JP2015020994A - 2'-o-carbamoyl modified nucleoside triphosphate - Google Patents
2'-o-carbamoyl modified nucleoside triphosphate Download PDFInfo
- Publication number
- JP2015020994A JP2015020994A JP2013152578A JP2013152578A JP2015020994A JP 2015020994 A JP2015020994 A JP 2015020994A JP 2013152578 A JP2013152578 A JP 2013152578A JP 2013152578 A JP2013152578 A JP 2013152578A JP 2015020994 A JP2015020994 A JP 2015020994A
- Authority
- JP
- Japan
- Prior art keywords
- rna
- nucleoside triphosphate
- population
- group
- general formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
本発明は、新規なヌクレオシド三リン酸誘導体及びその製造方法に関する。また、本発明は、この誘導体を利用したアプタマー、アプタマーの製造方法、RNA誘導体の製造方法に関する。 The present invention relates to a novel nucleoside triphosphate derivative and a method for producing the same. The present invention also relates to an aptamer utilizing this derivative, a method for producing an aptamer, and a method for producing an RNA derivative.
アプタマーは、標的物質と特異的に結合する核酸分子であり、近年、抗体に代わる生体物質として注目され、医療への応用に向け、研究開発が進められている。アプタマーは、SELEX(Systematic Evolution of Ligands by EXponential enrichment)法と呼ばれる方法により作製され、例えば、RNAアプタマーは、RNAの集団（RNAライブラリー）の逆転写、cDNAの集団（cDNAライブラリー）の増幅、増幅したDNA集団からRNA集団の合成、標的物質と結合するRNAの選択（選択されたRNAに対して再度前述した逆転写が行われる）、といった操作を複数回繰り返すことにより得られる。 Aptamers are nucleic acid molecules that specifically bind to a target substance. In recent years, aptamers have attracted attention as biological substances that can replace antibodies, and research and development have been promoted for medical applications. Aptamers are produced by a method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. For example, RNA aptamers are reverse transcription of RNA population (RNA library), amplification of cDNA population (cDNA library), It is obtained by repeating a plurality of operations such as synthesis of an RNA population from the amplified DNA population and selection of RNA that binds to the target substance (reverse transcription described above is performed again on the selected RNA).
未修飾の核酸分子は生体内で分解され易いため、アプタマーには分解酵素に耐性を持つ修飾核酸分子が用いられる。このような修飾核酸分子をSELEX法に適用するためには、その核酸分子を鋳型として逆転写酵素によりcDNAが合成されること、及び修飾基を持つヌクレオシド三リン酸がポリメラーゼの基質となり得る範囲の大きさであること（修飾基が大きすぎると取込まれない。）が必要である。 Since unmodified nucleic acid molecules are easily degraded in vivo, modified nucleic acid molecules resistant to degrading enzymes are used as aptamers. In order to apply such a modified nucleic acid molecule to the SELEX method, cDNA is synthesized by reverse transcriptase using the nucleic acid molecule as a template, and nucleoside triphosphate having a modifying group can be used as a substrate for polymerase. It must be large (it cannot be incorporated if the modifying group is too large).
上記の制約から、これまでにアプタマーに使われている2'-修飾核酸分子としては、2'-O-メチル基で修飾された核酸分子（非特許文献１）及び2'-フルオロ基で修飾された核酸分子（非特許文献２）など比較的小さな修飾基が知られている。これらの修飾核酸分子は、上記の必須条件に対しては問題ないが、修飾基自身の物理化学的性質から、標的物質との結合力がやや不十分であるという問題があった。 Due to the above restrictions, 2'-modified nucleic acid molecules used for aptamers so far are modified with 2'-O-methyl group (Non-patent Document 1) and 2'-fluoro group. Relatively small modifying groups such as a prepared nucleic acid molecule (Non-patent Document 2) are known. These modified nucleic acid molecules have no problem with the above-mentioned essential conditions, but have a problem that the binding force with the target substance is somewhat insufficient due to the physicochemical properties of the modifying group itself.
最近、本発明者らは、2'位をカルバモイル基で修飾したウリジン（2'-O-carbamoyluridine、以下「Ucm」という。）を合成し、この修飾ヌクレオシドを導入した二重鎖核酸は、二重鎖の安定性が低下すること、及びUcmの2'-O-カルバモイル基によってUcmとグアノシンとのミスマッチ塩基対が安定化することを報告している（非特許文献３）。非特許文献３には、Ucmのほか、Ucmを含む核酸分子、及びそれを合成するためのホスホロアミダイトユニットが記載されているが、2'位がカルバモイル基で修飾されたウリジン三リン酸は記載されておらず、また、Ucmを含む核酸分子をアプタマーとして用いることも記載されていない。 Recently, the present inventors synthesized a uridine modified with a carbamoyl group at the 2 ′ position (2′-O-carbamoyluridine, hereinafter referred to as “U cm ”), and a double-stranded nucleic acid into which this modified nucleoside has been introduced, the duplex stability decreases, and mismatched base pairs between the U cm and guanosine by 2'-O- carbamoyl group U cm is reported to stabilize (non-Patent Document 3). Non-patent document 3 describes a nucleic acid molecule containing U cm in addition to U cm , and a phosphoramidite unit for synthesizing it, but uridine trilin is modified at the 2 ′ position with a carbamoyl group. acid is not described, also not described the use of a nucleic acid molecule comprising a U cm as aptamers.
上記のようにアプタマーに使われている修飾核酸分子の種類は非常に少ない。本発明は、このような技術的背景の下、アプタマーに利用可能な新規な修飾核酸分子を提供することを目的とする。 As described above, there are very few types of modified nucleic acid molecules used for aptamers. An object of the present invention is to provide a novel modified nucleic acid molecule that can be used for aptamers under such technical background.
本発明者は、上記課題を解決するため鋭意検討を重ねた結果、2'-O-カルバモイル基がUcmとグアノシンとのミスマッチ塩基対を安定化することから、2'-O-カルバモイル基で修飾された核酸分子が多様な高次構造を取り、アプタマーとして有用であるという発想を得た。しかし、ミスマッチ塩基対の安定化によって、Ucmを含むRNAを鋳型とした逆転写において、多くのミスマッチを含むDNAが生成すると予想された。SELEX法によりRNAアプタマーを作製する際、逆転写を行うことが必要であるが、このようなミスマッチを含むDNAの生成は効率的なアプタマーの作製の大きな障害になる。そこで、実際に、本発明者は、Ucmを含むRNAを鋳型とした逆転写を行ってみたところ、前記予想に反し、ミスマッチは起きず、Ucmはアデノシンと正確に塩基対を形成することを見出した。 As a result of intensive studies to solve the above problems, the present inventor has stabilized the mismatch base pair between U cm and guanosine, so that the 2′-O-carbamoyl group The idea was that the modified nucleic acid molecule has a variety of higher-order structures and is useful as an aptamer. However, the stabilization of mismatched base pairs, in the reverse transcription and the RNA containing U cm as a template, it was expected that a DNA containing a number of mismatches to produce. When RNA aptamers are prepared by the SELEX method, it is necessary to perform reverse transcription, but the generation of DNA containing such mismatches is a major obstacle to the efficient production of aptamers. Therefore, in practice, the present inventor tried reverse transcription using RNA containing U cm as a template, and contrary to the above-mentioned prediction, no mismatch occurred, and U cm accurately forms a base pair with adenosine. I found.
