (CDCl3): δ8.19 (d, 1H), 7.32 (t, 1H), 6.91 (dd, 1H), 5.03 (s, 2H), 4.82 (s, 2H), 4.25 (t, 2H), 3.66 (t, 2H), 2.21 (s, 3H)

[0171] Next, compound 38 was synthesized according to the following scheme.

[0172]

[0173] Compound 37 (2.50 g, 4.20 mmol) and compound 36 (1.88 g, 6.29 mmol) were dissolved in tetrahydrofuran (16.8 mL), followed by the addition of molecular sieves 3A (2.50 g) and cooling to -40°C. Trifluoromethanesulfonic acid (557 μL, 6.29 mmol) and N-iodosuccinimide (1.42 g, 6.29 mmol) were added sequentially, and the mixture was stirred at the same temperature for 7 hours. Saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate were added, followed by stirring, and then the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (1-2 wt% methanol-chloroform) and then vacuum-dried to obtain compound 38 (2.89 g, 3.42 mmol, 81.4%). 1 H-NMR (DMSO-d6): δ11.97 (s, 1H), 11.49 (s, 1H), 8.09 (d, 1H), 8.04 (s, 1H), 7.01 ( dd, 1H), 6.96 (d, 1H), 5.83 (d, 1H), 5.14 (d, 1H), 5.04 (d, 1H), 5.02 (d, 1H), 4.95 (d , 1H), 4.60 (dd, 1H), 4.49 (dd, 1H), 4.19 (t, 2H), 4.09 (dd, 1H), 4.01 (dt, 1H), 3.94 (dd, 1H), 3.68 (t, 2H), 2.67 (m, 1H), 1.13 (d, 1H), 1.10 (d, 1H), 1.06-0.86 (m, 28H)

[0174] Next, compound 39 was synthesized according to the following scheme.

[0175]

[0176] Compound 38 (2.89 g, 3.42 mmol) was dissolved in acetonitrile (11.4 mL) and tetrahydrofuran (11.4 mL), and triethylamine trihydrofluoride (557 μL, 3.42 mmol) was added, followed by stirring at room temperature for 20 hours. The reaction solution was diluted with chloroform and washed with saturated aqueous sodium bicarbonate. After extraction with chloroform from a water bath, the organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (2-5 wt% methanol-chloroform) and then vacuum dried to obtain compound 39 (1.87 g, 3.10 mmol, 90.7%).1 H-NMR (DMSO-d6): δ11.77 (s, 1H), 11.50 (s, 1H), 8.26 (s, 1H), 8.04 (d, 1H), 6.9 9 (dd, 1H), 6.70 (d, 1H), 5.94 (d, 1H), 5.38 (d, 1H), 5.14 (t, 1H), 4.91 (d, 1H), 4 .. 88 (d, 1H), 4.77 (d, 1H), 4.69 (d, 1H), 4.68 (t, 1H), 4.30 (m, 1H), 4.23 (m, 2H), 3.96 (m, 1H), 3.73 (m, 2H), 3.60 (m, 2H), 2.68 (m, 1H), 1.14 (d, 3H), 1.09 (d, 3H)

[0177] Next, compound 41 was synthesized according to the following scheme.

[0178]

[0179] Compound 39 (1.87 g, 2.87 mmol) was azeotroped three times with pyridine, and the residue was dissolved in pyridine (9.6 mL). 4,4'-Dimethoxytrityl chloride (1.17 g, 3.44 mmol) was added, and the mixture was stirred at room temperature for 15 hours. Acetic anhydride (678 μL, 7.71 mmol) was added to the reaction solution, and the mixture was further stirred at room temperature for 16 hours. Methanol (5 mL) was added to the reaction solution, and the mixture was concentrated. Ethyl acetate was added to the concentrate, and the concentrate was washed with saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous magnesium sulfate and then concentrated. The concentrate was azeotroped three times with toluene to obtain crude compound 40. Next, crude compound 40 was dissolved in chloroform (28.7 mL) and cooled to 0°C. A methanol solution (9.57 mL) of 4-toluenesulfonic acid hydrate (998 mg) was added, and the mixture was stirred at the same temperature for 1 hour. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was stirred. The organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (2 to 5 w / w% methanol-chloroform) and then dried in vacuo to obtain Compound 41 (1.88 g, 2.71 mmol, 94.4%). 1H-NMR (DMSO-d6): δ11.79 (s, 1H), 11.54 (s, 1H), 8.29 (d, 1H), 8.04 (d, 1H), 7.0 0 (dd, 1H), 6.68 (d, 1H), 5.90 (d, 1H), 5.42 (d, 1H), 5.37 (t, 1H), 4.91 (dd, 1H), 4 .. 82 (d, 1H), 4.78 (d, 1H), 4.66 (d, 1H), 4.61 (d, 1H), 4.25 (m, 2H), 4.17 (m, 1H), 3 73 (m, 2H), 3.67 (m, 2H), 2.71 (m, 1H), 2.12 (s, 3H), 1.15, (d, 3H), 1.09 (d, 3H)

[0180] Next, compound 42 was synthesized according to the following scheme.

