JP2005046073A - RNA transcription and / or amplification method using modified nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new RNA transcription method using a modified nucleic acid (DNA) having a very high resistance to exo- and endo-nucleases as a template.
Transcription and / or amplification of RNA comprising a modified DNA in which a hydroxyl group and / or oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group A method for transcription and / or amplification of RNA, wherein the modified DNA that can function as a template is used as a template.
[Selection] Figure 2

Description

The present invention relates to a method for transcription and / or amplification of RNA using a modified nucleic acid, a template for RNA transcription and / or amplification, a kit for preparing a template for RNA transcription and / or amplification, and a kit for RNA transcription and / or amplification. About.

The development of various modified nucleic acids (DNA) and their use in biological research have been extensively performed. There are roughly two types of these methods, which are divided into those for chemically modifying the nucleobase portion and those for modifying the backbone skeleton of the phosphate portion. Modification of the nucleic acid base mainly affects the recognition and activity expression of modified nucleic acids for functional biological substances such as enzymes and proteins, and modification of the phosphate skeleton mainly affects the stability of the nucleic acid itself and blood It affects the solubility in cell fluids. So far, modified nucleic acids in which the hydroxyl group of the nucleic acid phosphate is partially substituted with sulfur or methyl groups have been synthesized, and their properties and behavior as DNA have been studied. Similarly, synthesis of a boranophosphate nucleic acid in which the hydroxyl group of the phosphate moiety is partially substituted with boron has been achieved, and this boranophosphate DNA is present in an excessive amount in the living body compared to the conventional natural DNA. In vitro systems have shown that resistance to -and endo-nucleases is hundreds to tens of thousands of times higher (Non-patent Documents 1 and 2). However, there are few examples of this boranophosphate DNA actually used in biological research. One is Sha
have reported a non-radioactive sequencing method using boranophosphate (Non-patent Document 2). This utilizes the property that boranophosphate is more resistant to exo-nuclease than the phosphate of natural DNA. The other was reported by Shaw et al.
-Boranodeoxynucleoside triphosphates were chemically synthesized and DNA containing boranophosphate was prepared by a biochemical method by PCR reaction in the presence of them (Non-patent Document 3).
In this case, however, boranophosphate DNA was recognized by DNA polymerase and functioned as a template for DNA replication. However, including other examples used so far, they were only recognized as DNA sites with high nuclease resistance. It has not been developed and used.

On the other hand, many researchers around the world, including us, are actively conducting RNAi research using siRNA. When siRNAs are expressed in mammalian cells that are currently attracting much attention, it is essential to inoculate the affected site directly with a long gene that is expressed by intravenous infusion or infusion. A major problem here is the stability of the introduced gene (DNA) in vivo. In vivo life of genes introduced from outside the living body is generally very short, and they are cut into pieces so long that they do not perform any function shortly after the introduction. This is due to the basic defense mechanism of the living body itself against in vitro substances, and the above-mentioned exo- and endo-nucleases directly play the role.

JACS (1998) 120,9417-9427 Nucleic Acid Research (1997) 25, 1611-1617. Nucleic Acid Research (1999) 27, 1788-1794.

An object of the present invention is to provide a new RNA transcription method using a modified nucleic acid (DNA) having a very high resistance to exo- and endo-nucleases as a template.

The present inventors first replicated an arbitrary site as a dsDNA from circular DNA using PCR in the presence of natural dATP, dCTP, dGTP and α-boranothymidine triphosphate. In all thymidine 5′-side phosphate sites present in this dsDNA, the hydroxyl group of the phosphate site is completely substituted with boron. The base sequence of the replicated dsDNA was confirmed by sequence analysis.
Since the obtained dsDNA has a T7 RNA polymerase promoter, a transcription test using T7 RNA polymerase confirmed that RNA was transcribed for the first time from boranophosphate DNA. In addition, the 5'-side phosphate site of thymidine in the promoter site, regardless of whether it was a natural phosphate type or a boranophosphate type, transcription proceeded without any problem. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
[1] A modified DNA or RNA in which a hydroxyl group and / or oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group, and R
A method for transcription and / or amplification of RNA, wherein the modified DNA or RNA that can function as a template for transcription and / or amplification of NA or function as suppression of gene expression is used as a template.
[2] The method according to [1], wherein the modified DNA or RNA is DNA or RNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with a borano group.
[3] Modified chemically synthesized DNA or RNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with an optically active borano group in modified DNA or RNA
The method according to [1], which is NA.
[4] Modified DNA in which a hydroxyl group and / or an oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group, which is used for transcription and / or amplification of RNA. A template for RNA transcription and / or amplification, comprising the modified DNA that can function as a template.
[5] The template according to [4], wherein the modified DNA is a DNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with a borano group.
[6] A template for RNA transcription and / or amplification comprising a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus in a phosphate moiety directly bonded to a sugar is substituted with another atom or group Preparation kit.
[7] The modified deoxyribonucleoside triphosphate is 2′-deoxyribonucleoside triphosphate in which the hydroxyl group bonded to phosphorus in the α-phosphate moiety is substituted with a borano group [
6] The kit of description.
[8] An RNA transcription and / or amplification kit comprising a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus in a phosphate moiety directly bonded to a sugar is substituted with another atom or group.
[9] The modified deoxyribonucleoside triphosphate is 2′-deoxyribonucleoside triphosphate in which the hydroxyl group bonded to phosphorus in the α-phosphate moiety is substituted with a borano group [
8] The kit according to the above.
[10] Modified DNA in which a hydroxyl group and / or an oxygen atom bonded to phosphorus in a phosphate moiety directly bonded to a sugar is substituted with another atom and / or group, wherein RNA is transcribed and / or Alternatively, an RNA transcription and / or amplification kit comprising the modified DNA that can function as a template for amplification.
[11] The kit according to [10], wherein the modified DNA is a DNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with a borano group.
[12] A modified DNA or RNA in which a hydroxyl group and / or an oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group,
The modified DNA or RNA, which can function as a template for RNA transcription and / or amplification, or can function as gene expression suppression.
[13] The modified DNA according to [12], which can be introduced into a cell or tissue so that the functional nucleic acid can be expressed in the cell or tissue.
[14] The functional nucleic acid to be expressed is a double-stranded RNA containing siRNA or hairpin RNA [1]
3] The modified DNA according to the above.
[15] The modified DNA according to [13], wherein the functional nucleic acid to be expressed is a ribozyme.
[16] The modified DNA according to [13], wherein the functional nucleic acid to be expressed is an antisense RNA.
[17] The target of the functional nucleic acid to be expressed is a gene related to a virus or cancer [1]
[3] The modified DNA according to any one of [16].
[18] Modified D according to [17], wherein the virus is selected from the group consisting of HIV, HCV and HBV
NA.
[19] The modified DNA or RNA according to [12], which can be introduced into a cell or tissue to suppress gene expression.
[20] The modified RNA according to [19], wherein the modified RNA is an antisense RNA.
[21] The modified RNA according to [19], wherein the modified RNA is a double-stranded RNA containing siRNA and hairpin RNA.
[22] The modified DNA according to [19], wherein the modified DNA is a DNAzyme.
[23] A composition comprising the modified DNA or RNA according to any one of [12] to [22].
[24] A pharmaceutical composition comprising the modified DNA or RNA according to any one of [12] to [22].
[25] A method for producing the modified RNA according to any one of [19] to [21] by in vitro transcription.
[26] A method of synthesizing the modified DNA or RNA according to any one of [12] to [22] from a nucleotide containing an optically active borano group.

The technology of the present invention is the development of siRNA expression method using modified DNA as a template, modified siRN
A. It can be used to apply modified DNA to RNAi research. Furthermore, application to the expression of functional nucleic acids such as hairpin RNA, ribozyme, antisense RNA, antisense RNA, siRNA,
It can be applied to nucleic acids having gene expression-inhibiting action such as hairpin RNA and DNAzyme.

In the present specification, “modified DNA or RNA” means natural DNA or RN.
Compared with A, DNA having a modification in which a hydroxyl group and / or an oxygen atom bonded to phosphorus in at least one phosphate moiety in the phosphate skeleton is substituted with another atom and / or group Or RNA.

The “template” refers to a template DNA used for synthesizing sense RNA using double-stranded DNA as a template in RNA transcription and / or amplification.

“Modified deoxyribonucleoside triphosphate” means that it binds to phosphorus (α-phosphate) in the phosphate (α-phosphate) moiety that is directly bound to the sugar, compared to natural deoxyribonucleoside triphosphate. It means deoxyribonucleoside triphosphate which is modified such that the hydroxyl group is substituted with another atom or group.

“Functional nucleic acid” refers to a nucleic acid having a specific function in cells, tissues, and organs, such as physiological activity such as gene expression suppression, enzymatic activity such as RNA cleavage, protein, RNA, low molecular weight compound, etc. Nucleic acids having the ability to bind to, and the like.

“Hairpin RNA” means that both ends of the 5 ′ end and 3 ′ end of linear double-stranded RNA are 1
RNA connected by RNA loops with a single-stranded structure, including stem structures and stem and loop RNAs that have a loop structure connecting them. The stem structure may not be perfectly matched.

  “Ribozyme” is a general term for biocatalysts having RNA as a chemical entity.

“Antisense RNA” refers to an RNA molecule having a sequence complementary to a certain RNA, and includes an RNA molecule that binds complementarily to a certain target RNA and suppresses its expression.

  “DNAzyme” is a general term for biocatalysts having DNA as a chemical entity.

“Nucleotide containing an optically active borano group” refers to a nucleotide having an asymmetric center that is generated because the borano group is covalently bonded to the 5 ′ α-phosphorus atom.

In the present specification, “to” indicates a range including the numerical values described before and after that as a minimum value and a maximum value, respectively.

The present invention provides a new RNA transcription method using a modified nucleic acid (DNA) having a very high resistance to exo- and endo-nucleases as a template.

The present invention relates to a modified DNA or RNA in which a hydroxyl group and / or an oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group, Alternatively, the present invention provides a method for transcription and / or amplification of RNA, wherein the modified DNA or RNA that can function as a template for amplification or function as gene expression suppression is used as a template.

In addition, the present invention provides a hydroxyl group and / or bonded to phosphorus in at least one phosphate moiety.
Or modified DNA in which an oxygen atom is substituted with another atom and / or group, and RNA
Consisting of the modified DNA capable of functioning as a template for transcription and / or amplification of
A template for RNA transcription and / or amplification is provided.

In the present invention, in the DNA used as a template, a hydroxyl group and / or an oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group. This substitution is aimed at improving exo- and endo-nuclease resistance. Although the example of substitution is given below, it is not limited to these.

  The structure of natural DNA is shown below.