また、2'-O-カルバモイル基で修飾されたアプタマーをSELEX法で作製する際、RNA合成の基質として2'-O-カルバモイルヌクレオシド三リン酸が必要であるが、この物質を従来の方法で合成するのは困難であった。通常、ヌクレオシド三リン酸を合成する際、3'位のヒドロキシ基をアセチル基で保護し、アンモニア水で脱保護するが、アンモニア水が2'-O-カルバモイル基を分解してしまうからである。本発明者は、2'-O-カルバモイルヌクレオシド三リン酸の合成法について検討した結果、3'位のヒドロキシ基をtert-ブチルジメチルシリル（TBS）基などで保護し、酢酸のような弱酸で脱保護することにより、2'-O-カルバモイルヌクレオシド三リン酸を合成できることを見出した。TBS基の脱保護は、通常、フッ化テトラブチルアンモニウム（TBAF）などで行われており、酢酸で脱保護できるということは全く予想外のことであった。 In addition, when preparing aptamers modified with 2'-O-carbamoyl groups by the SELEX method, 2'-O-carbamoyl nucleoside triphosphate is required as a substrate for RNA synthesis. It was difficult to synthesize. Usually, when synthesizing a nucleoside triphosphate, the hydroxy group at the 3 ′ position is protected with an acetyl group and deprotected with aqueous ammonia, because the aqueous ammonia decomposes the 2′-O-carbamoyl group. . As a result of examining the synthesis method of 2′-O-carbamoyl nucleoside triphosphate, the present inventor protected the hydroxy group at the 3 ′ position with a tert-butyldimethylsilyl (TBS) group, etc. It was found that 2′-O-carbamoyl nucleoside triphosphate can be synthesized by deprotection. The deprotection of the TBS group is usually performed with tetrabutylammonium fluoride (TBAF), and it was completely unexpected that it could be deprotected with acetic acid.
本発明は、以上の知見に基づき完成されたものである。 The present invention has been completed based on the above findings.
即ち、本発明は、以下の〔１〕〜〔１１〕を提供するものである。
〔１〕一般式（Ｉ）：
That is, the present invention provides the following [1] to [11].
[1] General formula (I):
〔式中、Ｒ１及びＲ２は水素原子又は炭素数１〜５のアルキル基を表し、Ｘは酸素原子又は硫黄原子を表し、Ｂは核酸塩基の残基を表す。〕
で表されるヌクレオシド三リン酸誘導体。
〔２〕一般式（Ｉ）においてＲ１及びＲ２が水素原子である〔１〕に記載のヌクレオシド三リン酸誘導体。
〔３〕一般式（Ｉ）においてＲ１及びＲ２の一方がメチル基であり、他方が水素原子である〔１〕に記載のヌクレオシド三リン酸誘導体。
〔４〕一般式（Ｉ）においてＢがチミン-1-イル、シトシン-1-イル、5-メチルシトシン-1-イル、ウラシル-1-イル、ウラシル-5-イル、アデニン-9-イル、又はグアニン-9-イルである〔１〕乃至〔３〕のいずれかに記載のヌクレオシド三リン酸誘導体。
〔５〕一般式（II）：
[Wherein, R 1 and R 2 represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, X represents an oxygen atom or a sulfur atom, and B represents a nucleobase residue. ]
A nucleoside triphosphate derivative represented by:
[2] The nucleoside triphosphate derivative according to [1], wherein R 1 and R 2 in the general formula (I) are hydrogen atoms.
[3] The nucleoside triphosphate derivative according to [1], wherein one of R 1 and R 2 in the general formula (I) is a methyl group and the other is a hydrogen atom.
[4] In the general formula (I), B is thymin-1-yl, cytosine-1-yl, 5-methylcytosin-1-yl, uracil-1-yl, uracil-5-yl, adenine-9-yl, Alternatively, the nucleoside triphosphate derivative according to any one of [1] to [3], which is guanine-9-yl.
[5] General formula (II):
〔式中、Ｒ１及びＲ２は水素原子又は炭素数１〜５のアルキル基を表し、Ｒ３はシリル系保護基を表し、Ｘは酸素原子又は硫黄原子を表し、Ｂは核酸塩基の残基を表す。〕
で表されるヌクレオシド三リン酸誘導体から弱酸性条件下でＲ３を脱離させる工程を含む〔１〕乃至〔４〕のいずれかに記載のヌクレオシド三リン酸誘導体の製造方法。
〔６〕弱酸性がpH 2〜6である〔５〕に記載のヌクレオシド三リン酸誘導体の製造方法。
〔７〕Ｒ３の脱離を酢酸の存在下で行う〔５〕又は〔６〕に記載のヌクレオシド三リン酸誘導体の製造方法。
〔８〕シリル系保護基がtert-ブチルジメチルシリル基である〔５〕乃至〔７〕のいずれかに記載のヌクレオシド三リン酸誘導体の製造方法。
〔９〕一般式（Ｖ）：
[Wherein, R 1 and R 2 represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R 3 represents a silyl protecting group, X represents an oxygen atom or a sulfur atom, and B represents the residue of a nucleobase. Represents a group. ]
Method for producing a nucleoside triphosphate derivative according to any one of comprising the step of the R 3 desorbed in the nucleoside triphosphate derivative represented by the mildly acidic conditions [1] to [4].
[6] The method for producing a nucleoside triphosphate derivative according to [5], wherein the weak acidity is pH 2 to 6.
[7] The method for producing a nucleoside triphosphate derivative according to [5] or [6], wherein R 3 is eliminated in the presence of acetic acid.
[8] The method for producing a nucleoside triphosphate derivative according to any one of [5] to [7], wherein the silyl protecting group is a tert-butyldimethylsilyl group.
[9] General formula (V):
〔式中、Ｂ１、Ｂ２及びＢ３は同一又は異なって核酸塩基の残基を表し、Ｂ２はｎの繰り返しにおいて異なっていてもよく、Ｙ１、Ｙ２及びＹ３は同一又は異なってヒドロキシ基、メトキシ基、フッ素原子、又は一般式（VI） [Wherein B 1 , B 2 and B 3 are the same or different and represent a nucleobase residue, B 2 may be different in the repetition of n, and Y 1 , Y 2 and Y 3 are the same or different. Hydroxy group, methoxy group, fluorine atom, or general formula (VI)
（式中、Ｒ１及びＲ２は水素原子又は炭素数１〜５のアルキル基を表し、Ｘは酸素原子又は硫黄原子を表す。）
で示される基を表し、Ｙ２はｎの繰り返しにおいて異なっていてもよく、ｎは１以上の整数を表す。但し、Ｙ１、Ｙ２、及びＹ３の少なくとも一つは一般式（VI）で示される基を表す。〕
で表されるRNAアプタマー。
〔１０〕以下の工程を有することを特徴とするRNA誘導体の製造方法、
（１）逆転写酵素をRNAの集団又はRNA誘導体の集団（このRNA誘導体の集団は、工程（４）で選択されたRNA誘導体の集団であってもよい。）に作用させ、DNAの集団を合成する工程、
（２）工程（１）で合成されたDNAの集団を、ポリメラーゼ連鎖反応により増幅させる工程、
（３）〔１〕乃至〔４〕のいずれかに記載のヌクレオシド三リン酸誘導体の存在下で、工程（２）で増幅されたDNAの集団にDNA依存RNAポリメラーゼを作用させ、前記ヌクレオシド三リン酸誘導体を残基として含むRNA誘導体の集団を合成する工程、
（４）工程（３）で合成したRNA誘導体の集団を標的物質と接触させ、標的物質に親和性を示すRNA誘導体の集団を選択する工程。
〔１１〕〔１〕乃至〔４〕のいずれかに記載のヌクレオシド三リン酸誘導体の存在下で、鋳型とするDNAにDNA依存RNAポリメラーゼを作用させ、前記ヌクレオシド三リン酸誘導体を残基として含むRNA誘導体を製造するRNA誘導体の製造方法。
(In the formula, R 1 and R 2 represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and X represents an oxygen atom or a sulfur atom.)