[0181]

[0182] Compound 41 (3.95 g, 6.12 mmol) and 2,6-lutidine (1.8 mL, 15.3 mmol) were dissolved in triethyl phosphate (19.0 mL) and cooled to -10°C. Phosphorus oxychloride (1.39 mL, 15.3 mmol) was added, and the mixture was stirred at the same temperature for 9 hours. 1.0 mol / L triethylammonium bicarbonate (30 mL) was added, and the mixture was stirred at the same temperature for 16 hours. Deionized water (50 mL) was added to the reaction mixture, followed by extraction five times with chloroform. The organic layers were combined, concentrated, and dissolved in 100 mmol / L triethylammonium acetate (50 mL). The mixture was purified by ODS silica gel column chromatography (100 mmol / L triethylammonium acetate to 50 wt% acetonitrile, 100 mmol / L triethylammonium acetate). The fraction containing the target product was concentrated to a small volume and then extracted three times with chloroform. The organic layers were combined and concentrated, and the concentrate was dissolved in methanol (30.6 mL). A 40 wt% methylamine methanol solution (10.2 mL) was added to this solution and stirred at room temperature for 24 hours. After concentrating the reaction mixture, the concentrate was dissolved in a 200 mmol / L triethylammonium acetate solution (500 mL) and purified by ODS silica gel column chromatography (YAMAZEN ODS-SM 30 μm, L, 3.0 × 16.5 cm, 37 g, 100 mmol / L triethylammonium acetate to 50 wt% acetonitrile, 100 mmol / L triethylammonium acetate). The fraction containing the target product was concentrated, and the concentrate was azeotroped ten times with deionized water. The resulting crystalline residue was dissolved in ethanol (75 mL) under heating, and the precipitated crystals were collected by filtration and dried to obtain compound 42 (2.41 g, 3.37 mmol, 55.1%). The crystal mother liquor was re-purified by ODS silica gel column chromatography (YAMAZEN ODS-SM 30 μm, L, 3.0 × 16.5 cm, 37 g, 100 mmol / L triethylammonium acetate to 50 w / w% acetonitrile, 100 mmol / L triethylammonium acetate) to obtain an additional 0.28 g of compound 42. 1H-NMR (DMSO-d6): δ10.51 (s, 1H), 8.10 (s, 1H), 7.94 (s, 1H), 7.00 (dd, 1H), 6.94 (d, 1H), 6.46 (s, 2H), 5.85 (d, 1H), 4.88 (d, 1H), 4.82 (d, 1H), 4.77 (d, 1H), 4.72 (m, 1H), 4.68 (d, 1H), 4.34 (m, 1H), 4.29 (m, 2H), 4.03 (m, 1H), 3.89 (m, 2H), 3.73 (m, 2H), 2.94 (q, 6H), 1.13 (t, 9H)

[0183] Next, compound 43 was synthesized according to the following scheme.

[0184]

[0185] Compound 42 (2.20 g, 3.08 mmol) was dissolved in N,N-dimethylacetamide (30.8 mL), iodomethane (959 μL) was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was concentrated, and deionized water was added to the concentrate. The precipitated solid was collected by filtration, washed with water, and then vacuum dried to obtain compound 43 (1.56 g, 2.49 mmol, 80.8%). 1 H-NMR (DMSO-d6): δ9.86 (s, 1H), 8.13 (d, 1H), 7.52 (s, 2H), 7.05-7.01 (m, 2H), 5.98 (d, 1H), 4.99 (s, 2H), 4.86 ( s, 2H), 4.69 (t, 1H), 4.41 (t, 1H), 4.29 (m, 2H), 4.16 (s, 1H), 4.05 (m, 1H), 3.94 (m, 1H), 3.90 (s, 3H), 3.72 (m, 2H)

[0186] Next, compound 45 was synthesized according to the following scheme.

[0187]

[0188] Compound 43 (400 mg, 0.637 mmol) was dissolved in N,N-dimethylformamide (3.19 mL), tributylamine (0.163 mL, 0.637 mmol), and 1,1'-carbonyldiimidazole (114 mg, 0.701 mmol) were added, and the mixture was stirred at room temperature for 5 hours. Deionized water (5 μL) was added, followed by stirring for 30 minutes. Di-tributylammonium phosphate (2.55 mmol) dissolved in N,N-dimethylformamide was added, and the mixture was stirred at room temperature for 19 hours. Deionized water (10 mL) was added, and the mixture was stirred at room temperature for 5 hours. The mixture was then purified by ODS silica gel column chromatography (YAMAZEN ODS-SM 30 μm, L, 3.0 × 16.5 cm, 37 g, deionized water to 50 w / w% acetonitrile). The fractions containing the target product were concentrated, and the concentrate was azeotroped three times with acetonitrile and dried under vacuum. Compound 45 (308 mg, 0.381 mmol, 59.7%) was obtained. HRMS (ESI) m / z:C 21 H 26 N9O 15 P2 - [M-H] - ion theoretical value 706.1029; measured value 706.0864

[0189] Next, compound 47 was synthesized according to the following scheme.