（１）リン酸部分のリンに結合している水酸基(-OH)（上式におけるX）が、ボラノ基(-BH
₃ ^-)、ホスホロ基(-S^-)、アミノ基(-NH₂)、低級アルキル基(-R)（Rは、例えば、メチル基
、エチル基など）およびアルコキシ基(-OR) （Rは、例えば、メチル基、エチル基など）
からなる群より選択される基に置換されている（Biochemistry (1979) 18, 5134.; Tetra
hedron Lett.
(1982) 23, 4289.)。
（２）リン酸部分のリンに結合しているオキソ基(=O)
（上式における=O¹）が、チオキソ基(=S)に置換されている（Tetrahedron
Lett. (1980) 21, 1121.; Biochemistry (1987) 26, 8237.）。
（３）リン酸部分のリンと糖の5’位の炭素に結合しているオキシ基(-O-)（上式における
-O²-）が、メチレン基(-CH₂-)、チオキシ基(-S-)およびアミノ基(-NH-)からなる群より選
択される基に置換されている（Nucleic Acids Res. (1997) 25, 830）。
（４）リン酸部分のリンと糖の3’位の炭素に結合しているオキシ基(-O-)（上式における
-O³-）が、メチレン基(-CH₂-)、チオキシ基(-S-)およびアミノ基(-NH-)からなる群より選
択される基に置換されている（Proc. Natl. Acad. Sci. USA (1995) 92, 5798）。
（５）（１）と（２）の組合せ（例えば、リン酸部分がホスホロジチオエート化されてい
る（上式において、XがS^-に置換し、=O¹が=Sに置換されている））など（Tetrahedron
Lett. (1988) 29, 2911.; JACS (1989) 111, 2321.）。

(1) A hydroxyl group (—OH) (X in the above formula) bonded to phosphorus in the phosphoric acid moiety is a borano group (—BH
₃ ^-), phosphoro group (-S ^-), amino group (-NH _2), a lower alkyl group (-R) (R is, for example, methyl group, ethyl group) and an alkoxy group (-OR) (R is , For example, methyl group, ethyl group, etc.)
Substituted with a group selected from the group consisting of (Biochemistry (1979) 18, 5134 .; Tetra
hedron Lett.
(1982) 23, 4289.).
(2) Oxo group (= O) bound to phosphorus in the phosphate moiety
(= O ¹ in the above formula) is substituted with a thioxo group (= S) (Tetrahedron
Lett. (1980) 21, 1121 .; Biochemistry (1987) 26, 8237.).
(3) Phosphoric acid moiety phosphorus and oxy group (-O-) bound to 5 'carbon of sugar (in the above formula
-O ^2- ) is substituted with a group selected from the group consisting of a methylene group (-CH _2- ), a thioxy group (-S-) and an amino group (-NH-) (Nucleic Acids Res. 1997) 25, 830).
(4) Phosphoric acid moiety phosphorus and oxy group (-O-) bonded to 3 'carbon of sugar (in the above formula
-O ^3- ) is substituted with a group selected from the group consisting of a methylene group (-CH _2- ), a thioxy group (-S-) and an amino group (-NH-) (Proc. Natl. Acad Sci. USA (1995) 92, 5798).
The combination of (5) (1) and (2) (e.g., in phosphate moiety is phosphorodithioate reduction (the above formula, X is S ^- substituted with, = O ¹ is = is replaced with S (Tetrahedron)
Lett. (1988) 29, 2911 .; JACS (1989) 111, 2321.).

In addition, substitution of a hydroxyl group and / or oxygen atom bonded to phosphorus in the phosphate portion of DNA used as a template with another atom and / or group (hereinafter, this substitution is referred to as “modification”) is at least 1 What is necessary is just to be made by the phosphoric acid part of the location. For example, certain types of bases (ie adenine (abbreviated as A), guanine (abbreviated as G), cytosine)
(Abbreviated as C) or thymine (abbreviated as T)) sugar (D-furanose)
Modification may be made on all or part of the phosphate moiety bound to the deoxyribonucleoside moiety bound to (ester bond), or all phosphate moieties may be modified. Alternatively, the modification may be made randomly with a plurality of phosphate moieties.

The modified DNA may be any DNA, for example, DNA expressing siRNA, ribozyme or aptamer, apoptosis-related gene such as p53, DNA useful as a pharmaceutical such as decoy DNA, and DNA of interest as a research object. However, it is not limited to these.

The modified DNA may be modified at a site other than the phosphate moiety. Examples of modifications other than the phosphate moiety include, but are not limited to, modification of the base moiety, modification of the sugar moiety, modification of the 5 ′ end, modification of the 3 ′ end, and the like.

Examples of modified bases include 2-aminopurine, 2'-amino-butyrylpyrene-uridine, 2'-
Aminouridine, 2'-deoxyuridine, 2'-fluoro-cytidine, 2'-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine, 5-bromo-uridine, 5-fluoro-cytidine, 5- Fluoro-uridine, 5-indo-uridine, 5-methyl-cytidine, inosine, N3-methyl
-Uridine, 7-deaza-guanine, 8-aminohexyl-amino-adenine, 6-thio-guanine, 4-thio-thymine, 2-thio-thymine, 5-iodo-uridine, 5-iodo-cytidine, 8- Bromo-
Examples include guanine, 8-bromo-adenine, 7-deaza-adenine, 7-deaza-guanine, 8-oxo-guanine, 5,6-dihydro-uridine, 5-hydroxymethyl-uridine, and the like. There is no limit. These synthesis units are commercially available (for example,
It can be introduced into DNA by chemical synthesis.

Examples of sugar moiety modifications include, but are not limited to, 3′-deoxylation, 2′-fluorination, arabanosidation, and the like. These can also be introduced into DNA by chemical synthesis.

Examples of 5'-terminal modifications include 5'-amination, 5'-biotinylation, 5'-fluoresceinization, 5'-tetrafluoro-fluoresceination, 5'-thiolation, 5'-dabsylation, etc. It can be mentioned, but is not limited to these.

Examples of 3 ′ terminal modifications include 3′-amination, 3′-biotinylation, 2,3-dideoxylation, 3 ′
-Thiolation, 3'-Dabsylation, 3'-carboxylation, 3'-cholestylation and the like can be mentioned, but are not limited thereto.

The modified DNA may be produced by any known method. An example of the manufacturing method will be briefly described.

Biological methods and chemical synthesis are possible to synthesize modified DNA having the substitution (1) described above.

In the biological method, first, a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus of a phosphate moiety directly bonded to a sugar is substituted with another atom or group is synthesized. The modified deoxyribonucleoside triphosphate may be racemic or have a desired configuration. The synthesis of racemic modified deoxyribonucleoside triphosphates is described in Biochemi
stry (1979) 18, 5134 .; Tetrahedron Lett.
(1982) 23, 4289. A method for synthesizing modified deoxyribonucleoside triphosphates in the R configuration is described in Example 8 below. A modified deoxyribonucleoside triphosphate having a desired configuration can also be obtained from a racemate by optical resolution. Using the obtained modified deoxyribonucleoside triphosphate, a known nucleic acid amplification method (for example, PCR)
) To synthesize the target modified DNA from the template DNA (Nucleic Acid Researc)
h (1997) 25, 1611-1617.)). In the nucleic acid amplification method, even when racemic modified deoxyribonucleoside triphosphate is used as a substrate, only the modified deoxyribonucleoside triphosphate having the S configuration is consumed by the enzyme, and the modified DNA having the R configuration is constructed. .
When this method is used, it is not necessary to synthesize or separate a modified deoxyribonucleoside triphosphate serving as a substrate as an optically active substance, and the optical purity of the introduced modified deoxyribonucleoside triphosphate is 100%.

In the chemical synthesis method, a chiral auxiliary group that restricts the direction of bond formation is bonded to the phosphorus atom of phosphoric acid, which is a reaction point, and phosphoric acid having an arbitrary stereochemistry is synthesized by this effect.
Since this chiral auxiliary group itself can be synthesized in both the front and back configurations, this also makes it possible to synthesize non-natural DNA having a binding mode opposite to that of natural pair stereochemistry. An example of a chemical synthesis method is described in Examples 6 and 7 below.

In order to synthesize modified DNA having the substitutions (2) to (5) described above, two units of nucleosides were linked, the phosphate portion was modified, and then the third unit nucleoside was converted into 2 units.
After the introduction in the same way as the unit, the phosphate part is modified again. Repeat this operation as many times as necessary,
DNA with the desired base sequence can be synthesized (Tetrahedron Lett. (1980) 21, 1121 .;
Biochemistry (1987) 26,
8237 .; Nucleic Acids Res. (1997) 25, 830; Proc. Natl. Acad. Sci. USA (1995) 92,
5798; Tetrahedron Lett. (1988) 29, 2911 .; JACS (1989) 111, 2321.).

Whether or not the modified DNA can function as a template for RNA transcription and / or amplification can be confirmed by carrying out a method for RNA transcription and / or amplification described later.

An example of a method for transcription and / or amplification of RNA using modified DNA as a template is described below. In the presence of a modified DNA template (3-4 μg) dissolved in a buffer (11-12 μl), 10-11 μL of various bases of ribonucleoside triphosphates (hereinafter “rNTPs”)
". 25-27 mM each, Promega), T7 Transcription 5x Buffer (5-6 μL, Promega)
), Enzyme Mix (2-2.5 μL, Promega), and the transcription reaction is performed at 36.5-37.5 ° C. for 4-5 hours. It can be confirmed by a commercially available acrylamide gel that RNA having a desired size was obtained. The base sequence of the obtained RNA was obtained after reverse transcription of DNA using reverse transcriptase.
DNA can be amplified by PCR and confirmed by a commercially available sequencing apparatus.

  For RNA, the same modification can be made by the same method.

By using the modified DNA or RNA of the present invention, functional nucleic acids such as double-stranded RNA including siRNA or hairpin RNA, ribozyme, and antisense RNA are expressed in cells or tissues, or gene expression is suppressed. Can do. The target of the functional nucleic acid may be a virus such as HIV, HCV, HBV, an oncogene, or the like. When the modified DNA of the present invention is introduced into cells or tissues, this modified DNA can be used as a DNAzyme as one method for suppressing gene expression.

The composition containing the modified DNA or RNA of the present invention can be used for various applications such as pharmaceuticals, cosmetics, reagents and foods.

Furthermore, the present invention provides RNA transcription and / or amplification comprising a modified deoxyribonucleoside triphosphate in which the hydroxyl group bonded to the phosphorus of the phosphate moiety directly bonded to the sugar is replaced with another atom or group A template preparation kit is provided.

In the modified deoxyribonucleoside triphosphate, the hydroxyl group bonded to phosphorus of the phosphate moiety directly bonded to the sugar is substituted with another atom or group. This substitution is exo- and en
It is an object to make it possible to produce a modified DNA with improved do-nuclease resistance. As an example of the substitution, a hydroxyl group (—OH) bonded to phosphorus of the α-phosphate moiety is replaced with a borano group (—B
Examples thereof include those substituted with H ₃ ⁻ ). Other phosphoro groups (-S ^-), amino group (-NH _2),
Lower alkyl group (—R) (R is, for example, methyl group, ethyl group, etc.) and alkoxy group (—OR)
A substance substituted with a group selected from the group consisting of (for example, methyl group, ethyl group, etc.) can be used as a transcription template as long as it is recognized by RNA polymerase (Bioche
mistry (1979) 18, 5134 .; Tetrahedron Lett.
(1982) 23, 4289.).

The base of the base part of the modified deoxynucleotide triphosphate may be any of A, G, C, and T.