Y 2 may be different in the repetition of n, and n represents an integer of 1 or more. However, at least one of Y 1 , Y 2 , and Y 3 represents a group represented by the general formula (VI). ]
RNA aptamer represented by
[10] A method for producing an RNA derivative, comprising the following steps:
(1) A reverse transcriptase is allowed to act on a population of RNA or a population of RNA derivatives (this population of RNA derivatives may be a population of RNA derivatives selected in step (4)). The step of synthesizing,
(2) Amplifying the population of DNA synthesized in step (1) by polymerase chain reaction;
(3) In the presence of the nucleoside triphosphate derivative according to any one of [1] to [4], a DNA-dependent RNA polymerase is allowed to act on the population of DNA amplified in step (2), thereby Synthesizing a population of RNA derivatives containing acid derivatives as residues,
(4) A step of contacting the population of RNA derivatives synthesized in step (3) with a target substance and selecting a population of RNA derivatives showing affinity for the target substance.
[11] DNA-dependent RNA polymerase is allowed to act on the template DNA in the presence of the nucleoside triphosphate derivative according to any one of [1] to [4], and the nucleoside triphosphate derivative is contained as a residue. An RNA derivative production method for producing an RNA derivative.
本発明のヌクレオシド三リン酸誘導体により、標的物質との結合性の優れるアプタマーが製造できるようになる。 The nucleoside triphosphate derivative of the present invention makes it possible to produce an aptamer with excellent binding properties to a target substance.
また、本発明のヌクレオシド三リン酸誘導体の製造方法は、弱酸性条件下で脱保護を行うので、副生成物の生成を抑制し、この誘導体の生成を容易にする。 In addition, since the method for producing a nucleoside triphosphate derivative of the present invention performs deprotection under weakly acidic conditions, the production of this by-product is suppressed by suppressing the production of by-products.
以下、本発明を詳細に説明する。
〔ヌクレオシド三リン酸誘導体〕
本発明のヌクレオシド三リン酸誘導体は、一般式（Ｉ）で表される。
Hereinafter, the present invention will be described in detail.
[Nucleoside triphosphate derivatives]
The nucleoside triphosphate derivative of the present invention is represented by the general formula (I).
一般式（Ｉ）におけるＲ１及びＲ２は水素原子又は炭素数１〜５のアルキル基を表す。ここで、「炭素数１〜５のアルキル基」とは、例えば、メチル基、エチル基、n-プロピル基n-ブチル基、n-ペンチル基などの直鎖アルキル基やイソプロピル、イソブチル、sec-ブチル、tert-ブチル、イソペンチル、tert-ペンチルなどの分岐鎖アルキルキを意味する。Ｒ１及びＲ２は同一の基であってもよく、異なる基であってもよい。Ｒ１及びＲ２は少なくとも一方が水素原子であることが好ましく、両方が水素原子であること、又は一方が水素原子で他方がメチル基であることが更に好ましい。 R 1 and R 2 in the general formula (I) represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Here, the “alkyl group having 1 to 5 carbon atoms” means, for example, a linear alkyl group such as methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, isopropyl, isobutyl, sec- It means a branched alkyl group such as butyl, tert-butyl, isopentyl, tert-pentyl. R 1 and R 2 may be the same group or different groups. At least one of R 1 and R 2 is preferably a hydrogen atom, more preferably both are hydrogen atoms, or one is a hydrogen atom and the other is a methyl group.
一般式（Ｉ）におけるＸは酸素原子又は硫黄原子を表す。Ｘは酸素原子であることが好ましい。 X in the general formula (I) represents an oxygen atom or a sulfur atom. X is preferably an oxygen atom.
一般式（Ｉ）におけるＢは核酸塩基の残基を表す。核酸塩基は、天然型の核酸塩基、非天然型の核酸塩基のいずれであってもよい。ここで、「非天然型の核酸塩基」とは、天然型の核酸塩基の環外酸素原子や環外窒素原子や環内窒素原子や環内炭素原子に置換基を有するなどの化学修飾、もしくは環外酸素原子や環内窒素原子や環内炭素原子が他の原子に置換されるなどの化学修飾、又はこれらの化学修飾をともに有する核酸塩基の誘導体を意味する。天然型の核酸塩基の残基としては、例えば、チミン-1-イル、シトシン-1-イル、5-メチルシトシン-1-イル、ウラシル-1-イル、ウラシル-5-イル、アデニン-9-イル、グアニン-9-イルなどを挙げることができる。非天然型の核酸塩基の残基としては、2-チオチミン-1-イル、2-チオウラシル-1-イル、N2-アシル-3-デアザグアニン-9-イル、N2-カルバモイルグアニン-9-イル、N4-アシルシトシン-1-イル、N6-アシル-7-デアザアデニン-9-イル、N6-アシル-7-デアザ-8‐アザアデニン-9-イルなどを挙げることができる。これらの核酸塩基の残基の中で好ましい核酸塩基の残基としては、チミン-1-イル、シトシン-1-イル、5-メチルシトシン-1-イル、ウラシル-1-イル、ウラシル-5-イル、アデニン-9-イル、グアニン-9-イルを挙げることができる。 B in the general formula (I) represents a nucleobase residue. The nucleobase may be a natural nucleobase or a non-natural nucleobase. Here, the “non-natural nucleobase” means a chemical modification such as having a substituent on the exocyclic oxygen atom, exocyclic nitrogen atom, cyclin nitrogen atom or carbocyclic carbon atom of the natural nucleobase, or It means a chemical modification such as substitution of an exocyclic oxygen atom, a ring nitrogen atom or a ring carbon atom with another atom, or a nucleobase derivative having both of these chemical modifications. Examples of natural nucleobase residues include thymin-1-yl, cytosine-1-yl, 5-methylcytosin-1-yl, uracil-1-yl, uracil-5-yl, adenine-9- Yl, guanine-9-yl and the like. Non-natural nucleobase residues include 2-thiothymin-1-yl, 2-thiouracil-1-yl, N2-acyl-3-deazaguanine-9-yl, N2-carbamoylguanine-9-yl, N4 -Acylcytosin-1-yl, N6-acyl-7-deazaadenine-9-yl, N6-acyl-7-deaza-8-azaadenine-9-yl and the like. Among these nucleobase residues, preferred nucleobase residues include thymin-1-yl, cytosine-1-yl, 5-methylcytosin-1-yl, uracil-1-yl, uracil-5- Yl, adenine-9-yl, and guanine-9-yl.