[0190]

[0191] Compound 45 (121 mg, 0.150 mmol) was dissolved in N,N-dimethylformamide (0.75 mL), and tributylamine (0.163 mL, 0.637 mmol) and 1,1'-carbonyldiimidazole (36.5 mg, 0.225 mmol) were added, followed by stirring at room temperature for 5 hours. Deionized water (10 μL) was added, followed by stirring for 30 minutes. A solution of guanosine 5'-monophosphate monotributylamine salt (123 mg, 0.225 mmol) dissolved in N,N-dimethylformamide (0.50 mL) and a solution of magnesium chloride hexahydrate dissolved in N,N-dimethylformamide (0.50 mL) were then added, and the mixture was stirred at room temperature for 17 hours. The reaction mixture was diluted with deionized water (10 mL) and purified by ODS silica gel column chromatography (YAMAZEN ODS-SM 30 μm, L, 3.0 × 16.5 cm, 37 g, deionized water to 50 w / w% acetonitrile). The fractions containing the target compound were concentrated, and the concentrate was azeotroped three times with acetonitrile and dried in vacuo to obtain compound 47 (98 mg, 0.089 mmol, 60%). HRMS (ESI) m / z: C 31 H 38 N 14 O 22 P3 - [M-H] - Theoretical value for ions: 1051.1503; Measured value: 1051.1542

[0192] <Synthesis of mRNA Cap Analogue (Compound 47) 2> Compound 47 was synthesized by a method different from the above method. First, compound 50 was synthesized according to the following scheme.

[0193]

[0194] Tributylamine (163 μL, 637 μmol) was added to an aqueous solution of compound 48 (84.8 mmol / L, 4.51 mL, 382 μmol), concentrated, and the concentrate was azeotroped three times with N,N-dimethylformamide. The residue was dissolved in N,N-dimethylformamide (1.59 mL), and 1,1'-carbonyldiimidazole (129 mg, 0.797 mmol) was added and stirred at room temperature for 3 hours. Deionized water (5 μL) was added, followed by stirring for 30 minutes. Compound 43 (200 mg, 319 μmol) and a solution of magnesium chloride hexahydrate (77.8 mg, 382 μmol) in N,N-dimethylformamide (0.50 mL) were added sequentially, and the mixture was stirred at room temperature for 22 hours. Deionized water (5 mL) was added, and the precipitated solid was collected by filtration and washed with water. The precipitated solid in the filtrate was further collected by filtration and washed with water. The obtained solids were combined and dried under vacuum to obtain Compound 50 (313 mg, 290 μmol, 91.0%). HRMS (ESI) m / z: C 32 H 36 N 14 O 23 P3 - [M-H] - ion theoretical value 1077.1296; measured value 1077.1289

[0195] Next, compound 47 was synthesized according to the following scheme.

[0196] Compound 50 (200 mg, 185 μmol) was dissolved in pyridine (927 μL) and deionized water (927 μL) and heated with stirring at 60°C for 3 hours. Acetonitrile was added to the reaction mixture, and the resulting precipitate was collected by filtration and washed with acetonitrile. The resulting precipitate was dissolved in deionized water (30 mL) and purified by ODS silica gel column chromatography (YAMAZEN ODS-SM, 30 μm, M, 2.3 × 12.3 cm, 14 g, 100 mmol / L TEAA to 100 mmol / L TEAA, 50 w / w% acetonitrile). The fractions containing the target product were concentrated and then desalted by ODS silica gel column chromatography (YAMAZEN ODS-SM, 30 μm, M, 2.3 × 12.3 cm, 14 g, deionized water to 50 w / w% acetonitrile). The fractions containing the target compound were concentrated to a small volume and passed through a cation exchange column (Dowex 50w x 8, Na type, 10 mL) and washed with water. The effluent and washings were combined and concentrated, and the concentrate was dried in vacuo to obtain Compound 47 (101 mg, 185 μmol, 49.2%).

[0197] <Synthesis of mRNA cap analog (compound 54)> Compound 54 was synthesized according to the following scheme.

[0198]