In addition, modified deoxyribonucleoside triphosphate is a phosphate (α
-It may or may not be modified at a site other than the phosphoric acid moiety. Examples of modifications at sites other than the phosphate moiety directly bonded to the sugar include, but are not limited to, modification of the base moiety and modification of the sugar moiety. Examples of the modification of the base moiety, the modification of the sugar moiety, and the like can include the same examples as described above for the modification of the base moiety of the modified DNA, the modification of the sugar moiety, etc., but are not limited thereto. Absent. Examples of other modifications of the sugar moiety include the same examples as described above for the modification of the 3 ′ end of the modified DNA. Further, the phosphoric acid (β, γ-phosphate) moiety that is not directly bonded to the sugar may not be modified.

The modified deoxyribonucleoside triphosphate may be 2′-deoxyribonucleoside triphosphate.

The modified deoxyribonucleoside triphosphate may be racemic or have a desired configuration.

On the other hand, the modified deoxyribonucleoside triphosphate may be produced by any known method. The literature describing the production method of modified deoxyribonucleoside triphosphates is as described above.

The template preparation kit for RNA transcription and / or amplification of the present invention further comprises:
Inducing agents (for example, DNA polymerase, reverse transcriptase, etc.), other deoxyribonucleoside triphosphates (for example, modified deoxyribonucleoside triphosphates) that promote the synthesis of DNA as a template for transcription and / or amplification of RNA Deoxyribonucleoside triphosphates that contain a different type of base from the specific base that is a component of (a phosphate that is directly linked to the sugar, but not modified) Etc.), buffers (eg, Hepes, TRIS, glycerol, DTT, etc.), metal ions (eg, sodium, potassium, magnesium, etc.), instructions describing how to use the kit, etc. . Furthermore, the kit of the present invention transcribes RNA and / or
Alternatively, it may contain a primer pair for synthesizing DNA as a template for amplification, DNA as a template, etc. An example of how to use the kit for preparing a template for RNA transcription and / or amplification of the present invention is as follows: Explained. Modified deoxyribonucleotide triphosphates and natural deoxyribonucleotide triphosphates (for example, dATP) having various bases different from specific bases (for example, T) that are components of modified deoxyribonucleotide triphosphates , DGTP and dCTP), the target DNA (template for RNA transcription and / or amplification) is synthesized using a template DNA, a set of primers and a DNA polymerase. The molecular weight of the synthesized DNA can be confirmed with a commercially available acrylamide gel. The base sequence of the synthesized DNA can be confirmed with a commercially available sequencing apparatus.

Furthermore, the present invention includes a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus of a phosphate moiety directly bonded to a sugar is substituted with another atom or group.
A transcription and / or amplification kit (hereinafter, “RNA transcription and / or first aspect of the present invention”)
Or “amplification kit”. )I will provide a.

The kit for RNA transcription and / or amplification according to the first aspect of the present invention comprises (A) an element for producing DNA serving as a template for RNA transcription and / or amplification, and (B) RNA.
May be composed of elements for transcription and / or amplification.

The element of (A) includes a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus of a phosphate moiety directly bonded to a sugar is substituted with another atom or group, transcription of RNA and / or Inducing agents (for example, DNA polymerase, reverse transcriptase, etc.) that promote the synthesis of DNA as a template for amplification, other deoxyribonucleoside triphosphates (
For example, deoxyribonucleoside triphosphates containing a different type of base as a constituent element from a specific base that is a constituent element of a modified deoxyribonucleoside triphosphate (modified with a phosphate moiety directly attached to a sugar) Or it may be unmodified)
Etc.), buffers (eg, Hepes, TRIS, glycerol, DTT, etc.), metal ions (eg, sodium, potassium, magnesium, etc.) and the like.

The element of (B) includes an inducer that promotes transcription and / or amplification of RNA (for example,
RNA polymerase, etc.), ribonucleoside triphosphates (base part is A, respectively,
G, C, and U), buffers (eg, Hepes, TRIS, glycerol, etc.), metal ions (eg, sodium, potassium, magnesium, etc.), and the like.

Furthermore, the RNA transcription and / or amplification kit of the first aspect of the present invention may include instructions describing how to use the kit.

An example of a method for using the RNA transcription and / or amplification kit of the first aspect of the present invention will be described below. First, using the element (A), a DNA serving as a template for RNA transcription and / or amplification is prepared. The method is the same as the method of using the template preparation kit for RNA transcription and / or amplification of the present invention. Next, RNA is transcribed and / or amplified using the prepared template (DNA) and (B) elements.
An example of the method is described below. In the presence of a DNA template, transcription reaction is performed using rNTPs of various bases, buffers, and RNA polymerase. The molecular weight of the obtained RNA can be confirmed by a commercially available acrylamide gel. The base sequence of the obtained RNA can be confirmed with a commercially available sequencer after reverse transcription of the DNA using reverse transcriptase and amplifying the obtained DNA by PCR.

The present invention also provides a modified DNA in which a hydroxyl group and / or an oxygen atom bonded to phosphorus of a phosphate moiety directly bonded to a sugar is substituted with another atom and / or group,
Comprising the modified DNA capable of functioning as a template for transcription and / or amplification of A,
An RNA transcription and / or amplification kit (hereinafter referred to as “the RNA transcription and / or amplification kit of the second aspect of the present invention”) is provided.

The modified DNA is as described above. This modified DNA serves as a template for RNA transcription and / or amplification.

The kit for RNA transcription and / or amplification according to the second aspect of the present invention further comprises an inducing agent (for example, RNA polymerase) that promotes transcription and / or amplification of RNA, ribonucleoside triphosphate (base portion base). Are A, G, C and U, respectively), buffers (eg, Hepes, TRIS, glycerol, etc.), metal ions (eg, sodium, potassium, magnesium, etc.), instructions describing how to use the kit Etc. may be included.
The template at this time can also be synthesized by chemical synthesis using an optically active nucleoside.

An example of a method for using the RNA transcription and / or amplification kit of the second aspect of the present invention will be described below. RNTP, buffer, RN of various bases in the presence of DNA template (modified DNA)
Perform transcription reaction using A polymerase. The molecular weight of the obtained RNA can be confirmed by a commercially available acrylamide gel. The base sequence of the obtained RNA can be confirmed with a commercially available sequencer after reverse transcription of the DNA using reverse transcriptase and amplifying the obtained DNA by PCR. In addition, when optically active borano-modified rNTP is used in RNA transcription reaction, borano-modified RNA
Is prepared. Furthermore, supply by chemical synthesis is possible by using an optically active borano-modified RNA synthesis unit.

Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention, and do not limit the scope of the present invention.
[Example 1] α-boranothymidine triphosphate (dTTP-αB), α-boranoadenosine triphosphate (
dATP-αB), α-boranoguanosine triphosphate (dGTP-αB) and α-boranocytidine triphosphate (dCTP-αB)

（１）α-ボラノチミジン三リン酸（dTTP-αB）の合成（上式のB=チミン）
乾燥させた市販の3’-O-アセチルチミジン（Sigma-Aldrich社）（0.25 mmol）をアルゴ
ン雰囲気下、無水ピリジン（0.1
mL）と無水DMF（0.75 mL）に溶かした。そこへ1Mの2-クロロ-4H-1,3,2-ベンゾジオキサホ
スフォリン-4-オン（Aldrich社）（0.75 mL）を加え攪拌した。15分後に0.5Mのビス（ト
リ-n-ブチルアンモニウム）ピロリン酸（Sigma-Aldrich社）（0.75 mL）と無水n-トリブ
チルアミン（東京化成）（0.125
mL）を加えてさらに10分間攪拌後、N,N-ジイソプロピルエチルアミン-ボラン錯体（Aldri
ch社）（1 mL）を加えて室温で9時間攪拌した。反応液を水（25 mL）で希釈して1.5時間
攪拌後、ロータリーエバポレーターで濃縮して固体を得た。アンモニア水-メタノール/2:
1（40 mL）を加えて室温で68時間攪拌後、ロータリーエバポレーターで濃縮して固体を得
た。残査を水（25 mL）に溶解し、ジクロロメタン（10
mLx2）で洗浄後、水溶液を凍結乾燥して固体を得た。粗生成物はイオン交換クロマトグラ
フィー（UNO-Q12、BIORAD社）によって炭酸水素アンモニウム（溶出濃度0.45M-0.5M）を
用いて精製した。溶出液の凍結乾燥を３度繰り返して生成物（dTTP-αB）を得た。収率36
％。dTTP-αBの質量分析スペクトルを図５に示す。dTTP-αBの質量分析値(M)は479.0364
であった。

（２）α-ボラノアデノシン三リン酸（dATP-αB）の合成（上式のB=アデニン）
2’,3’-O-ジアセチル-5’-O-[(2R,5R)-3-メチル-5-フェニル-1,3,2-オキサザホスホリジ
ン-2-イル]アデノシンの合成
3’-O-ジアセチルアデノシン（1.5mmol）をTHF（7.5mL）とトリエチルアミン（7.5mmol
）に溶解して-78℃に冷却した。0.22M溶液の(5R)-1（7.5mL, 1.65mmol）を滴下し、室温
まで上昇させて30分間攪拌した。飽和重曹水（75mL）とクロロホルム（75mL）を加えて有
機層を分離後、有機層を飽和重曹水で洗浄した。水層をクロロホルムで逆抽出して混合後
、無水硫酸ナトリウムで乾燥させた。溶媒を減圧留去後、粗生成物をシリカゲルカラムク
ロマトグラフを用いて精製した（ヘキサン-酢酸エチル-トリエチルアミン=50:50:2）。溶
出物を飽和重曹水で洗浄して無水硫酸ナトリウムで乾燥させ、濃縮して純粋な生成物（2
’,3’-O-ジアセチル-5’-O-[(2R,5R)-3-メチル-5-フェニル-1,3,2-オキサザホスホリジ
ン-2-イル] アデノシン）（1.2 mmol）を得た。

アデノシン 5’-[α-(Rp)-ボラノ]三リン酸の合成
0.2Mアセトニトリル溶液のN-シアノメチルピロリジニウムトリフラート（500μL, 100
μmol）と2,6-ジニトロフェノール（50μmol）に2’,3’-O-ジアセチル-5’-O-[(2R,5R)-
3-メチル-5-フェニル-1,3,2-オキサザホスホリジン-2-イル] アデノシン（39.6mg, 60μm
ol）を加えた。５分後にボラン-ジメチルスルフィド錯体（60μmol）を加え、８分後に無
水ピリジン（500μmol）と無水酢酸（100μmol）を加えた。さらに３分後にテトラ（トリ
-n-ブチルアンモニウム）ピロリン酸（60μmol）を加えて１時間攪拌した。反応溶液をク
ロロホルム（3mL）で希釈し、0.2Mのリン酸緩衝液（pH7.0,
3mL）で洗浄し、水層をクロロホルムで逆抽出した。有機層を混合後、無水硫酸ナトリウ
ムで乾燥させた。残査を25%アンモニア水-ピリジン（9:1）中55℃で30分反応させてキラ
ル補助基と保護基を除去し、冷却後に濃縮した。残査を水に溶解してエーテルで洗浄し、
水を減圧留去後、粗生成物をイオン交換クロマトグラフHPLCを用いて精製して純粋な生成
物(アデノシン 5’-[α-(Rp)-ボラノ]三リン酸)（35.4 μmol）を得た。