一般式（Ｉ）で表されるヌクレオシド三リン酸誘導体の具体例としては、2'-O-カルバモイルウリジン三リン酸、2'-O-カルバモイルアデノシン三リン酸、2'-O-カルバモイルチミジン三リン酸、2'-O-カルバモイルグアノシン三リン酸、2'-O-カルバモイルシチジン三リン酸、2'-O-カルバモイル-5-メチルシチジン三リン酸、2'-O-チオカルバモイルウリジン三リン酸、2'-O-チオカルバモイルアデノシン三リン酸、2'-O-チオカルバモイルチミジン三リン酸、2'-O-チオカルバモイルグアノシン三リン酸、2'-O-チオカルバモイルシチジン三リン酸、2'-O-チオカルバモイル-5-メチルシチジン三リン酸、2'-O-N-メチルカルバモイルウリジン三リン酸、2'-O-N-メチルカルバモイルアデノシン三リン酸、2'-O-N-メチルカルバモイルチミジン三リン酸、2'-O-N-メチルカルバモイルグアノシン三リン酸、2'-O-N-メチルカルバモイルシチジン三リン酸、2'-O-N-メチルカルバモイル-5-メチルシチジン三リン酸、2'-O-N-メチルチオカルバモイルウリジン三リン酸、2'-O-N-メチルチオカルバモイルアデノシン三リン酸、2'-O-N-メチルチオカルバモイルチミジン三リン酸、2'-O-N-メチルチオカルバモイルグアノシン三リン酸、2'-O-N-メチルチオカルバモイルシチジン三リン酸、2'-O-N-メチルチオカルバモイル-5-メチルシチジン三リン酸などを挙げることができる。 Specific examples of the nucleoside triphosphate derivative represented by the general formula (I) include 2′-O-carbamoyluridine triphosphate, 2′-O-carbamoyladenosine triphosphate, and 2′-O-carbamoylthymidine triphosphate. Phosphate, 2'-O-carbamoylguanosine triphosphate, 2'-O-carbamoylcytidine triphosphate, 2'-O-carbamoyl-5-methylcytidine triphosphate, 2'-O-thiocarbamoyluridine triphosphate Acid, 2'-O-thiocarbamoyladenosine triphosphate, 2'-O-thiocarbamoylthymidine triphosphate, 2'-O-thiocarbamoylguanosine triphosphate, 2'-O-thiocarbamoylcytidine triphosphate, 2'-O-thiocarbamoyl-5-methylcytidine triphosphate, 2'-ON-methylcarbamoyluridine triphosphate, 2'-ON-methylcarbamoyl adenosine triphosphate, 2'-ON-methylcarbamoylthymidine triphosphate Acid, 2'-ON-methylcarbamoylguano Syn triphosphate, 2'-ON-methylcarbamoylcytidine triphosphate, 2'-ON-methylcarbamoyl-5-methylcytidine triphosphate, 2'-ON-methylthiocarbamoyluridine triphosphate, 2'-ON- Methylthiocarbamoyladenosine triphosphate, 2'-ON-methylthiocarbamoylthymidine triphosphate, 2'-ON-methylthiocarbamoylguanosine triphosphate, 2'-ON-methylthiocarbamoylcytidine triphosphate, 2'-ON-methylthiocarbamoyl And -5-methylcytidine triphosphate.
〔ヌクレオシド三リン酸誘導体の製造方法〕
本発明のヌクレオシド三リン酸誘導体の製造方法は、上記のヌクレオシド三リン酸誘導体を製造する方法であって、一般式（II）で表されるヌクレオシド三リン酸誘導体から弱酸性条件下でＲ３を脱離させる工程を含むものである。
[Method for producing nucleoside triphosphate derivative]
The method for producing a nucleoside triphosphate derivative according to the present invention is a method for producing the above-described nucleoside triphosphate derivative, wherein R 3 is produced under weakly acidic conditions from a nucleoside triphosphate derivative represented by the general formula (II). A step of desorbing.
一般式（II）におけるＲ１、Ｒ２、Ｘ、及びＢは上記と同じ意味である。 R 1 , R 2 , X, and B in the general formula (II) have the same meaning as described above.
一般式（II）におけるＲ３はシリル系保護基を表す。ここで「シリル系保護基」とは、ヒドロキシ基（特にリボースの3'ヒドロキシ基）を保護し、ケイ素を含む基であり、例えば、プロテクティブ・グループス・イン・オーガニック・シンセシス(PROTECTIVE GROUPS IN ORGANIC SYNTHESIS)、第３版、ジョン・ウィリー・アンド・サンズ(JOHN WILLY&SONS)出版(1999年)等に記載されている任意のシリル系保護基である。具体的にはトリメチルシリル基、トリエチルシリル基、tert-ブチルジメチルシリル（TBS）基、tert-ブチルジフェニルシリル基などを挙げることができる。 R 3 in the general formula (II) represents a silyl protecting group. Here, the “silyl protecting group” is a group that protects a hydroxy group (particularly the 3′-hydroxy group of ribose) and contains silicon. For example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS), 3rd edition, JOHN WILLY & SONS publication (1999), etc. are arbitrary silyl type protecting groups. Specific examples include a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl (TBS) group, and a tert-butyldiphenylsilyl group.
弱酸性条件は、Ｒ３を脱離させることができる条件であればよい。具体的なpHは2〜6とするのが好ましく、4〜5とするのが更に好ましい。 The weakly acidic conditions may be any conditions as long as R 3 can be eliminated. The specific pH is preferably 2 to 6, and more preferably 4 to 5.
このような弱酸性条件は、反応系に酸性物質を加えることで達成できる。酸性物質としては、酢酸、プロピオン酸、ピリジニウムp-トルエンスルホン酸などの有機酸、希塩酸、硫酸水素カリウムなどの無機酸などを例示できる。 Such weakly acidic conditions can be achieved by adding an acidic substance to the reaction system. Examples of the acidic substance include organic acids such as acetic acid, propionic acid and pyridinium p-toluenesulfonic acid, and inorganic acids such as dilute hydrochloric acid and potassium hydrogen sulfate.
この脱離反応は、反応を阻害しない溶媒中で行う。このような溶媒としては、水、メタノール、エタノール、プロパノール、テトラヒドロフラン、1,4-ジオキサンやこれらの混合溶媒などを例示できる。 This elimination reaction is performed in a solvent that does not inhibit the reaction. Examples of such a solvent include water, methanol, ethanol, propanol, tetrahydrofuran, 1,4-dioxane and a mixed solvent thereof.
反応温度は特に限定されないが、10〜50℃の範囲内であることが好ましい。 Although reaction temperature is not specifically limited, It is preferable to exist in the range of 10-50 degreeC.
反応時間は特に限定されないが、6〜24時間とすることが好ましい。 Although reaction time is not specifically limited, It is preferable to set it as 6 to 24 hours.
一般式（II）で表されるヌクレオシド三リン酸誘導体は、ヌクレオシド三リン酸の合成においてよく知られた方法に従い、下記の一般式（IV）で表されるヌクレオシド誘導体から一般式（III）で表されるヌクレオシド誘導体を合成し、この化合物から合成することができる。 The nucleoside triphosphate derivative represented by the general formula (II) is obtained from the nucleoside derivative represented by the following general formula (IV) by the general formula (III) according to a well-known method in the synthesis of nucleoside triphosphate. The represented nucleoside derivative can be synthesized and synthesized from this compound.
ここで、Ｒ４は、ヒドロキシ基の保護基、例えば、プロテクティブ・グループス・イン・オーガニック・シンセシス(PROTECTIVE GROUPS IN ORGANIC SYNTHESIS)、第３版、ジョン・ウィリー・アンド・サンズ(JOHN WILLY&SONS)出版(1999年)等に記載されている任意の保護基である。具体的にはジメチルトリチル（DMTr）基、メトキシトリチル基、トリチル基、ジフェニル-tert-ブチルシリル基、ジメチル-tert-ブチルシリル基などを表す。 Here, R 4 is a protecting group for a hydroxy group, such as PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd edition, published by JOHN WILLY & SONS ( 1999)) and the like. Specifically, it represents a dimethyltrityl (DMTr) group, a methoxytrityl group, a trityl group, a diphenyl-tert-butylsilyl group, a dimethyl-tert-butylsilyl group, and the like.