[0199] Compound 45 (20 mg, 0.025 mmol) was dissolved in N,N-dimethylformamide (0.99 mL), and 1,1'-carbonyldiimidazole (6.0 mg, 0.037 mmol) was added, followed by stirring at room temperature for 3 hours. Deionized water (10 μL) was added, followed by stirring for 30 minutes, and the oligonucleotide derivative (compound 53; 0.025 mmol; sequence 5'-G OMe G OMe G AGA CGC GUG UUA AAU AAC-3';G OMeA solution of 2'OMe-G (2'OMe-G) in dimethyl sulfoxide (2 mL) and a solution of magnesium chloride hexahydrate (50 mg, 0.25 mmol) in N,N-dimethylformamide (1 mL) were added sequentially and stirred at room temperature for 20 hours. A separately prepared solution of compound 46 (equivalent to 0.037 mmol) in N,N-dimethylformamide (1 mL) was added and stirred for an additional 24 hours. The reaction solution was diluted with deionized water to 300 mL and purified by ODS silica gel column chromatography (YAMAZEN ODS-AQ, 30 μm, 2 L, 3.0 × 20.0 cm, 51 g, 100 mmol / L TEAA to 100 mmol / L TEAA, 50 w / w% acetonitrile). The fractions containing the target product were concentrated and then desalted using ODS silica gel column chromatography (YAMAZEN ODS-AQ, 30 μm, 2 L, 3.0 × 20.0 cm, 51 g, deionized water to 50 w / w% acetonitrile). The fractions containing the target product were concentrated to obtain compound 54 as an aqueous solution (6.86 μmol, 27.4%). HRMS (ESI) m / z: C 226 H 278 N 96 O 159 P 23 - [M-H] - Theoretical value for ions: 7592.0590; Measured value: 7592.0747

[0200] <Synthesis of Insoluble Carrier Having Cyclooctyne Group (Compound 60)> An insoluble carrier having a cyclooctyne group (Compound 60) was synthesized according to the following scheme.

[0201] First, compound 57 was synthesized according to the following scheme.

[0202]

[0203] Commercially available compound 56 (388 mg, 1.27 mmol) and N,N-diisopropylethylamine (681 μmol) were dissolved in N,N-dimethylformamide (6.35 mL) and cooled on ice. 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (530 mg, 1.40 mmol) was added and stirred at room temperature for 1 hour. After cooling the reaction mixture on ice, an N,N-dimethylformamide solution (2 mL) of compound 55 (Organic & Biomolecular Chemistry (2014), 12(14), 2263-2272) (500 mg, 1.27 mmol) was added and stirred at room temperature for 18 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate, and the organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (50-75-100 w / w% ethyl acetate-hexane) to obtain Compound 57 (0.61 g, 0.90 mmol, 71%). HRMS (ESI) m / z: C 43 H 39 N2O6 - [M-H] - ion theoretical value 679.2814; measured value 679.2819; C 44 H 41 N2O8 - [M + HCOO] - ion theoretical value 725.2868; measured value 725.2890; C 45 H 43 N2O8 - [M + CH3COO] - Theoretical value for ions: 739.3025; Measured value: 739.2993

[0204] Next, compound 59 was synthesized according to the following scheme.

[0205]

[0206] Compound 57 (0.49 g, 0.72 mmol) was dissolved in pyridine (3.6 mL), and succinic anhydride (0.14 g, 1.4 mmol) and N,N-dimethylaminopyridine (44 mg, 0.36 mmol) were added, followed by stirring at room temperature for 18 hours. The reaction mixture was concentrated, and then ethyl acetate was added to the concentrate and washed with water. The organic layer was dried over anhydrous magnesium sulfate, and the residue was dissolved in acetonitrile. Di(N-succinimidyl)carbonate (0.22 g, 0.86 mmol) and triethylamine (0.24 mL, 1.7 mmol) were added, followed by stirring at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (75-100 w / w% ethyl acetate-hexane) to obtain compound 59 (0.54 g, 0.62 mmol, 85%). HRMS (ESI) m / z: C 51 H 46 N3O 11 - [M-H] - Theoretical value for ions: 876.3138; Measured value: 876.3094

[0207] Next, compound 60 was synthesized according to the following scheme.

[0208]

[0209] A suspension of EAH Sepharose® 4B (7-12 μmol / mL) was centrifuged at 2,000 rpm for 5 minutes (volume after centrifugation: 10 mL), and the supernatant was removed by decantation. The precipitate was suspended in acetonitrile (10 mL), centrifuged at 2,000 rpm for 5 minutes, and the supernatant was removed by decantation. This procedure was repeated five times. The precipitate was suspended in acetonitrile (10 mL), and N,N-diisopropylethylamine (25 μL, 0.14 mmol) and compound 59 (61 mg, 70 μmol) were added and stirred at room temperature for 15 hours. N,N-diisopropylethylamine (25 μL, 0.14 mmol) and compound 59 (61 mg, 70 μmol) were then added and stirred at room temperature overnight. This procedure was repeated twice. N-acetylimidazole (7.7 mg, 70 μmol) was added to the suspension and stirred at room temperature for 24 hours. The suspension was centrifuged at 2,000 rpm for 5 minutes, and the supernatant was removed by decantation. The precipitate was suspended in acetonitrile (10 mL), centrifuged at 2,000 rpm for 5 minutes, and the supernatant was removed by decantation. This procedure was repeated five times to obtain a precipitate (compound 60). The precipitate was then made up to 10 mL with deionized water.

[0210] <Evaluation of the amount of cyclooctyne units supported on compound 60> Compound 60 was treated according to the following scheme.