（３）α-ボラノグアノシン三リン酸（dGTP-αB）の合成（上式のB=グアニン）
上記の最初の原料にジアセチルグアノシンを使用して、上記の方法により、α-ボラノ
グアノシン三リン酸を得た。



（４）α-ボラノシチジン三リン酸（dCTP-αB）の合成（上式のB=シトシン）
上記の最初の原料にジアセチルシチジンを使用して、上記の方法により、α-ボラノシ
チジン三リン酸を得た。

(1) Synthesis of α-boranothymidine triphosphate (dTTP-αB) (B = thymine in the above formula)
A dried commercial 3′-O-acetylthymidine (Sigma-Aldrich) (0.25 mmol) was added in anhydrous pyridine (0.1 mmol) under an argon atmosphere.
mL) and anhydrous DMF (0.75 mL). 1M 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (Aldrich) (0.75 mL) was added thereto and stirred. After 15 minutes, 0.5M bis (tri-n-butylammonium) pyrophosphate (Sigma-Aldrich) (0.75 mL) and anhydrous n-tributylamine (Tokyo Kasei) (0.125
mL) and stirring for another 10 minutes, followed by N, N-diisopropylethylamine-borane complex (Aldri
ch) (1 mL) was added and stirred at room temperature for 9 hours. The reaction solution was diluted with water (25 mL), stirred for 1.5 hours, and then concentrated by a rotary evaporator to obtain a solid. Ammonia water-methanol / 2:
1 (40 mL) was added, and the mixture was stirred at room temperature for 68 hours, and then concentrated by a rotary evaporator to obtain a solid. Dissolve the residue in water (25 mL) and add dichloromethane (10
After washing with mLx2), the aqueous solution was lyophilized to obtain a solid. The crude product was purified by ion exchange chromatography (UNO-Q12, BIORAD) using ammonium bicarbonate (elution concentration 0.45M-0.5M). The eluate was freeze-dried three times to obtain a product (dTTP-αB). Yield 36
%. A mass spectrum of dTTP-αB is shown in FIG. The mass spectrometric value (M) of dTTP-αB is 479.0364.
Met.

(2) Synthesis of α-boranoadenosine triphosphate (dATP-αB) (B = adenine in the above formula)
Synthesis of 2 ', 3'-O-diacetyl-5'-O-[(2R, 5R) -3-methyl-5-phenyl-1,3,2-oxazaphospholidin-2-yl] adenosine
3'-O-diacetyladenosine (1.5mmol) with THF (7.5mL) and triethylamine (7.5mmol)
) And cooled to -78 ° C. A 0.22 M solution of (5R) -1 (7.5 mL, 1.65 mmol) was added dropwise, and the mixture was allowed to rise to room temperature and stirred for 30 minutes. Saturated aqueous sodium hydrogen carbonate (75 mL) and chloroform (75 mL) were added to separate the organic layer, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate. The aqueous layer was back extracted with chloroform, mixed, and dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the crude product was purified using silica gel column chromatography (hexane-ethyl acetate-triethylamine = 50: 50: 2). The eluate is washed with saturated aqueous sodium bicarbonate, dried over anhydrous sodium sulfate, and concentrated to give pure product (2
', 3'-O-diacetyl-5'-O-[(2R, 5R) -3-methyl-5-phenyl-1,3,2-oxazaphospholidin-2-yl] adenosine) (1.2 mmol) Got.

Synthesis of adenosine 5 '-[α- (Rp) -borano] triphosphate
N-cyanomethylpyrrolidinium triflate in 0.2M acetonitrile solution (500μL, 100
μmol) and 2,6-dinitrophenol (50 μmol) to 2 ', 3'-O-diacetyl-5'-O-[(2R, 5R)-
3-Methyl-5-phenyl-1,3,2-oxazaphospholidin-2-yl] adenosine (39.6mg, 60μm
ol) was added. After 5 minutes, borane-dimethyl sulfide complex (60 μmol) was added, and after 8 minutes, anhydrous pyridine (500 μmol) and acetic anhydride (100 μmol) were added. After another 3 minutes, tetra (tri
-n-Butylammonium) pyrophosphate (60 μmol) was added and stirred for 1 hour. The reaction solution was diluted with chloroform (3 mL), and 0.2 M phosphate buffer (pH 7.0,
3 mL), and the aqueous layer was back extracted with chloroform. The organic layers were mixed and then dried over anhydrous sodium sulfate. The residue was reacted in 25% aqueous ammonia-pyridine (9: 1) at 55 ° C. for 30 minutes to remove the chiral auxiliary and protecting group, and concentrated after cooling. Dissolve the residue in water and wash with ether,
After distilling off water under reduced pressure, the crude product was purified by ion exchange chromatography HPLC to obtain a pure product (adenosine 5 ′-[α- (Rp) -borano] triphosphate) (35.4 μmol). It was.


(3) Synthesis of α-boranoguanosine triphosphate (dGTP-αB) (B = guanine in the above formula)
Using diacetylguanosine as the first raw material, α-boranoguanosine triphosphate was obtained by the above method.



(4) Synthesis of α-boranocytidine triphosphate (dCTP-αB) (B = cytosine in the above formula)
Using diacetylcytidine as the first raw material, α-boranocytidine triphosphate was obtained by the above method.

[Example 2] Preparation of boron-containing oligo DNA by PCR Commercially available natural 2'-deoxyadenosine triphosphate (Takara Bio), 2'-deoxycytidine triphosphate (Takara Bio), 2'-deoxyguanosine Triphosphate (Takara Bio)
(0.25 mM each) and in the presence of α-boranothymidine triphosphate (0.25 mM) synthesized in Example 1, two kinds of primers (TTAATACGACTCACTATAGAAGGAGACATATGGATGTTCAGCTGCAGG (SEQ ID NO: 1) and GCTCTAGATTAGTGGTGGTGGTGGTGG (SEQ ID NO: 2)) (0.5 μM each) PCR was performed using a template (base sequence is shown in SEQ ID NO: 3) (0.5 pM) and ExTaq polymerase (Takara Bio) (1 μL) (35 cycles (94 ° C.-1 min, 55 ° C.- 1 minute, 72 ° C-1 minute.)).
The product was purified using 2% agarose (Takara Bio) to obtain the desired dsDNA (Figure 1).
). The base sequence of the obtained dsDNA is a sequencing device (Applied Biosystems, ABI-PRISM377)
Was used to confirm the sequence.
Instead of natural dATP + dCTP + dGTP and dTTP-αB, natural dATP + dCTP + dGTP and dTTP-
αB, natural dCTP + dGTP and dATP-aB + dTTP-αB, natural dTTP and dATP-aB + dGTP-aB + dCT
P-αB, natural dCTP and dATP-aB + dGTP-aB + dTTP-αB, natural dGTP and dATP-aB + dCTP-
aB + dTTP-αB, native dATP and dGTP-aB + dCTP-aB + dTTP-αB, or dATP-aB + dGTP-aB +
The above operation was repeated using dCTP-aB + dTTP-αB. The results are shown in FIG. DsD obtained
The nucleotide sequence of NA was confirmed using a sequencing device (Applied Biosystems, ABI-PRISM377).

[Example 3]
A sequence containing the human H1 promoter and human U6 promoter was amplified by PCR based on the DNA on the chromosome. The primer sequences used for PCR are as follows.
U6 (90bp)
5'-TGCACTAGTATTTCCCATGATTCCTTCATATTTGCATCTTACCGTAACTTGAA-3 '(SEQ ID NO: 4)
and
5′-GTGGGGCCCTTCGAACGCGAATTCCTTTCCACAGATA-3 ′ (SEQ ID NO: 5).
H1 (100bp)
5'-TGCACTAGTATTTGCATGTCGCTA-3 '(SEQ ID NO: 6)
and
5′-GTGGGGCCCTTCGAACGCGAATTCGTC-3 ′ (SEQ ID NO: 7).

DNA amplified by PCR was transferred to plasmid pPUR (CLONTECH / CLONTECH Laboratories, In
c. California, USA) and named pRU6 (90) and pRH1 (100), respectively.

Functional RNA (double-stranded RNA) sequences were incorporated into these constructed plasmid DNAs. The arrangement is as follows.
5'-AATTAACACCGCAGAAGCTATGAAACGATTTGCTTCCTGTCACAAATCGTTTCATAGCTTCTGCTTTTT-3 '(SEQ ID NO: 8)
and
5′-AATTAAAAAGCAGAAGCTATGAAACGATTTGTGACAGGAAGCAAATCGTTTCATAGCTTCTGCGGTGTT-3 ′ (SEQ ID NO: 9).

Two kinds of DNAs having the above sequences were mixed, heated, and then gradually cooled to room temperature. The resulting D
NA was incorporated into the above four types of plasmids and named pRU6 (90) -L23 and pRH1 (100) -L23, respectively. The expressed double-stranded RNA has homology to the gene for the luciferase enzyme.

The procedure of Example 2 was repeated using a plasmid having a U6 promoter (pRU6 (90) -L23) instead of the template DNA having the base sequence shown in SEQ ID NO: 3. The results are shown in FIG. Boron-containing PCR products were obtained even when a plasmid having a U6 promoter was used as a template.

Furthermore, when the enzyme was changed from ExTaq to Taq from Fermentas, a PCR product in which boron was introduced in all base combinations was obtained (FIG. 3).

[Example 4] T7 RNA transcription reaction In the presence of the DNA template (3 μg / 12 μL) prepared in Example 2, 10 μL of rNTPs (Promeg
Company a) (25 mM each), T7
Transcription 5x Buffer (6 μL, Promega) and Enzyme Mix (2 μL, Promega) were used for transcription reaction at 37 ° C. for 4 hours. It was confirmed by 5% acrylamide gel that RNA having the desired size was obtained (FIG. 4).

[Example 5] RNAi experiment
Plasmids with U6 promoter or H1 promoter (pRU6 (90) -L23, pRH1 (100) -L
23), the procedure of Example 2 was repeated to prepare various boranophosphate DNAs.
RNAi experiments using these various boranophosphate DNAs as templates were conducted. The experimental procedure was performed according to Nature Biotechnology (2002) 20, 497-500. Transfection was performed using Lipofectamine 2000 (Life Technology) according to the reagent operation manual.
The luciferase assay consists of 20ng Renilla luciferase expression vector in HeLa S3 cells,
A 50 ng firefly luciferase expression vector and a 66 ng siRNA expression vector were used.
24 hours after dual
Using the luciferase system (Promega), the operation was performed according to the kit. Remil
The firefly expression level was converted from the expression level (measured value) of la and compared. The results are shown in FIG.
The RNAi effect was observed for both the PCR product in which boron was introduced for one type of base and the PCR product in which boron was introduced for all combinations of bases.