一般式（IV）で表されるヌクレオシド誘導体から一般式（III）で表されるヌクレオシド誘導体の合成（5'位のヒドロキシ基の脱保護と3'位のヒドロキシ基の保護）は、例えば、 Patil, S. V. et al., Syn. Commun., 1994 , 24, 2423やFriedel, M. G.et al.,Chemistry. 2006, 12, 6081.に記載の方法に従って行うことができ、一般式（III）で表されるヌクレオシド誘導体から一般式（II）で表されるヌクレオシド三リン酸誘導体の合成（三リン酸の付加）は、例えば、Ludwig,J.; Eckstein, F. J. Org. Chem., 1989, 54, 631に記載の方法に従って行うことができる。また、一般式（IV）で表されるヌクレオシド誘導体はSeio, K. et al., Bioorg. Med. Chem. 2009, 17, 7275-7280（非特許文献３）に記載の方法に従って合成することができる。 Synthesis of the nucleoside derivative represented by the general formula (III) from the nucleoside derivative represented by the general formula (IV) (deprotection of the hydroxy group at the 5 ′ position and protection of the hydroxy group at the 3 ′ position) is, for example, Patil , SV et al., Syn. Commun., 1994, 24, 2423 and Friedel, MGet al., Chemistry. 2006, 12, 6081. and represented by the general formula (III) Synthesis of a nucleoside triphosphate derivative represented by the general formula (II) (addition of triphosphate) from, for example, Ludwig, J .; Eckstein, FJ Org. Chem., 1989, 54, 631 This can be done according to the methods described. The nucleoside derivative represented by the general formula (IV) can be synthesized according to the method described in Seio, K. et al., Bioorg. Med. Chem. 2009, 17, 7275-7280 (Non-patent Document 3). it can.
〔アプタマー〕
本発明のアプタマーは、上記のヌクレオシド三リン酸誘導体を利用して製造され、一般式（Ｖ）で表される。
[Aptamer]
The aptamer of the present invention is produced using the above nucleoside triphosphate derivative and is represented by the general formula (V).
一般式（Ｖ）及び（VI）におけるＲ１、Ｒ２、Ｘは上記と同じ意味であり、また、Ｂ１、Ｂ２及びＢ３は、上記Ｂと同様の核酸塩基の残基を表す。 In the general formulas (V) and (VI), R 1 , R 2 and X have the same meanings as described above, and B 1 , B 2 and B 3 represent the same nucleobase residues as in B above.
本発明のアプタマーは、標的物質と特異的に結合する構造、例えば、ヘアピン構造、バルジ構造、シュードノット構造、グアニンカルテット構造などを持つことが好ましい。 The aptamer of the present invention preferably has a structure that specifically binds to a target substance, for example, a hairpin structure, a bulge structure, a pseudoknot structure, a guanine quartet structure, or the like.
一般式（Ｖ）におけるｎは、１以上であれば特に限定されないが、ヌクレオチド数（ｎ＋２）がアプタマーとして好適な数になるようにするのが好ましい。具体的には、ｎは10〜80であることが好ましく、20〜40であることが更に好ましい。 Although n in general formula (V) will not be specifically limited if it is 1 or more, It is preferable to make it the number of nucleotides (n + 2) suitable for an aptamer. Specifically, n is preferably 10 to 80, and more preferably 20 to 40.
〔SELEX法〕
本発明のRNA誘導体の製造方法は、上記のヌクレオシド三リン酸誘導体を用いたSELEX法である。この方法は、（１）逆転写酵素をRNAの集団又はRNA誘導体の集団（このRNA誘導体の集団は、工程（４）で選択されたRNA誘導体の集団であってもよい。）に作用させ、DNAの集団を合成する工程、（２）工程（１）で合成されたDNAの集団を、ポリメラーゼ連鎖反応により増幅させる工程、（３）上記ヌクレオシド三リン酸誘導体の存在下で、工程（２）で増幅されたDNAの集団にDNA依存RNAポリメラーゼを作用させ、上記ヌクレオシド三リン酸誘導体を残基として含むRNA誘導体の集団を合成する工程、（４）工程（３）で合成したRNA誘導体の集団を標的物質と接触させ、標的物質に親和性を示すRNA誘導体の集団を選択する工程を有する。
[SELEX method]
The method for producing an RNA derivative of the present invention is the SELEX method using the above nucleoside triphosphate derivative. In this method, (1) reverse transcriptase is allowed to act on a population of RNA or a population of RNA derivatives (this population of RNA derivatives may be a population of RNA derivatives selected in step (4)). A step of synthesizing a population of DNA, (2) a step of amplifying the population of DNA synthesized in step (1) by polymerase chain reaction, (3) in the presence of the nucleoside triphosphate derivative, step (2) A step of causing a DNA-dependent RNA polymerase to act on the population of DNA amplified in step 1 to synthesize a population of RNA derivatives containing the nucleoside triphosphate derivative as a residue; (4) a population of RNA derivatives synthesized in step (3) Is contacted with the target substance, and a group of RNA derivatives showing affinity for the target substance is selected.
上記方法は、RNA誘導体の集団を合成する際、基質として上記ヌクレオシド三リン酸誘導体を使用する点を除き、通常のSELEX法と同様に行うことができる。 The above method can be performed in the same manner as the normal SELEX method, except that the nucleoside triphosphate derivative is used as a substrate when a population of RNA derivatives is synthesized.
上記の（１）〜（４）の工程は、複数回繰り返して行う。繰り返す回数は特に限定されないが、1〜30回とするのが好ましく、5〜15回とするのが更に好ましい。 The steps (1) to (4) are repeated a plurality of times. The number of repetitions is not particularly limited, but is preferably 1 to 30 times, and more preferably 5 to 15 times.
逆転写酵素としては、通常のSELEX法で使用されているものでよく、市販の逆転写酵素、例えば、SuperScriptIII（Invitrogen）、SuperScriptII（Invitrogen）、ThermoScript（Invitrogen）、ReverTra Ace（東洋紡）などを使用することができる。DNA依存RNAポリメラーゼとしては、通常のSELEX法で使用されているものでよく、T7ファージ由来のRNAポリメラーゼ、T3ファージ由来のRNAポリメラーゼ、SP6 RNA ポリメラーゼなどを使用することができる。逆転写反応、ポリメラーゼ連鎖反応、転写反応（RNA合成反応）は、通常のSELEX法における反応と同様に行うことができる。また、標的物質に親和性を示すRNAの選択も、通常のSELEX法と同様に行うことができる。 The reverse transcriptase may be the one used in the normal SELEX method, and a commercially available reverse transcriptase such as SuperScriptIII (Invitrogen), SuperScriptII (Invitrogen), ThermoScript (Invitrogen), ReverTra Ace (Toyobo), etc. is used. can do. As the DNA-dependent RNA polymerase, those used in the usual SELEX method may be used, and RNA polymerase derived from T7 phage, RNA polymerase derived from T3 phage, SP6 RNA polymerase and the like can be used. The reverse transcription reaction, polymerase chain reaction, and transcription reaction (RNA synthesis reaction) can be carried out in the same manner as in the usual SELEX method. In addition, RNA having affinity for the target substance can be selected in the same manner as in the normal SELEX method.