[0211]

[0212] To a suspension of compound 60 (0.5 mL), 28 wt% aqueous ammonia (1 mL) was added and heated at 60°C for 17 hours. After removing unnecessary materials by filtration, the filtrate and washings were concentrated and made up to 10 mL with 50 wt% acetonitrile containing 1 wt% triethylamine. The resulting compound 57 was analyzed by mass spectrometry (AcO - Theoretical value for the adduct: 739.3025; measured value: 739.3027.) Furthermore, as a result of quantification using a calibration curve, the amount of cyclooctyne units supported on the insoluble carrier was 3.19 μmol / mL.

[0213] <Conjugation (Click Reaction) of mRNA Cap Analogue (Compound 47) with Insoluble Carrier Having a Cyclooctyne Group (Compound 60)> According to the following scheme, a click reaction was carried out between the mRNA cap analogue (Compound 47) and the insoluble carrier having a cyclooctyne group (Compound 60) to obtain a conjugate (Compound 61).

[0214]

[0215] A suspension of compound 60 (0.50 mL, 1.60 μmol) and a solution of compound 47 (1 mmol / mL, 1.60 mL, 1.60 μmol) were mixed and stirred at room temperature for 4 days. During this time, the reaction solution was analyzed over time, and the progress of the click reaction was confirmed by HPLC by confirming the decrease in the peak of compound 47 in the reaction solution. The results are shown in Figure 7.

[0216] <Cleavage test of compound 61> After the click reaction, the suspension was centrifuged at 40,000 rpm for 5 minutes, and the supernatant was removed by decantation. The precipitate was suspended in deionized water (3 mL), then centrifuged at 40,000 rpm for 5 minutes, and the supernatant was removed by decantation. This procedure was repeated five times. The precipitate was suspended in deionized water (5 mL), cooled to 5°C, and irradiated with ultraviolet light at a wavelength of 365 nm while stirring. The progress of the following photocleavage reaction was investigated by HPLC.

[0217]

[0218] As a result, it was confirmed that a new component was produced in the supernatant of the reaction solution, as shown in Figure 8. Mass spectrometry analysis identified this as compound 62. This confirmed that the cleavage reaction due to irradiation with ultraviolet light at a wavelength of 365 nm had progressed.

[0219] <Conjugation (Click Reaction) of mRNA Cap Analogue (Compound 54) with Insoluble Carrier Having a Cyclooctyne Group (Compound 60)> According to the following scheme, a click reaction was carried out between the mRNA cap analogue (Compound 54) and the insoluble carrier having a cyclooctyne group (Compound 60) to obtain a conjugate (Compound 63).

[0220]

[0221] A suspension of compound 60 (0.50 mL, 1.60 μmol), a solution of compound 54 (0.5 mL, 0.083 μmol), and compound 53 as an internal standard were mixed and stirred at room temperature for 4 days. During this time, the reaction solution was analyzed over time, and the progress of the click reaction was confirmed by confirming the decrease in the peak of compound 54 in the reaction solution ( FIG. 9 ).

[0222] <Cleavage test of compound 63> After the click reaction, the suspension was centrifuged at 40,000 rpm for 5 minutes, and the supernatant was removed by decantation. The precipitate was suspended in deionized water (3 mL), and then centrifuged at 40,000 rpm for 5 minutes. The supernatant was removed by decantation. This procedure was repeated five times. The precipitate was suspended in deionized water (3 mL), cooled to 5°C, and irradiated with ultraviolet light at a wavelength of 365 nm while stirring. The progress of the following photocleavage reaction was investigated by HPLC.

[0223]

[0224] As a result, as shown in Figure 10, a new peak appeared in HPLC after UV irradiation, and analysis by LC / MS confirmed that this was Compound 64. This confirmed that the cleavage reaction had progressed due to UV irradiation at a wavelength of 365 nm.

[0225] <Synthesis of Insoluble Carrier Having Cyclooctyne Group (Compound 72)> First, Compound 65 was synthesized according to the following scheme.

[0226]

[0227] 11-Azido-3,6,9-trioxaundecanoic acid (1.00 g, 4.29 mmol) and N,N-diisopropylethylamine (2.30 mL, 12.9 mmol) were dissolved in N,N-dimethylformamide (21.4 mL) and the solution was ice-cooled. 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (1.79 g, 4.72 mmol) was added and stirred at room temperature for 1 hour. After ice-cooling the reaction solution, an N,N-dimethylformamide solution (10 mL) of compound 55 (1.69 g, 4.29 mmol) was added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (85 w / w% ethyl acetate-hexane to 2 w / w% methanol-ethyl acetate) to obtain Compound 65 (2.47 g, 4.06 mmol, 94.6%). 1 H-NMR (DMSO-d6): δ7.45-6.87 (m, 14H), 5.05 (d, 1H), 3.84 (s, 1H), 3.74 (s, 7H ), 3.60-3.49 (m, 10H), 3.37 (t, 2H), 3.09 (dt, 1H), 2.93 (dd, 1H), 2.84 (dd, 1H)

[0228] Next, compound 67 was synthesized according to the following scheme.