[Example 6] Synthesis for controlling the configuration of oligonucleoside phosphorothioate
Synthesis of N- (cyanomethyl) pyrrolidinium trifluoromethanesulfonate (1f)

N-(シアノメチル)ピロリジン（2.2g, 20mmol）溶液（ジクロロメタン20mL）にトリフルオ
ロメタンスルホン酸（1.77mL,
20mmol）を0℃で加えた。反応液を室温まで上昇させてエーテル（40mL）で希釈後、不溶
物を濾過してエーテルで洗浄した。濾取物を減圧乾燥して純粋な生成物1f（5.2g, 20mmol
）を得た。
融点67.0-67.5℃. IR (KBr) v_max 2996, 2841, 2651,2477, 2347, 2282, 1637, 1462, 14
37, 1269, 1228, 1168, 1033, 985, 911, 849, 761,641 cm^-1. ¹H NMR (300 MHz, CD₃CN)
δ 8.14 (br,1H), 4.28 (s, 2H), 3.48 (br, 4H), 2.09 (m, 4H). ¹³C NMR (75 MHz, CD
₃CN)δ 121.5 (q, ¹J_CF = 320 Hz), 112.6, 56.2, 42.3, 23.9.Anal. Calcd for C₇H₁₁F₃
N₂O₃S:C, 32.31; H, 4.26; N, 10.76. Found: C, 32.31; H, 4.22; N, 10.81.

1a-e,g-nは同様の方法で合成した。

(5R)-2-クロロ-3-メチル-5-フェニル-1,3,2-オキサザホスホリジン[(5R)-3a]の合成

N- (Cyanomethyl) pyrrolidine (2.2 g, 20 mmol) solution (dichloromethane 20 mL) was added to trifluoromethanesulfonic acid (1.77 mL,
20 mmol) was added at 0 ° C. The reaction mixture was warmed to room temperature and diluted with ether (40 mL), and the insoluble material was filtered and washed with ether. The filter cake was dried under reduced pressure to give pure product 1f (5.2 g, 20 mmol).
)
Melting point 67.0-67.5 ℃ IR (KBr) v _max 2996, 2841, 2651, 2477, 2347, 2282, 1637, 1462, 14
37, 1269, 1228, 1168, 1033, 985, 911, 849, 761,641 cm ^-1 . ¹ H NMR (300 MHz, CD ₃ CN)
δ 8.14 (br, 1H), 4.28 (s, 2H), 3.48 (br, 4H), 2.09 (m, 4H). ¹³ C NMR (75 MHz, CD
₃ CN) δ 121.5 (q, ¹ J _CF = 320 Hz), 112.6, 56.2, 42.3, 23.9.Anal.Calcd for C ₇ H ₁₁ F ₃
N ₂ O ₃ S: C, 32.31; H, 4.26; N, 10.76.Found: C, 32.31; H, 4.22; N, 10.81.

1a-e and gn were synthesized by the same method.

Synthesis of (5R) -2-chloro-3-methyl-5-phenyl-1,3,2-oxazaphosphoridine [(5R) -3a]

N-メチルモルホリン（1.1mL, 10mmol）を(R)-2a（756mg, 5mmol）のトルエン溶液（2.5mL
）に溶かし、三塩化リン（435μL, 5mmol）溶液（トルエン2.5mL）を0°Cで加えた。反応
液を室温まで上昇させて30分攪拌後、不溶物を-78°Cで濾過して濾液を濃縮して(5R)-3a
の粗生成物（1.07g, 5mmol）を得た。粗生成物は精製せずに次の反応に用いた。¹³C NMR
(75 MHz, CDCl₃) δ 138.1, 128.5, 128.5,126.5, 85.4 (br), 56.3 (d, ²J_PC = 6.6 Hz)
, 31.0 (d, ²J_PC= 13.0 Hz).

(5S)-3a, 3b-fは同様の方法で合成した。(5S)-3a, 3b-fの精製法およびスペクトルデート
を以下に示す。(5S)-3aは蒸留によって精製した。20mmolから2.1g, 9.7mmol。

(5S)-2-クロロ-3-メチル-5-フェニル-1,3,2-オキサザホスホリジン[(5S)-3a]の合成
(S)-2aから(5S)-3aの粗生成物を得、蒸留によって精製した。20mmolから2.22g, 12mmol。

(4S,5R)-2-クロロ-3,4-ジメチル-5-フェニル-1,3,2-オキサザホスホリジン(3b)の合成
出発物質として(1R,2S)-エフェドリン(2b)（496 mg, 3.0 mmol）を用い、得られた粗生成
物をクーゲロール蒸留によって精製して、3bを得た。3mmolから356mg, 1.6mmol。
¹H NMR (300 MHz, CDCl₃).δ 7.40-7.23(m, 5H), 5.85 (d, ³J_HH = 6.3 Hz, 1H), 3.72-3
.61 (m, 1H),2.72 (d, ³J_PH = 15.6 Hz, 3H), 0.72 (d, ³J_HH= 4.2 Hz, 3H). ¹³C NMR (7
5 MHz, CDCl₃) δ 136.4 (d, ³J_PC= 2.6 Hz), 128.3, 128.3, 126.9, 87.7 (d, ²J_PC = 8
.9 Hz),57.7 (br), 29.0 (d, ²J_PC = 13.8 Hz), 14.4. ³¹PNMR (121 MHz, CDCl₃).δ 171
.5.

(4S,5R)-2-クロロ-3-メチル-4,5-ジフェニル-1,3,2-オキサザホスホリジン(3c)の合成
(1R,2S)-2-メチル-1,2-ジフェニルエタノール(2c)(2.27 g, 10 mmol)から3cの粗生成物を
得た。10mmolから3.17g, 10mmol。
¹H NMR (300 MHz, CDCl₃). δ 7.08-7.05(m, 6H), 6.91-6.81 (m, 4H), 6.15 (d, ³J_HH =
8.3 Hz, 1H),4.64 (dd, ³J_HH = 8.3 Hz, ³J_PH = 4.2Hz, 1H), 2.64 (d, ³J_PH = 15.3 Hz
, 3H). ¹³C NMR(75 MHz, CDCl₃) δ 135.8 (d, ³J_PC = 3.2 Hz),134.5 (d, ³J_PC = 5.4 H
z), 128.2, 127.9, 127.7, 127.6,127.5, 126.5, 88.0 (d, ²J_PC = 8.6 Hz), 68.1 (d, ²
J_PC= 6.3 Hz), 29.6 (d, ²J_PC = 14.1 Hz). ³¹P NMR(121 MHz, CDCl₃). δ 171.7.

(5R)-2-クロロ-3-イソプロピル-5-フェニル-1,3,2-オキサザホスホリジン(3d)の合成
(R)-2-イソプロピル-1-フェニルエタノール(2d)(896 mg, 5.0 mmol)から3dの粗生成物を
得た。5mmolから1.22g, 5mmol。
¹H NMR (300 MHz, CDCl₃) δ 7.45-7.34(m, 5H), 5.94-5.24 (br, 1H), 3.62-3.47 (m, 1
H), 3.18 (dd, ²J_HH= 18.3 Hz, ³J_HH = 9.3 Hz, 1H), 1.35 (d, ³J_HH= 6.6 Hz, 6H). ¹³C
NMR (75 MHz, CDCl₃) δ 138.5 (br),128.6, 128.5, 126.5 (br), 84.7 (br), 51.3 (br
), 47.3 (d, ²J_PC= 10.6 Hz), 22.0 (d, ³J_PC = 8.9 Hz), 21.8 (d, ³J_PC= 9.5 Hz). ³¹P
NMR (121 MHz, CDCl₃) δ 170.6 (br).

(5R)-2-クロロ-3-メチル-5-イソプロピル-1,3,2-オキサザホスホリジン(3e)の合成
出発物質として(R)-1-メチルアミノ-3-メチル-2-ブタノール(2e)(1.17
g, 10 mmol)を用い、得られた粗生成物を蒸留によって精製して、3eを得た。10mmolから1
.09g, 6mmol。
¹H NMR (300 MHz, CDCl₃). δ 4.68, 4.18(br, br, 1H), 3.16, 2.95 (m, m, 2H), 2.72
(d, ³J_PH = 15.0Hz, 3H), 2.01, 1.90 (br, br, 1H), 1.06-0.99 (m, 3H), 0.94 (d, ³J_H
H= 6.6 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 90.7, 88.7 (br,br), 53.0, 51.8 (br, b
r), 32.9 (br), 31.1 (d, ²J_PC = 14.3Hz), 18.3, 18.2 (br, br). ³¹P NMR (121 MHz, C
DCl₃). δ 173.7,172.5 (br, br).

(4S)-2-クロロ-3-メチル-4-イソプロピル-1,3,2-オキサザホスホリジン(3f)の合成
出発物質として(S)-2-メチルアミノ-3-メチル-1-ブタノール(2f)(1.17
g, 10 mmol)を用い、得られた粗生成物を蒸留によって精製して、3fを得た。10mmolから1
.09g, 6mmol。
¹H NMR (300 MHz, CDCl₃). δ 4.57 (d, ²J_HH= 8.7 Hz, ³J_HH = 8.7 Hz, 1H), 4.21 (m,
1H), 3.25 (m, 1H),2.68 (d, ³J_PH = 16.2 Hz, 3H), 2.03 (m, 1H), 0.92 (d, ³J_HH= 7.2
Hz, 3H), 0.84 (d, ³J_HH = 6.6 Hz, 3H). ¹³CNMR (75 MHz, CDCl₃) δ 71.5 (d, ²J_PC =
9.2 Hz),63.5 (br), 29.1 (d, ²J_PC = 14.1 Hz), 26.7 (d, ³J_PC= 4.9 Hz), 19.3, 14.5
. ³¹P NMR (121 MHz, CDCl₃) δ 176.8.

5’-O-(tert-ブチルジメチルシリル)-3’-O-[(2R,5R)-3-メチル-5-フェニル-1,3,2-オキ
サザホスホリジン-2-イル]チミジン [(Rp)-5a]の合成.

N-methylmorpholine (1.1 mL, 10 mmol) was added to a toluene solution (2.5 mL) of (R) -2a (756 mg, 5 mmol)
), And a solution of phosphorus trichloride (435 μL, 5 mmol) (toluene 2.5 mL) was added at 0 ° C. The reaction solution was allowed to rise to room temperature and stirred for 30 minutes, then the insoluble material was filtered at -78 ° C, and the filtrate was concentrated (5R) -3a.
Of crude product (1.07 g, 5 mmol) was obtained. The crude product was used in the next reaction without purification. ¹³ C NMR
(75 MHz, CDCl ₃ ) δ 138.1, 128.5, 128.5, 126.5, 85.4 (br), 56.3 (d, ² J _PC = 6.6 Hz)
, 31.0 (d, ² J _PC = 13.0 Hz).

(5S) -3a and 3b-f were synthesized by the same method. The purification method and spectral date of (5S) -3a, 3b-f are shown below. (5S) -3a was purified by distillation. 20 mmol to 2.1 g, 9.7 mmol.

Synthesis of (5S) -2-chloro-3-methyl-5-phenyl-1,3,2-oxazaphosphoridine [(5S) -3a]
The crude product of (5S) -3a was obtained from (S) -2a and purified by distillation. 20 mmol to 2.22 g, 12 mmol.