〔RNA誘導体の製造方法〕
本発明のRNA誘導体の製造方法は、上記ヌクレオシド三リン酸誘導体の存在下で、鋳型とするDNAにDNA依存RNAポリメラーゼを作用させ、上記ヌクレオシド三リン酸誘導体を残基として含むRNA誘導体を製造するものである。この方法は、基質として上記ヌクレオシド三リン酸誘導体を使用する点を除き、通常のDNA依存RNAポリメラーゼを用いたRNAの製造方法と同様に行うことができる。
[Method for producing RNA derivative]
In the method for producing an RNA derivative of the present invention, a DNA-dependent RNA polymerase is allowed to act on a template DNA in the presence of the nucleoside triphosphate derivative to produce an RNA derivative containing the nucleoside triphosphate derivative as a residue. Is. This method can be performed in the same manner as the method for producing RNA using a normal DNA-dependent RNA polymerase, except that the nucleoside triphosphate derivative is used as a substrate.
以下、実施例に沿って本発明を更に詳細に説明するが、これら実施例は本発明の範囲を何ら限定するものではない。また、本発明において使用する試薬や装置、材料は特に言及されない限り、商業的に入手可能である。また、本明細書において、略号で表示する場合、各表示は、IUPAC-IUB Commission on Biochemical Nomenclatureによる略号あるいは当該分野における慣用略号に基づくものである。
〔参考例〕 2'-O-カルバモイルウリジンホスホロアミダイトユニットの合成
（１）2'-O-フェノキシカルボニル-3',5'-O-(1,1,3,3,-テトライソプロピイルジシロキサン-1,3,-ジイル)ウリジンの合成
EXAMPLES Hereinafter, although this invention is demonstrated further in detail along an Example, these Examples do not limit the scope of the present invention at all. In addition, the reagents, devices, and materials used in the present invention are commercially available unless otherwise specified. Further, in this specification, when abbreviations are used, each indication is based on an abbreviation by the IUPAC-IUB Commission on Biochemical Nomenclature or a common abbreviation in the field.
[Reference Example] Synthesis of 2'-O-carbamoyluridine phosphoramidite unit (1) 2'-O-phenoxycarbonyl-3 ', 5'-O- (1,1,3,3, -tetraisopropiyldisiloxane Synthesis of (-1,3, -diyl) uridine
アルゴン雰囲気下、3',5'-O-1,1,3,3,-テトライソプロピイルジシロキサン-1,3,-ジイル)ウリジン (6.1 g, 13 mmol)を脱水トルエン(126mL)に溶解させ、ピリジン(1.2 mL, 13 mmol)加えた。クロロギ酸フェニルをゆっくり滴加し、室温にて３時間撹拌した。反応終了後、精製水（10 mL）加えた後、酢酸エチル (200 mL)にて希釈、飽和食塩水で３回洗浄した。得られた有機層を無水硫酸ナトリウムで乾燥し濾過したのち、減圧下留去した。つづいてシリカゲルカラムクロマトグラフィー(クロロホルム-ヘキサン、６：４-４：６, v/v)にて精製し、減圧乾燥させて表題の化合物（6.1g、79％）を得た。
1H NMR (500 MHz, CDCl3) δ 0.97-1.12 (28H, m), 4.03 (1H, dd, J = 2.6 Hz, 11.0 Hz), 4.11 (1H, dd, J = 1.3 Hz, 8.1 Hz), 4.26 (1H, d, J=3.4Hz), 4.47 (1H, dd, J=4.4Hz, 4.9Hz), 5.31 (1H, d, J=4.6Hz), 5.71 (1H, d, J = 8.0 Hz), 5.93 (1H, s), 7.17 (2H, m), 7.26 (1H, m), 7.38 (2H, m), 7.73 (1H, d, J = 8.0 Hz), 8.28 (1H, s);
13C NMR (500 MHz, CDCl3) δ13.1, 17.4, 59.6, 68.1, 79.7, 82.2, 88.6, 102.5, 121.1, 126.5, 129.8, 139.5, 149.7, 151.3, 152.4, 162.8.
MS m/z calculated for C28H43N2O9Si2 + [M+H]+: 607.2502, found: 607.2596.
In an argon atmosphere, 3 ', 5'-O-1,1,3,3, -tetraisopropiyldisiloxane-1,3, -diyl) uridine (6.1 g, 13 mmol) was dissolved in dehydrated toluene (126 mL). Pyridine (1.2 mL, 13 mmol) was added. Phenyl chloroformate was slowly added dropwise and stirred at room temperature for 3 hours. After completion of the reaction, purified water (10 mL) was added, diluted with ethyl acetate (200 mL), and washed 3 times with saturated brine. The obtained organic layer was dried over anhydrous sodium sulfate and filtered, and then evaporated under reduced pressure. Subsequently, the residue was purified by silica gel column chromatography (chloroform-hexane, 6: 4-4: 6, v / v) and dried under reduced pressure to obtain the title compound (6.1 g, 79%).
1 H NMR (500 MHz, CDCl 3 ) δ 0.97-1.12 (28H, m), 4.03 (1H, dd, J = 2.6 Hz, 11.0 Hz), 4.11 (1H, dd, J = 1.3 Hz, 8.1 Hz), 4.26 (1H, d, J = 3.4Hz), 4.47 (1H, dd, J = 4.4Hz, 4.9Hz), 5.31 (1H, d, J = 4.6Hz), 5.71 (1H, d, J = 8.0 Hz) , 5.93 (1H, s), 7.17 (2H, m), 7.26 (1H, m), 7.38 (2H, m), 7.73 (1H, d, J = 8.0 Hz), 8.28 (1H, s);
13 C NMR (500 MHz, CDCl 3 ) δ 13.1, 17.4, 59.6, 68.1, 79.7, 82.2, 88.6, 102.5, 121.1, 126.5, 129.8, 139.5, 149.7, 151.3, 152.4, 162.8.
MS m / z calculated for C 28 H 43 N 2 O 9 Si 2 + [M + H] + : 607.2502, found: 607.2596.
（２）2'-O-カルバモイル-3',5'-O-(1,1,3,3-テトライソプロピイルジシロキサン-1,3,-ジイル)ウリジンの合成 (2) Synthesis of 2'-O-carbamoyl-3 ', 5'-O- (1,1,3,3-tetraisopropiyldisiloxane-1,3, -diyl) uridine
2'-O-フェノキシカルボニル-3',5'-O-(1,1,3,3,-テトライソプロピイルジシロキサン-1,3,-ジイル)ウリジン（1.8 g, 3.0 mmol）を無水ピリジン(30 mL)に溶解し、2 M アンモニア-エタノール (8.9 mL, 17.8 mmol)を加え、室温にて16時間撹拌した。反応終了後、酢酸エチル (100 mL)にて希釈し、飽和食塩水にて３回洗浄した。得られた有機層を無水硫酸ナトリウムで乾燥させた後に濾過し、溶媒を減圧下留去した。つづいてN-Hシリカゲルを用いてシリカゲルクロマトグラフィー（クロロホルム-メタノール, 100:0-100:2, v/v）にて精製し、減圧乾燥させて表題の化合物 (1.4g, 89%)を得た。
1H NMR (500 MHz, CDCl3) δ 0.93-1.11 (28H, m), 4.00 (2H, m), 4.20 (1H, d, J = 3.5 Hz), 4.39 (1H, dd, J = 3.9 Hz, 5.2 Hz), 4.90 (2H, s), 5.27 (1H, d, J = 5.1 Hz), 5.69 (1H, dd, J = 8.3 Hz), 5.83 (1H, s), 7.64 (1H, d, J = 8.3 Hz), 8.76 (1H, s);
13C NMR (CDCl3) δ 13.2, 17.3, 60.0, 68.1, 76.3, 82.4, 89.0, 102.5, 139.7, 149.9, 155.2, 163.1,
MS m/z calculated for C22H40N3O8Si2 + [M+H]+: 530.2348, found: 530.2370.