[0229]

[0230] Compound 65 (0.87 g, 1.4 mmol) was dissolved in tetrahydrofuran (7.1 mL) and deionized water (0.71 mL), and triphenylphosphine (0.41 g, 1.6 mmol) was added and stirred at room temperature for 15 hours. The reaction mixture was concentrated, and the concentrate was azeotroped three times with acetonitrile. The residue was dissolved in acetonitrile (7.1 mL), and triethylamine (0.44 mL, 3.1 mmol) and ethyl trifluoroacetate (0.34 mL, 2.9 mmol) were added, followed by stirring at room temperature for 1 hour. The reaction mixture was concentrated, and the concentrate was purified by silica gel column chromatography (75-85-100 w / w% ethyl acetate-hexane to 2 w / w% methanol-ethyl acetate) to obtain compound 67 (0.80 g, 1.2 mmol, 82%). 1H-NMR (DMSO-d6): δ9.48 (t, 1H), 7.46-6.87 (m, 14H), 5.05 (d, 1H), 3.84 (s, 2H), 3.74 (s , 7H), 3.51-3.48 (m, 10H), 3.38-3.35 (m, 1H), 3.08 (dt, 1H), 2.93 (dd, 1H), 2.84 (dd, 1H)

[0231] Next, compound 69 was synthesized according to the following scheme.

[0232]

[0233] Compound 67 (0.58 g, 0.85 mmol) was placed in ethanol (3.9 mL), and 33 wt% methylamine ethanol solution (1.6 mL) was added, followed by stirring at room temperature for 24 hours. The reaction mixture was concentrated, and the concentrate was azeotroped three times with acetonitrile. The residue was dissolved in acetonitrile (3.9 mL), and triethylamine (0.13 mL, 0.93 mmol) and succinimidyl (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl carbonate (0.23 g, 0.78 mmol) were added, followed by stirring at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (ethyl acetate to 1 wt% methanol-ethyl acetate) to obtain compound 69 (0.44 g, 0.58 mmol, 75%). HRMS (ESI) m / z: C 43 H 53 N2O 10 - [M-H] - ion theoretical value 757.3706; measured value 757.3727

[0234] Next, compound 71 was synthesized according to the following scheme.

[0235]

[0236] Compound 69 (0.70 g, 0.92 mmol) was dissolved in pyridine (4.6 mL), and succinic anhydride (0.18 g, 1.8 mmol) and N,N-dimethylaminopyridine (56 mg, 0.46 mmol) were added, followed by stirring at room temperature for 19 hours. The reaction mixture was concentrated, and then ethyl acetate was added to the concentrate and washed with 1 mol / L aqueous citric acid. The organic layer was dried over anhydrous magnesium sulfate, concentrated, and then dissolved in acetonitrile (4.6 mL). Di(N-succinimidyl)carbonate (0.28 g, 1.1 mmol) and triethylamine (0.31 mL, 2.2 mmol) were added, followed by stirring at room temperature for 3 hours. The reaction mixture was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate, and the organic layer was dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (2-5-10-30 wt% acetone-ethyl acetate) to obtain compound 71 (0.161 g, 0.168 mmol, 18%). HRMS (ESI) m / z:C 51 H 60 N3O 15 - [M-H] - Theoretical value as ion: 954.4030; Measured value: 954.3997

[0237] Next, compound 72 was synthesized according to the following scheme.

[0238]

[0239] A suspension of EAH Sepharose® 4B (7-12 μmol / mL) was centrifuged at 2,000 rpm for 5 minutes (volume after centrifugation: 10 mL), and the supernatant was removed by decantation. The precipitate was suspended in acetonitrile (10 mL) and centrifuged at 2,000 rpm for 5 minutes. The supernatant was removed by decantation; this procedure was repeated five times. The precipitate was suspended in acetonitrile (10 mL), and N,N-diisopropylethylamine (37 μL, 0.21 mmol) and Compound 71 (67 mg, 70 μmol) were added, followed by stirring at room temperature for 16 hours. N,N-diisopropylethylamine (37 μL, 0.21 mmol) and Compound 71 (67 mg, 70 μmol) were then added, followed by stirring at room temperature for 24 hours. N-acetylimidazole (27 mg, 0.21 mmol) was then added, followed by stirring at room temperature for 24 hours. The suspension was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was removed by decantation. The precipitate was suspended in 20 mL of 100 mmol / mL aqueous triethylammonium acetate solution, followed by centrifugation at 4,000 rpm for 10 minutes. The supernatant was removed by decantation. This procedure was repeated three times to obtain a precipitate (compound 72). The precipitate was then diluted to 10 mL with deionized water.

[0240] <Evaluation of the amount of cyclooctyne units supported in compound 72>

[0241] Compound 72 was treated according to the following scheme.

[0242] To a suspension of compound 72 (0.5 mL), 28 wt% aqueous ammonia (1 mL) was added and heated at 60°C for 17 hours. After removing unnecessary materials by filtration, the filtrate and washings were concentrated and made up to 10 mL with 60 wt% acetonitrile containing 1 wt% triethylamine. The resulting compound 69 was analyzed by mass spectrometry (theoretical value: 757.3706, 803.3760 (HCO - ), 817.3917 (AcO - ); Measured value: 757.3727, 803.3773 (HCO - ), 817.3928 (AcO - )) Furthermore, as a result of quantification using a calibration curve, the amount of cyclooctyne units supported on the insoluble carrier was 4.22 μmol / mL.