Synthesis of (4S, 5R) -2-chloro-3,4-dimethyl-5-phenyl-1,3,2-oxazaphosphoridine (3b) (1R, 2S) -ephedrine (2b) (496 mg, 3.0 mmol) was used to purify the resulting crude product by Kugelol distillation to give 3b. 3 mmol to 356 mg, 1.6 mmol.
¹ H NMR (300 MHz, CDCl ₃ ) .δ 7.40-7.23 (m, 5H), 5.85 (d, ³ J _HH = 6.3 Hz, 1H), 3.72-3
.61 (m, 1H), 2.72 (d, ³ J _PH = 15.6 Hz, 3H), 0.72 (d, ³ J _HH = 4.2 Hz, 3H). ¹³ C NMR (7
5 MHz, CDCl ₃ ) δ 136.4 (d, ³ J _PC = 2.6 Hz), 128.3, 128.3, 126.9, 87.7 (d, ² J _PC = 8
.9 Hz), 57.7 (br) , 29.0 (d, 2 J PC = 13.8 Hz), 14.4. 31 PNMR (121 MHz, CDCl 3) .δ 171
.Five.

Synthesis of (4S, 5R) -2-chloro-3-methyl-4,5-diphenyl-1,3,2-oxazaphosphoridine (3c)
The crude product of 3c was obtained from (1R, 2S) -2-methyl-1,2-diphenylethanol (2c) (2.27 g, 10 mmol). 10 mmol to 3.17 g, 10 mmol.
¹ H NMR (300 MHz, CDCl ₃ ) .δ 7.08-7.05 (m, 6H), 6.91-6.81 (m, 4H), 6.15 (d, ³ J _HH =
8.3 Hz, 1H), 4.64 (dd, ³ J _HH = 8.3 Hz, ³ J _PH = 4.2Hz, 1H), 2.64 (d, ³ J _PH = 15.3 Hz
¹³ C NMR (75 MHz, CDCl ₃ ) δ 135.8 (d, ³ J _PC = 3.2 Hz), 134.5 (d, ³ J _PC = 5.4 H
z), 128.2, 127.9, 127.7, 127.6,127.5, 126.5, 88.0 (d, ² J _PC = 8.6 Hz), 68.1 (d, ²
_{J PC = 6.3 Hz), 29.6} (d, 2 J PC = 14.1 Hz). 31 P NMR (121 MHz, CDCl 3). Δ 171.7.

Synthesis of (5R) -2-chloro-3-isopropyl-5-phenyl-1,3,2-oxazaphosphoridine (3d)
The crude product of 3d was obtained from (R) -2-isopropyl-1-phenylethanol (2d) (896 mg, 5.0 mmol). 5 mmol to 1.22 g, 5 mmol.
¹ H NMR (300 MHz, CDCl ₃ ) δ 7.45-7.34 (m, 5H), 5.94-5.24 (br, 1H), 3.62-3.47 (m, 1
^{H), 3.18 (dd, 2} J HH = 18.3 Hz, 3 J HH = 9.3 Hz, 1H), 1.35 (d, 3 J HH = 6.6 Hz, 6H). 13 C
NMR (75 MHz, CDCl ₃ ) δ 138.5 (br), 128.6, 128.5, 126.5 (br), 84.7 (br), 51.3 (br
), 47.3 (d, ² J _PC = 10.6 Hz), 22.0 (d, ³ J _PC = 8.9 Hz), 21.8 (d, ³ J _PC = 9.5 Hz). ³¹ P
NMR (121 MHz, CDCl ₃ ) δ 170.6 (br).

Synthesis of (5R) -2-chloro-3-methyl-5-isopropyl-1,3,2-oxazaphosphoridine (3e) (R) -1-methylamino-3-methyl-2-butanol (2e) (1.17
g, 10 mmol) was used to purify the resulting crude product by distillation to give 3e. 10mmol to 1
.09g, 6mmol.
¹ H NMR (300 MHz, CDCl ₃ ) .δ 4.68, 4.18 (br, br, 1H), 3.16, 2.95 (m, m, 2H), 2.72
(d, ³ J _PH = 15.0Hz, 3H), 2.01, 1.90 (br, br, 1H), 1.06-0.99 (m, 3H), 0.94 (d, ³ J _H
H = 6.6 Hz, 3H). ¹³ C NMR (75 MHz, CDCl ₃ ) δ 90.7, 88.7 (br, br), 53.0, 51.8 (br, b
r), 32.9 (br), 31.1 (d, 2 J PC = 14.3Hz), 18.3, 18.2 (br, br). 31 P NMR (121 MHz, C
DCl ₃ ) .δ 173.7, 172.5 (br, br).

Synthesis of (4S) -2-chloro-3-methyl-4-isopropyl-1,3,2-oxazaphosphoridine (3f) (S) -2-methylamino-3-methyl-1-butanol (2f) (1.17
g, 10 mmol) was used to purify the resulting crude product by distillation to give 3f. 10mmol to 1
.09g, 6mmol.
¹ H NMR (300 MHz, CDCl ₃ ) .δ 4.57 (d, ² J _HH = 8.7 Hz, ³ J _HH = 8.7 Hz, 1H), 4.21 (m,
1H), 3.25 (m, 1H), 2.68 (d, ³ J _PH = 16.2 Hz, 3H), 2.03 (m, 1H), 0.92 (d, ³ J _HH = 7.2
Hz, 3H), 0.84 (d, ³ J _HH = 6.6 Hz, 3H). ¹³ CNMR (75 MHz, CDCl ₃ ) δ 71.5 (d, ² J _PC =
9.2 Hz), 63.5 (br), 29.1 (d, ² J _PC = 14.1 Hz), 26.7 (d, ³ J _PC = 4.9 Hz), 19.3, 14.5
³¹ P NMR (121 MHz, CDCl ₃ ) δ 176.8.

5'-O- (tert-butyldimethylsilyl) -3'-O-[(2R, 5R) -3-methyl-5-phenyl-1,3,2-oxazaphospholidin-2-yl] thymidine [ Synthesis of (Rp) -5a].

5’-O-(tert-ブチルジメチルシリル)チミジン（721mg, 1.5mmol）をTHF（7.5mL）とトリ
エチルアミン（7.5mmol）に溶解して-78℃に冷却する。0.22MTHF溶液の(5R)-3a（7.5mL,
1.65mmol）を滴下し、室温まで上昇させて30分間攪拌した。飽和重曹水（75mL）とクロロ
ホルム（75mL）を加えて有機層を分離後、有機層を飽和重曹水で洗浄した。水層をクロロ
ホルムで逆抽出して混合後、無水硫酸ナトリウムで乾燥させた。溶媒を減圧留去後、粗生
成物をシリカゲルカラムクロマトグラフを用いて精製した（ヘキサン-酢酸エチル-トリエ
チルアミン=50:50:2）。溶出物を飽和重曹水で洗浄して無水硫酸ナトリウムで乾燥させ、
濃縮して純粋な生成物5b（765mg, 1.2mmol）を得た。³¹P NMR(121 MHz, CDCl₃) δ 144.6
. FAB-HRMS: calcd for C₃₅H₄₃N₃O₆PBi⁺(M + H⁺) 660.2659, found 660.2658.

5’-O-(tert-ブチルジフェニルシリル)-3’-O-[(2R,4S,5R)-3-メチル-4,5-ジフェニル-1,
3,2-オキサザホスホリジン-2-イル]チミジン [5c]の合成.
5’-O-(tert-ブチルジメチルシリル)チミジン（721mg, 1.5mmol）をTHF（7.5mL）とトリ
エチルアミン（7.5mmol）に溶解して-78℃に冷却した。0.22MTHF溶液の3c（7.5mL, 1.65m
mol）を滴下し、室温まで上昇させて30分間攪拌した。飽和重曹水（75mL）とクロロホル
ム（75mL）を加えて有機層を分離後、有機層を飽和重曹水で洗浄した。水層をクロロホル
ムで逆抽出して混合後、無水硫酸ナトリウムで乾燥させた。溶媒を減圧留去後、粗生成物
をシリカゲルカラムクロマトグラフを用いて精製した（ヘキサン-酢酸エチル-トリエチル
アミン=67:33:2から0:100:2）。溶出物を飽和重曹水で洗浄して無水硫酸ナトリウムで乾
燥させ、濃縮して純粋な生成物5c（1.41g, 1.9mmol）を得た。³¹P NMR(121 MHz, CDCl₃)
δ 142.3. FAB-HRMS: calcd for C₄₁H₄₇N₃O₆PBi⁺(M + H⁺) 736.2972, found 736.2975.

(Sp)-1,8-ジアザビシクロ[5.4.0]ウンデク-7-エニウム 5’-O-(tert-ブチルジフェニルシ
リル)チミジン-3’-イル3’-O-(tert-ブチルジメチルシリル)チミジン-5’-イル ホスホ
ロチオエート [(Sp)-11]
の合成.

5′-O- (tert-butyldimethylsilyl) thymidine (721 mg, 1.5 mmol) is dissolved in THF (7.5 mL) and triethylamine (7.5 mmol) and cooled to −78 ° C. 0.22MTHF solution (5R) -3a (7.5mL,
1.65 mmol) was added dropwise, the temperature was raised to room temperature, and the mixture was stirred for 30 minutes. Saturated aqueous sodium hydrogen carbonate (75 mL) and chloroform (75 mL) were added to separate the organic layer, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate. The aqueous layer was back extracted with chloroform, mixed, and dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the crude product was purified using silica gel column chromatography (hexane-ethyl acetate-triethylamine = 50: 50: 2). The eluate was washed with saturated aqueous sodium hydrogen carbonate, dried over anhydrous sodium sulfate,
Concentration gave pure product 5b (765 mg, 1.2 mmol). ³¹ P NMR (121 MHz, CDCl ₃ ) δ 144.6
FAB-HRMS: calcd for C ₃₅ H ₄₃ N ₃ O ₆ PBi ⁺ (M + H ⁺ ) 660.2659, found 660.2658.

5'-O- (tert-butyldiphenylsilyl) -3'-O-[(2R, 4S, 5R) -3-methyl-4,5-diphenyl-1,
Synthesis of 3,2-oxazaphospholidin-2-yl] thymidine [5c].
5′-O- (tert-butyldimethylsilyl) thymidine (721 mg, 1.5 mmol) was dissolved in THF (7.5 mL) and triethylamine (7.5 mmol) and cooled to −78 ° C. 0.22MTHF solution 3c (7.5mL, 1.65m
mol) was added dropwise, and the mixture was raised to room temperature and stirred for 30 minutes. Saturated aqueous sodium hydrogen carbonate (75 mL) and chloroform (75 mL) were added to separate the organic layer, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate. The aqueous layer was back extracted with chloroform, mixed, and dried over anhydrous sodium sulfate. After the solvent was distilled off under reduced pressure, the crude product was purified using silica gel column chromatography (hexane-ethyl acetate-triethylamine = 67: 33: 2 to 0: 100: 2). The eluate was washed with saturated aqueous sodium hydrogen carbonate, dried over anhydrous sodium sulfate, and concentrated to give pure product 5c (1.41 g, 1.9 mmol). ³¹ P NMR (121 MHz, CDCl ₃ )
δ 142.3. FAB-HRMS: calcd for C ₄₁ H ₄₇ N ₃ O ₆ PBi ⁺ (M + H ⁺ ) 736.2972, found 736.2975.