2'-O-phenoxycarbonyl-3 ', 5'-O- (1,1,3,3, -tetraisopropiyldisiloxane-1,3, -diyl) uridine (1.8 g, 3.0 mmol) was added to anhydrous pyridine ( 30 mL), 2 M ammonia-ethanol (8.9 mL, 17.8 mmol) was added, and the mixture was stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was diluted with ethyl acetate (100 mL) and washed 3 times with saturated brine. The obtained organic layer was dried over anhydrous sodium sulfate and then filtered, and the solvent was distilled off under reduced pressure. It was then purified by silica gel chromatography (chloroform-methanol, 100: 0-100: 2, v / v) using NH silica gel and dried under reduced pressure to give the title compound (1.4 g, 89%).
1 H NMR (500 MHz, CDCl 3 ) δ 0.93-1.11 (28H, m), 4.00 (2H, m), 4.20 (1H, d, J = 3.5 Hz), 4.39 (1H, dd, J = 3.9 Hz, 5.2 Hz), 4.90 (2H, s), 5.27 (1H, d, J = 5.1 Hz), 5.69 (1H, dd, J = 8.3 Hz), 5.83 (1H, s), 7.64 (1H, d, J = 8.3 Hz), 8.76 (1H, s);
13 C NMR (CDCl 3 ) δ 13.2, 17.3, 60.0, 68.1, 76.3, 82.4, 89.0, 102.5, 139.7, 149.9, 155.2, 163.1,
MS m / z calculated for C 22 H 40 N 3 O 8 Si 2 + [M + H] + : 530.2348, found: 530.2370.
（３）2'-O-カルバモイル-5'-O-(4,4'-ジメトキシトリチル)ウリジンの合成 (3) Synthesis of 2'-O-carbamoyl-5'-O- (4,4'-dimethoxytrityl) uridine
2'-O-カルバモイル-3',5'-O-(1,1,3,3-テトライソプロピイルジシロキサン-1,3,-ジイル)ウリジン(6.0 g、11 mmol)を無水THF(57 mL)に溶解させ、トリエチルアミン(2.7 mL, 20 mmol)および三フッ化水素トリエチルアミン(6.3 mL, 38 mmol)を加えた後、室温にて2時間撹拌した。溶媒を減圧下留去し、トルエンおよびピリジンにて共沸を３回行った。無水ピリジン(17 mL)に溶解させ、ジメトキトリチルクロライド(5.7g, 17 mmol)、トリエチルアミン(2.4 mL, 17 mmol)、ジクロロ酢酸(1.4 mL, 17 mmol)を加え室温下23時間撹拌した。メタノール(5 mL)加え反応を停止した後、酢酸エチル(200 mL)にて希釈し、飽和食塩水にて三回洗浄した。得られた有機層を無水硫酸ナトリウムにて乾燥させた後に濾過し、溶媒を減圧下留去した。つづいてシリカゲルカラムクロマトグラフィー(クロロホルム-メタノール-トリエチルアミン, 100:0:0.5-100:2:0.5, v/v/v)にて精製し、表題の化合物(5.5g, 84%)を得た。
1H NMR (500 MHz, CDCl3) δ 3.44 (2H, m), 3.73 (6H, s), 3.96 (1H, m), 4.16 (1H, d, J = 3.9 Hz), 4.57 (1H, d, J = 4.2 Hz), 5.26 (1H, m), 5.34 (1H, d, J = 8.1 Hz), 5.65 (2H, s), 6.14 (1H, d, J = 5.1 Hz), 6.80 (4H, m) 7.15-7.37 (9H, m), 7.76 (1H, d, J = 8.1 Hz), 9.71 (1H, s);
13C NMR (500 MHz, CDCl3) δ 55.5, 62.8, 70.4, 84.1, 86.8, 87.4, 103.1, 113.6, 127.4, 128.3, 128.4, 130.4, 135.3, 135.5, 140.3, 144.4, 151.2, 156.5, 158.9, 163.6.
MS m/z calculated for C31H31N3NaO9 + [M+Na]+: 612.1925, found: 612.2367.
2'-O-carbamoyl-3 ', 5'-O- (1,1,3,3-tetraisopropiyldisiloxane-1,3, -diyl) uridine (6.0 g, 11 mmol) in anhydrous THF (57 mL ), Triethylamine (2.7 mL, 20 mmol) and hydrogen trifluoride triethylamine (6.3 mL, 38 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure, and azeotropy was performed three times with toluene and pyridine. It was dissolved in anhydrous pyridine (17 mL), dimethoxytrityl chloride (5.7 g, 17 mmol), triethylamine (2.4 mL, 17 mmol) and dichloroacetic acid (1.4 mL, 17 mmol) were added, and the mixture was stirred at room temperature for 23 hours. Methanol (5 mL) was added to stop the reaction, and the mixture was diluted with ethyl acetate (200 mL) and washed three times with saturated brine. The obtained organic layer was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. Subsequently, the residue was purified by silica gel column chromatography (chloroform-methanol-triethylamine, 100: 0: 0.5-100: 2: 0.5, v / v / v) to obtain the title compound (5.5 g, 84%).
1 H NMR (500 MHz, CDCl 3 ) δ 3.44 (2H, m), 3.73 (6H, s), 3.96 (1H, m), 4.16 (1H, d, J = 3.9 Hz), 4.57 (1H, d, J = 4.2 Hz), 5.26 (1H, m), 5.34 (1H, d, J = 8.1 Hz), 5.65 (2H, s), 6.14 (1H, d, J = 5.1 Hz), 6.80 (4H, m) 7.15-7.37 (9H, m), 7.76 (1H, d, J = 8.1 Hz), 9.71 (1H, s);
13 C NMR (500 MHz, CDCl 3 ) δ 55.5, 62.8, 70.4, 84.1, 86.8, 87.4, 103.1, 113.6, 127.4, 128.3, 128.4, 130.4, 135.3, 135.5, 140.3, 144.4, 151.2, 156.5, 158.9, 163.6 .
MS m / z calculated for C 31 H 31 N 3 NaO 9 + [M + Na] + : 612.1925, found: 612.2367.