[0243] <Conjugation (Click Reaction) of mRNA Cap Analogue (Compound 54) with Insoluble Carrier Having a Cyclooctyne Group (Compound 72)> According to the following scheme, a click reaction was carried out between the mRNA cap analogue (Compound 54) and the insoluble carrier having a cyclooctyne group (Compound 72) to obtain a conjugate (Compound 73).

[0244]

[0245] A suspension of compound 72 (0.50 mL, 2.11 μmol), a solution of compound 54 (0.5 mL, 0.083 μmol), and compound 53 as an internal standard were mixed and stirred at room temperature for 4 days. During this time, the reaction solution was analyzed over time, and the progress of the click reaction was confirmed by confirming the decrease in the peak of compound 54 in the reaction solution ( FIG. 11 ).

[0246] <Cleavage test of compound 73> The suspension after the click reaction was centrifuged at 40,000 rpm for 5 minutes, and the supernatant was removed by decantation. The precipitate was suspended in deionized water (3 mL), then centrifuged at 40,000 rpm for 5 minutes, and the supernatant was removed by decantation. This procedure was repeated five times. The precipitate was suspended in deionized water (3 mL), cooled to 5°C, and irradiated with 365 nm ultraviolet light while stirring. The progress of the following photocleavage reaction was investigated by HPLC.

[0247]

[0248] As a result, as shown in Figure 12, a new peak appeared in HPLC after UV irradiation, and analysis by LC / MS confirmed that this was Compound 64. This confirmed that the cleavage reaction had progressed due to UV irradiation at a wavelength of 365 nm.

[0249] The above results demonstrated that it is possible to bind insoluble carriers (compounds 60 and 72) to mRNA cap analogs (compounds 47 and 54) by click reaction, and to cleave the linker by ultraviolet irradiation.

[0250] SEQ ID NO: 1: Oligonucleotide derivative (21-mer) synthesized in the example SEQ ID NO: 2: Oligonucleotide derivative (compound 53) used in the example

Claims

1. An mRNA cap analogue comprising a linker having a functional group selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group, and having a structure that can be cleaved by light or a fluoride ion, at the sugar portion or base portion of a nucleotide.

2. The mRNA cap analog of claim 1, wherein the linker has an o-nitrobenzyl ether structure or a silyl ether structure.

3. The mRNA cap analog of claim 1 or 2, represented by the following general formula (1) or (2): 【Chemistry 1】 【Chemistry 2】 In the formula, R 1 OH, OCH 3 Or the following general formula (3): 【Chemistry 3】 is a group represented by R 2 is OH or OCH 3 and R 3 is O, OH, NH 2 , N.H.H. 3 or N(CH 3 ) 2 and R 3 If is O, then R 3 C and R bonded with 3 The bond with R is a double bond. 3 and the bond between the C bonded to R and the adjacent N is a single bond, 3 OH, NH 2 , N.H.H. 3 or N(CH 3 ) 2 In the case of R 3 C and R bonded with 3 The bond with R is a single bond. 3 and the bond between the C bonded to the N adjacent thereto is a double bond, R 4 NH 2 , N.H.H. 3 , N(CH 3 ) 2 or H, R 5 is a propargyloxy group or an azidoethoxy group, R 6 and R 7 may be the same or different, and are OH or OCH 3 and a is 3 to 6 b is 0 to 19; Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosine-1-yl, 5-methylcytosine-1-yl, uracil-1-yl, uracil-5-yl or 1-methyluracil-5-yl.

4. R 3 is O, and R 4 NH 2 The mRNA cap analog of claim 3,

5. A step of mixing an mRNA cap analog having a linker at the sugar or base moiety of a nucleotide, the linker including a functional group A and having a structure cleavable by light or a fluoride ion, a nucleoside triphosphate, a template DNA, and an RNA polymerase, and obtaining a preparation including an mRNA having a 5' cap structure by in vitro transcription; mixing an insoluble carrier having a functional group B capable of reacting with the functional group A with the preparation to generate a conjugate between the mRNA and the insoluble carrier; filtering and washing the conjugate; applying light or fluoride ions to the conjugate to cleave the linker and isolate the mRNA having the 5' cap structure; A method for producing an mRNA having a 5' cap structure, comprising:

6. The method according to claim 5, wherein the functional groups A and B are a combination of an amino group and a carboxylic acid active ester, a combination of an aminooxy group or a hydrazino group and a carbonyl group, a combination of a thiol group and a thiol group, a haloacetyl group or a Michael acceptor functional group, or a combination of an alkynyl group and a 1,3-dipole functional group.

7. The method according to claim 5 or 6, wherein the linker has an o-nitrobenzyl ether structure or a silyl ether structure.