(Sp) -1,8-diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldiphenylsilyl) thymidine-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine -5'-yl phosphorothioate [(Sp) -11]
Synthesis of.

0.22Mアセトニトリル溶液の1f（500μL, 100μmol）に(Rp)-5a（39.6mg, 60μmol）を加
えた。５分後に無水ピリジン（500μmol）と無水酢酸（100μmol）を加え、さらに３０秒
後に3H-1,2-ベンゾジチオール-3-オン 1,1-ジオキシド（12mg,60μmol）を加えた。さら
に３分後、1,8-ジアザビシクロ[5.4.0]ウンデク-7-エン（74.6μL,500μmol）を加えて50
℃で３０分攪拌した。反応溶液を冷却後クロロホルム（3mL）で希釈し、0.2Mのリン酸緩
衝液（pH7.0,3mL）で洗浄し、水層をクロロホルムで逆抽出した。有機層を混合後、無水
硫酸ナトリウムで乾燥させる。残査をPTLCによって精製後、残査をクロロホルム（3mL）
に溶解して0.2M炭酸水素1,8-ジアザビシクロ[5.4.0]ウンデク-7-エニウム溶液で洗浄し、
濃縮して生成物11（50.4mg, 47μmol）を得た。

(Rp)-1,8-ジアザビシクロ[5.4.0]ウンデク-7-エニウム 5’-O-(tert-ブチルジフェニルシ
リル)チミジン-3’-イル3’-O-(tert-ブチルジメチルシリル)チミジン-5’-イル ホスホ
ロチオエート [(Rp)-11]の合成.
出発物質として(Sp)-5aを用い、上記と同様に合成した。60μmolから39.6mg, 60μmol。

1,8-ジアザビシクロ[5.4.0]ウンデク-7-エニウム 5’-O-(tert-ブチルジフェニルシリル)
チミジン-3’-イル3’-O-(tert-ブチルジメチルシリル)チミジン-5’-イル ホスホロチオ
エート (11)の合成
常用のH-ホスホネート法（Kers, A.; Kers, I.;Kraszewski, A.; Sobkowski, M.; Szabo
´, T.; Thelin, M.; Zain,R.;Stawinski,J.Nucleosides Nucleotides 1996,15(1-3),361
-378.）に従い、ジアステレオマーの混合物[(Rp)-11:(Sp)-11=46:54]の混合物として11を
合成した。

(Sp)-アンモニウム チミジン-3’-イル チミジン-5’-イル ホスホロチオエート （（Sp)
-12a）の合成
(Sp)-11（50.4mg, 47μmol）をトリエチルアミントリヒドロフルオライド（500μL）に溶
かし室温で１５時間攪拌した。0.1Mの酢酸アンモニウム緩衝液（3mL）を加えてエーテル
で洗浄し、有機層を濃縮した。残査を逆送クロマトで精製し、（Sp)-12aの精製物（20.5m
g, 35.4μmol）を得た。

(Rp)-アンモニウム チミジン-3’-イル チミジン-5’-イル ホスホロチオエート （(Rp)-
12a）の合成
(Rp)-11(52.9 mg, 49μmol)を出発物質として用いて、(Rp)-12aを得た。49μmolから20.6
mg, 36μmol。

アンモニウムチミジン-3’-イルチミジン-5’-イル ホスホロチオエート (12a)の合成
常用のH-ホスホネート法（Kers, A.; Kers, I.;Kraszewski, A.; Sobkowski, M.; Szabo
´, T.; Thelin, M.; Zain,R.;Stawinski,J.Nucleosides Nucleotides 1996,15(1-3),361
-378.）に従い、ジアステレオマーの混合物[(Rp)-12a:(Sp)-12a=41:59]の混合物として12
aを得た。


マニュアル固相合成

(Rp) -5a (39.6 mg, 60 μmol) was added to 1f (500 μL, 100 μmol) of 0.22 M acetonitrile solution. After 5 minutes, anhydrous pyridine (500 μmol) and acetic anhydride (100 μmol) were added, and further 30 seconds later, 3H-1,2-benzodithiol-3-one 1,1-dioxide (12 mg, 60 μmol) was added. After an additional 3 minutes, 1,8-diazabicyclo [5.4.0] undec-7-ene (74.6 μL, 500 μmol) was added and 50
Stir at 30 ° C. for 30 minutes. The reaction solution was cooled, diluted with chloroform (3 mL), washed with 0.2 M phosphate buffer (pH 7.0, 3 mL), and the aqueous layer was back-extracted with chloroform. The organic layer is mixed and then dried over anhydrous sodium sulfate. After the residue was purified by PTLC, the residue was chloroform (3 mL)
And then washed with 0.2M 1,8-diazabicyclo [5.4.0] undec-7-enium bicarbonate solution,
Concentration gave the product 11 (50.4 mg, 47 μmol).

(Rp) -1,8-diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldiphenylsilyl) thymidine-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidine Synthesis of -5'-yl phosphorothioate [(Rp) -11].
Synthesis was performed in the same manner as described above, using (Sp) -5a as a starting material. 60μmol to 39.6mg, 60μmol.

1,8-diazabicyclo [5.4.0] undec-7-enium 5'-O- (tert-butyldiphenylsilyl)
Synthesis of thymidine-3'-yl 3'-O- (tert-butyldimethylsilyl) thymidin-5'-yl phosphorothioate (11) Conventional H-phosphonate method (Kers, A .; Kers, I .; Kraszewski, A .; Sobkowski, M .; Szabo
´, T .; Thelin, M .; Zain, R .; Stawinski, J. Nucleosides Nucleotides 1996, 15 (1-3), 361
-378.) 11 was synthesized as a mixture of diastereomeric mixtures [(Rp) -11: (Sp) -11 = 46: 54].

(Sp) -Ammonium thymidine-3'-yl thymidine-5'-yl phosphorothioate ((Sp)
-12a)
(Sp) -11 (50.4 mg, 47 μmol) was dissolved in triethylamine trihydrofluoride (500 μL) and stirred at room temperature for 15 hours. 0.1M ammonium acetate buffer (3 mL) was added and washed with ether, and the organic layer was concentrated. The residue was purified by reverse chromatography and purified (Sp) -12a (20.5m
g, 35.4 μmol).

(Rp) -Ammonium thymidine-3'-yl thymidine-5'-yl phosphorothioate ((Rp)-
Synthesis of 12a)
(Rp) -11 (52.9 mg, 49 μmol) was used as starting material to give (Rp) -12a. 49 μmol to 20.6
mg, 36 μmol.

Synthesis of ammonium thymidine-3'-ylthymidin-5'-yl phosphorothioate (12a) Conventional H-phosphonate method (Kers, A .; Kers, I .; Kraszewski, A .; Sobkowski, M .; Szabo
´, T .; Thelin, M .; Zain, R .; Stawinski, J. Nucleosides Nucleotides 1996, 15 (1-3), 361
-378.) As a mixture of diastereomers [(Rp) -12a: (Sp) -12a = 41: 59]
got a.


Manual solid phase synthesis

上記反応式の13a-dにおいて、B¹は、それぞれ、Th(チミン)、Ad（アデニン）、Cy（シト
シン）及びGu（グアニン）残基である。

3%TCAによる脱トリチル、ジクロロメタンおよびアセトニトリルによる洗浄、カップリン
グ（0.2M モノマー13a-d及び1.0M 1fのアセトニトリル溶液、９秒）、アセトニトリルに
よる洗浄、キャッピング[Ac₂O-N-メチルイミダゾール-THF(1:2:7, v/v/v)、３０秒]、ア
セトニトリルによる洗浄、硫化（ボカージ試薬）、アセトニトリルによる洗浄を繰り返し
た。各ステップの平均収率は97-98%であった。固相坦体をアンモニア水-ピリジン（9:1）
で５５℃、３０分処理してキラル補助基と核酸塩基の保護基を除去し、生成物を逆相クロ
マトによって分析、精製した。

In 13a-d of the above reaction formula, B ¹ is Th (thymine), Ad (adenine), Cy (cytosine), and Gu (guanine) residues, respectively.

Detrityl with 3% TCA, washing with dichloromethane and acetonitrile, coupling (0.2M monomer 13a-d and 1.0M 1f in acetonitrile, 9 seconds), washing with acetonitrile, capping [Ac ₂ ON-methylimidazole-THF (1 : 2: 7, v / v / v), 30 seconds], washing with acetonitrile, sulfurization (Bocardi reagent), and washing with acetonitrile were repeated. The average yield for each step was 97-98%. Solid phase carrier is ammonia water-pyridine (9: 1)
At 55 ° C. for 30 minutes to remove the chiral auxiliary and nucleobase protecting group, and the product was analyzed and purified by reverse phase chromatography.

Hereinafter, “T” represents thymidine phosphate and “T _PS ” represents thymidine phosphorothioate.

Synthesis of All- (Rp)-[T _PS ] ₃ T (15).
According to the above method, 15 crude products were obtained from 5′-O- (DMTr) thymidine 3′-O-succinate bound to HCP. The average yield for each step was 98%. The product was confirmed by MALDI-TOF-MS (m / z 1201 (M-H +)-). The product was purified by reverse phase chromatography. Yield 80%.

Synthesis of All- (Sp)-[T _PS ] ₃ T (16).
According to the above method, 16 crude products were obtained from 5'-O- (DMTr) thymidine 3'-O-succinate conjugated to HCP. The average yield for each step was 98%. Yield 78%.

all- (Rp) -d [C _PS A _PS G _PS ] T
Synthesis of (17).
According to the method described above, 17 crude products were obtained from 5′-O- (DMTr) thymidine 3′-O-succinate bound to HCP. The average yield for each step was 98%. The product was confirmed by MALDI-TOF-MS (m / z 1220 (M-H +)-). Yield 75%.

Synthesis of All- (Rp)-[T _PS ] ₉ T (18).
According to the above method, 18 crude products were obtained from 5′-O- (DMTr) thymidine 3′-O-succinate conjugated to HCP. The average yield for each step was 98%. The product was confirmed by MALDI-TOF-MS (m / z 3121 (M-H +)-). Yield 75%.

[Example 7] Synthesis for controlling the configuration of oligonucleoside boranophosphate
In the process of synthesizing (Sp) -11, instead of 3H-1,2-benzodithiol-3-one 1,1-dioxide, BH ₃ · SMe ₂ is used instead of sulfidation by Bocage reagent in manual solid phase synthesis The oligonucleoside boranophosphate is synthesized according to the procedure of Example 6 except that boronation with BH ₃ · SMe ₂ is performed.