8. The method of claim 5, wherein the mRNA cap analog is represented by the following general formula (1) or (2): 【Chemistry 4】 【Chemistry 5】 In the formula, R 1 OH, OCH 3 Or the following general formula (3): 【Chemistry 6】 is a group represented by R 2 is OH or OCH 3 and R 3 is O, OH, NH 2 , N.H.H. 3 or N(CH 3 ) 2 and R 3 If is O, then R 3 C and R bonded with 3 The bond with R is a double bond. 3 and the bond between the C bonded to R and the adjacent N is a single bond, 3 OH, NH 2 , N.H.H. 3 or N(CH 3 ) 2 In the case of R 3 C and R bonded with 3 The bond with R is a single bond. 3 and the bond between the C bonded to the adjacent N is a double bond, R 4 NH 2 , N.H.H. 3 , N(CH 3 ) 2 or H, R 5 is a propargyloxy group or an azidoethoxy group, R 6 and R 7 may be the same or different, and are OH or OCH 3 and a is 3 to 6 b is 0 to 19; Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosine-1-yl, 5-methylcytosine-1-yl, uracil-1-yl, uracil-5-yl or 1-methyluracil-5-yl.

9. R 3 is O, and R 4 NH 2 The method according to claim 8, wherein

10. R 5 The method according to claim 8 or 9, wherein when is a propargyloxy group, the insoluble carrier is represented by the following formula (4): 【Chemistry 7】 In the formula, Sup is an insoluble carrier structural portion.

11. R 5 The method according to claim 8 or 9, wherein when is an azidoethoxy group, the insoluble carrier is represented by the following formula (5), (6) or (7). 【Chemistry 8】 In the formula, Sup is an insoluble carrier structural portion.

12. A step of introducing a linker having a functional group selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group, and having a structure that can be cleaved by light or a fluoride ion, into the sugar moiety or the base moiety of the first nucleotide; Ligating the first nucleotide having the linker introduced therein with a second nucleotide in the presence of magnesium chloride; A method for producing an mRNA cap analog, comprising:

13. A kit for use in producing an mRNA having a 5' cap structure, comprising: an mRNA cap analogue comprising a functional group A selected from an amino group, a carboxylic acid active ester group, an aminooxy group, a hydrazino group, a carbonyl group, a thiol group, a haloacetyl group, a Michael acceptor functional group, an alkynyl group, and a 1,3-dipole functional group, and a linker having a structure that can be cleaved by light or a fluoride ion, at a sugar moiety or a base moiety of a nucleotide; an insoluble carrier having a functional group B capable of reacting with the functional group A; Kit including:

14. The kit of claim 13, wherein the functional groups A and B are a combination of an amino group and a carboxylic acid active ester, a combination of an aminooxy group or a hydrazino group and a carbonyl group, a combination of a thiol group and a thiol group, a haloacetyl group or a Michael acceptor functional group, or a combination of an alkynyl group and a 1,3-dipole functional group.

15. The kit according to claim 13 or 14, wherein the linker has an o-nitrobenzyl ether structure or a silyl ether structure.

16. The kit of claim 13 , wherein the mRNA cap analog is represented by the following general formula (1) or (2): 【Chemistry 9】 【Chemistry 10】 In the formula, R 1 OH, OCH 3 Or the following general formula (3): 【Chemistry 11】 is a group represented by R 2 is OH or OCH 3 and R 3 is O, OH, NH 2 , N.H.H. 3 or N(CH 3 ) 2 and R 3 If is O, then R 3 C and R bonded with 3 The bond with R is a double bond. 3 and the bond between the C bonded to R and the adjacent N is a single bond, 3 OH, NH 2 , N.H.H. 3 or N(CH 3 ) 2 In the case of R 3 C and R bonded with 3 The bond with R is a single bond. 3 and the bond between the C bonded to the N adjacent thereto is a double bond, R 4 NH 2 , N.H.H. 3 , N(CH 3 ) 2 or H, R 5 is a propargyloxy group or an azidoethoxy group, R 6 and R 7 may be the same or different, and are OH or OCH 3 and a is 3 to 6 b is 0 to 19; Base is adenine-9-yl, N6-methyladenine-9-yl, guanine-9-yl, cytosine-1-yl, 5-methylcytosine-1-yl, uracil-1-yl, uracil-5-yl or 1-methyluracil-5-yl.

17. R 3 is O, and R 4 NH 2 The kit of claim 16 ,

18. R 5 The kit according to claim 16 or 17, wherein, when is a propargyloxy group, the insoluble carrier is represented by the following formula (4): 【Chemistry 12】 In the formula, Sup is an insoluble carrier structural portion.

19. R 5 The kit according to claim 16 or 17, wherein, when is an azidoethoxy group, the insoluble carrier is represented by the following formula (5), (6) or (7). 【Chemistry 13】 In the formula, Sup is an insoluble carrier structural portion.

20. The kit according to claim 13 or 14, further comprising one or more selected from the group consisting of nucleoside triphosphates, template DNA, and RNA polymerase.

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