[Example 8] Production of 2'-deoxythymidine 5 '-[α- (Rp) -borano] triphosphate

3’-O-アセチル-5’-O-[(2R,5R)-3-メチル-5-フェニル-1,3,2-オキサザホスホリジン-2-
イル]チミジン[(Rp)-2]の合成
3’-O-アセチルチミジン（1.5mmol）をTHF（7.5mL）とトリエチルアミン（7.5mmol）に溶
解して-78℃に冷却する。0.22M溶液の(5R)-1（7.5mL, 1.65mmol）を滴下し、室温まで上
昇させて30分間攪拌する。飽和重曹水（75mL）とクロロホルム（75mL）を加えて有機層を
分離後、有機層を飽和重曹水で洗浄する。水層をクロロホルムで逆抽出して混合後、無水
硫酸ナトリウムで乾燥させる。溶媒を減圧留去後、粗生成物をシリカゲルカラムクロマト
グラフを用いて精製した（ヘキサン-酢酸エチル-トリエチルアミン=50:50:2）。溶出物を
飽和重曹水で洗浄して無水硫酸ナトリウムで乾燥させ、濃縮して純粋な生成物(Rp)-2（1.
2 mmol）を得る。

2’-デオキシチミジン 5’-[α-(Rp)-ボラノ]三リン酸の合成
0.2Mアセトニトリル溶液のN-シアノメチルピロリジニウムトリフラート（500μL, 100μm
ol）と2,6-ジニトロフェノール（50μmol）に(Rp)-2（39.6mg, 60μmol）を加える。５分
後にボラン-ジメチルスルフィド錯体（60μmol）を加え、８分後に無水ピリジン（500μm
ol）と無水酢酸（100μmol）を加える。さらに３分後にテトラ（トリ-n-ブチルアンモニ
ウム）ピロリン酸（60μmol）を加えて１時間攪拌する。反応溶液をクロロホルム（3mL）
で希釈し、0.2Mのリン酸緩衝液（pH7.0,
3mL）で洗浄し、水層をクロロホルムで逆抽出する。有機層を混合後、無水硫酸ナトリウ
ムで乾燥させる。残査を25%アンモニア水-ピリジン（9:1）中55℃で30分反応させてキラ
ル補助基と保護基を除去し、冷却後に濃縮する。残査を水に溶解してエーテルで洗浄し、
水を減圧留去後、粗生成物をイオン交換クロマトグラフHPLCを用いて精製して純粋な生成
物(Rp)-7（35.4 μmol）を得る。

3'-O-acetyl-5'-O-[(2R, 5R) -3-methyl-5-phenyl-1,3,2-oxazaphosphoridine-2-
Synthesis of [Il] thymidine [(Rp) -2]
3′-O-acetylthymidine (1.5 mmol) is dissolved in THF (7.5 mL) and triethylamine (7.5 mmol) and cooled to −78 ° C. Add a 0.22M solution of (5R) -1 (7.5 mL, 1.65 mmol) dropwise, warm to room temperature and stir for 30 minutes. Saturated aqueous sodium bicarbonate (75 mL) and chloroform (75 mL) are added to separate the organic layer, and the organic layer is washed with saturated aqueous sodium bicarbonate. The aqueous layer is back-extracted with chloroform, mixed and dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the crude product was purified using silica gel column chromatography (hexane-ethyl acetate-triethylamine = 50: 50: 2). The eluate was washed with saturated aqueous sodium bicarbonate, dried over anhydrous sodium sulfate, and concentrated to give pure product (Rp) -2 (1.
2 mmol) is obtained.

Synthesis of 2'-deoxythymidine 5 '-[α- (Rp) -borano] triphosphate
0.2M N-cyanomethylpyrrolidinium triflate in acetonitrile solution (500μL, 100μm
ol) and 2,6-dinitrophenol (50 μmol) add (Rp) -2 (39.6 mg, 60 μmol). After 5 minutes, borane-dimethyl sulfide complex (60 μmol) was added, and after 8 minutes anhydrous pyridine (500 μm)
ol) and acetic anhydride (100 μmol). Further, after 3 minutes, tetra (tri-n-butylammonium) pyrophosphate (60 μmol) is added and stirred for 1 hour. The reaction solution was chloroform (3 mL)
Diluted with 0.2 M phosphate buffer (pH 7.0,
3 mL) and back-extract the aqueous layer with chloroform. The organic layer is mixed and then dried over anhydrous sodium sulfate. The residue is reacted in 25% aqueous ammonia-pyridine (9: 1) at 55 ° C. for 30 minutes to remove the chiral auxiliary and protecting group, and concentrated after cooling. Dissolve the residue in water and wash with ether,
After distilling off water under reduced pressure, the crude product is purified using ion exchange chromatography HPLC to obtain a pure product (Rp) -7 (35.4 μmol).

Further, by improving the above-described method, that is, by using a commercially available 2′-protected ribonucleoside, it is possible to synthesize optically active borano-modified RNA.

The structure of borano-modified ribonucleoside triphosphate is shown below. In the following structure, Base
Are adenine, cytosine, guanine, uracil and the like.

2 is an electrophoresis photograph of dsDNA prepared in Example 1. 1 and 10 are natural DNA lanes, 2 are natural dATP + dCTP + dGTP and dTTP-αB (with NH ₄ HCO ₃ added) lanes, 3 are natural dATP + dCTP + dGTP and dTTP-αB lanes , M is a DNA marker, 4 is a lane of natural dCTP + dGTP and dATP-aB + dTTP-αB, 5 is a lane of natural dTTP and dATP-aB + dGTP-aB + dCTP-αB, 6 is a natural dCTP and dATP -aB + dGTP-aB + dTTP-αB lane, 7 is natural dGTP and dATP-aB + dCTP-aB + dTTP-αB lane, 8 is natural dATP and dGTP-aB + dCTP-aB + dTTP-αB lane, 9 is a lane of dATP-aB + dGTP-aB + dCTP-aB + dTTP-αB.

Similarly, a boron-containing PCR product was obtained with a plasmid having a U6 promoter. 1A, 2G, 3C, 4T, 5AG, 6AC, 7AT, 8GC, 9GT, 10CT, 11AGC, 12AGT, 13ACT, 14GCT, 15AGCT, 16natural

As a result of some conditions, a PCR product in which boron was introduced in all combinations of bases was obtained. 1AT, 2AGT, 3ACT, 4GCT, 5AGCT,

# 1 shows the result of SYBRGold staining, and # 2 shows the result of phosphor contrast medium (RI). A is a lane for RNA markers (500 nt and 200 nt), B is a lane for control RNA (1800 nt), C is a lane for natural dNTP, and D is a lane for natural dATP + dCTP + dGTP and dTTP-αB.

It is a mass spectrometry spectrum of dTTP-αB.

As a result of the RNAi experiment, boron-containing DNA has an excellent activity comparable to that of a natural product in terms of intracellular effects. After culturing HeLaS3 cells, a PCR product (100 ng) is allowed to act in the presence of OPTI-MEM (50 mL), Lipofectamine 2000 (1 mg), pRL-RSV (20 ng), and pGL3 (50 ng). After 24 hours of action at 37 ° C., the expression level was measured using Dual Luciferase Reporter Assay System (Promega). 1: H1 promoter control2: H1 promoterPCR-natural3: H1 promoter PCR-CTaB4: H1 promoter PCR-fullaB6: pU6-90 promotercontrol7: pU6-90 promoterPCR-natural8: pU6-90 promoterPCR-CTaB9: pU6-90 promoterPCR-fullaB

  SEQ ID NO: 1 shows the base sequence of primer 1 used in Example 2.

  SEQ ID NO: 2 shows the base sequence of primer 2 used in Example 2.

  SEQ ID NO: 3 shows the base sequence of the template used in Example 2.

  SEQ ID NO: 4 shows the base sequence of the primer used for PCR.

  SEQ ID NO: 5 shows the nucleotide sequence of a primer used for PCR.

  SEQ ID NO: 6 shows the base sequence of the primer used for PCR.

  SEQ ID NO: 7 shows the base sequence of the primer used for PCR.

  SEQ ID NO: 8 shows the base sequence of functional RNA.

SEQ ID NO: 9 shows the base sequence of functional RNA.

Claims (26)

A modified DNA or RNA in which a hydroxyl group and / or oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group, and a template for transcription and / or amplification of RNA A method for transcription and / or amplification of RNA, wherein the modified DNA or RNA that can function as a gene or function as a gene expression suppressor is used as a template. The method according to claim 1, wherein the modified DNA or RNA is a DNA or RNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with a borano group. The method according to claim 1, wherein the modified DNA or RNA is a completely chemically synthesized DNA or RNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with an optically active borano group. A modified DNA in which a hydroxyl group and / or oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group, and functions as a template for RNA transcription and / or amplification RNA transcription and / or consisting of said modified DNA
Or a template for amplification. The template according to claim 4, wherein the modified DNA is a DNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with a borano group. A template preparation kit for RNA transcription and / or amplification comprising a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus of a phosphate moiety directly bonded to a sugar is substituted with another atom or group . The kit according to claim 6, wherein the modified deoxyribonucleoside triphosphate is 2'-deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus in the α-phosphate moiety is substituted with a borano group. A kit for RNA transcription and / or amplification comprising a modified deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus of a phosphate moiety directly bonded to a sugar is substituted with another atom or group. The kit according to claim 8, wherein the modified deoxyribonucleoside triphosphate is 2'-deoxyribonucleoside triphosphate in which a hydroxyl group bonded to phosphorus in the α-phosphate moiety is substituted with a borano group. A modified DNA in which a hydroxyl group and / or an oxygen atom bonded to phosphorus of a phosphate moiety directly bonded to a sugar is replaced with another atom and / or group, and the transcription and / or amplification of RNA RNA transcription and / or comprising said modified DNA that can function as a template
Or amplification kit. The kit according to claim 10, wherein the modified DNA is a DNA in which a hydroxyl group bonded to phosphorus in at least one phosphate moiety is substituted with a borano group. A modified DNA or RNA in which a hydroxyl group and / or oxygen atom bonded to phosphorus in at least one phosphate moiety is substituted with another atom and / or group, and a template for transcription and / or amplification of RNA The modified DNA or RNA, which can function as a gene or can suppress the expression of a gene. The modified DNA according to claim 12, which can be introduced into a cell or tissue to allow the functional nucleic acid to be expressed in the cell or tissue. The modified DNA according to claim 13, wherein the functional nucleic acid to be expressed is a double-stranded RNA containing siRNA or hairpin RNA. The modified DNA according to claim 13, wherein the functional nucleic acid to be expressed is a ribozyme. The modified DNA according to claim 13, wherein the functional nucleic acid to be expressed is an antisense RNA. The functional nucleic acid target to be expressed is a gene related to a virus or cancer.
6. The modified DNA according to any one of 6. The modified DNA according to claim 17, wherein the virus is selected from the group consisting of HIV, HCV and HBV. The modified DNA or RNA according to claim 12, which can be introduced into a cell or tissue to suppress gene expression. The modified RNA according to claim 19, wherein the modified RNA is an antisense RNA. The modified RNA according to claim 19, wherein the modified RNA is a double-stranded RNA containing siRNA and hairpin RNA. The modified DNA according to claim 19, wherein the modified DNA is a DNAzyme. A composition comprising the modified DNA or RNA according to any one of claims 12 to 22. A pharmaceutical composition comprising the modified DNA or RNA according to any one of claims 12 to 22. A method for producing the modified RNA according to any one of claims 19 to 21 by in vitro transcription. A method for synthesizing the modified DNA or RNA according to any one of claims 12 to 22 from a nucleotide containing an optically active borano group.

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