（４）2'-O-カルバモイル-5'-O-(4,4'-ジメトキシトリチル)ウリジン 3'-(2-シアノエチル N,N-ジイソプロピルホスホロアミダイト)の合成 (4) Synthesis of 2'-O-carbamoyl-5'-O- (4,4'-dimethoxytrityl) uridine 3 '-(2-cyanoethyl N, N-diisopropyl phosphoramidite)
2'-O-カルバモイル-5'-O-(4,4'-ジメトキシトリチル)ウリジン(1.3g, 2.2 mmol)を無水ピリジン、無水トルエンで共沸した後、無水ジクロロメタン(22 mL)へ溶解した。ジイソプロピルアミン(155 μL, 1.1 mmol)、1H-テトラゾール(77 mg, 1.1 mol)および2-シアノエチル N,N,N’,N’-テトライソプロピルホスホロジアミダイト(839 μL, 2.6 mmol)を加え室温下２時間撹拌した。反応終了後、水 (1mL)加え、酢酸エチル(50 mL)にて希釈した。有機層を5%炭酸ナトリウム水溶液で5回洗浄した後に、無水硫酸ナトリウムにて乾燥し、濾過、減圧下溶媒を留去した。シリカゲルシリカゲルカラムクロマトグラフィー（クロロホルム-メタノール-トリエチルアミン、100:2:0.5-100:4:0.5, v/v/v）にて精製し、表題の化合物(0.89g, 51%)を得た。
1H NMR (CDCl3) δ 1.06-1.27 (14H, m), 2.42 (1H, m), 2.69 (1H, m), 3.42-3.53 (2H, m), 3.57-3.70 (2H, m), 3.79 (6H, d, J = 2.9 Hz), 4.12 (1H, m), 4.21- 4.31 (1H, m), 4.67 (1H, m), 4.69 (2H, d, J = 3.2 Hz), 5.30-5.43 (2H, m), 6.22 (1H, m), 6.84 (4H, m), 7.24-7.40 (9H, m), 7.70 (1H m), 8.11 (1H, m);
13C NMR (500 MHz, CDCl3) δ 14.5, 20.4, 24.8, 43.5, 55.5, 58.2, 60.6, 63.1, 71.3, 75.5, 84.5, 86.2, 87.5, 103.1, 113.6, 127.5, 128.5, 130.5, 135.2, 140.3, 144.3, 150.7, 155.5, 159.0, 162.8;
31P NMR (CDCl3) δ 151.2, 151.5.
MS m/z calculated for C40H49N5O10P+ [M+H]+: 790.2312, found: 790.3297.
2'-O-carbamoyl-5'-O- (4,4'-dimethoxytrityl) uridine (1.3 g, 2.2 mmol) was azeotroped with anhydrous pyridine and anhydrous toluene, and then dissolved in anhydrous dichloromethane (22 mL) . Add diisopropylamine (155 μL, 1.1 mmol), 1H-tetrazole (77 mg, 1.1 mol) and 2-cyanoethyl N, N, N ', N'-tetraisopropyl phosphorodiamidite (839 μL, 2.6 mmol) at room temperature Stir for 2 hours. After completion of the reaction, water (1 mL) was added and diluted with ethyl acetate (50 mL). The organic layer was washed 5 times with 5% aqueous sodium carbonate solution, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure. Purification by silica gel column chromatography (chloroform-methanol-triethylamine, 100: 2: 0.5-100: 4: 0.5, v / v / v) gave the title compound (0.89 g, 51%).
1 H NMR (CDCl 3 ) δ 1.06-1.27 (14H, m), 2.42 (1H, m), 2.69 (1H, m), 3.42-3.53 (2H, m), 3.57-3.70 (2H, m), 3.79 (6H, d, J = 2.9 Hz), 4.12 (1H, m), 4.21- 4.31 (1H, m), 4.67 (1H, m), 4.69 (2H, d, J = 3.2 Hz), 5.30-5.43 ( 2H, m), 6.22 (1H, m), 6.84 (4H, m), 7.24-7.40 (9H, m), 7.70 (1H m), 8.11 (1H, m);
13 C NMR (500 MHz, CDCl 3 ) δ 14.5, 20.4, 24.8, 43.5, 55.5, 58.2, 60.6, 63.1, 71.3, 75.5, 84.5, 86.2, 87.5, 103.1, 113.6, 127.5, 128.5, 130.5, 135.2, 140.3 , 144.3, 150.7, 155.5, 159.0, 162.8;
31 P NMR (CDCl 3 ) δ 151.2, 151.5.
MS m / z calculated for C 40 H 49 N 5 O 10 P + [M + H] + : 790.2312, found: 790.3297.
〔実施例１〕 2'-O-カルバモイルウリジン三リン酸の合成
（１）2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)ウリジンの合成
[Example 1] Synthesis of 2'-O-carbamoyluridine triphosphate (1) Synthesis of 2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) uridine
2'-O-カルバモイル-5'-O-(4,4'-ジメトキシトリチル)ウリジン (2.12 g, 3.60 mmol) を脱水ピリジン(1 ml, ×3), 脱水トルエン (1 ml, ×3), 脱水アセトニトリル (1 ml, ×3) で共沸し、脱水DMF (7.2 ml)に溶解させた。イミダゾール(0.74 g, 10.8 mmol) を加えたのち、tert-ブチルジメチルクロロシラン(0.81 g, 5.40 mmol) を加え、室温で14時間、アルゴン存在下で反応させた。反応終了後、反応系に、酢酸エチル (30 ml) を加え、水、飽和炭酸水素ナトリウム水、水の順で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下留去した。4% トリフルオロ酢酸-ジクロロメタン溶液 (36 ml) を加え、室温で30分反応させたのち、氷冷下、反応溶液をピリジン：メタノール (1 : 1, v/v) 混合溶媒 (100 ml) に滴加し1時間撹拌した。そののち、溶媒を減圧下で留去し、得られた残渣をジクロロメタン (1 ml) で溶解し、ヘキサン (10 ml) を加えた。そうして析出した沈殿物をろ過により精製し、表題の化合物 (0.91 g, 63%) を得た。
1H NMR (500 MHz, DMSO-d6) δ 11.39 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H), 6.81 (s, 1H), 6.62 (s, 1H), 5.95 (d, J = 6.3 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 5.26 (t, J = 4.9 Hz, 1H), 5.04 (dd, J = 6.3, 5.1 Hz, 1H), 4.38 (dd, J = 5.1, 3.3 Hz, 1H), 3.87 (q, J = 3.3 Hz, 1H), 3.65 (dt, J = 12.0, 4.1 Hz, 1H), 3.54 (dt, J = 12.0, 4.1 Hz, 1H), 0.88 (s, 9H), 0.08 (s, 6H).
MS m/z calculated for C16H27N3NaO7Si+ [M+H]+: 424.1510, found: 424.1511.
2'-O-carbamoyl-5'-O- (4,4'-dimethoxytrityl) uridine (2.12 g, 3.60 mmol) was added to dehydrated pyridine (1 ml, × 3), dehydrated toluene (1 ml, × 3), It was azeotroped with dehydrated acetonitrile (1 ml, × 3) and dissolved in dehydrated DMF (7.2 ml). Imidazole (0.74 g, 10.8 mmol) was added, then tert-butyldimethylchlorosilane (0.81 g, 5.40 mmol) was added, and the reaction was carried out at room temperature for 14 hours in the presence of argon. After completion of the reaction, ethyl acetate (30 ml) was added to the reaction system, and extraction was performed three times in the order of water, saturated aqueous sodium hydrogen carbonate, and water. The organic layer was collected, dried using sodium sulfate, and the solvent was distilled off under reduced pressure. 4% trifluoroacetic acid-dichloromethane solution (36 ml) was added and reacted at room temperature for 30 minutes. The reaction solution was then mixed with pyridine: methanol (1: 1, v / v) mixed solvent (100 ml) under ice cooling. Added dropwise and stirred for 1 hour. After that, the solvent was distilled off under reduced pressure, the resulting residue was dissolved in dichloromethane (1 ml), and hexane (10 ml) was added. The precipitate thus precipitated was purified by filtration to obtain the title compound (0.91 g, 63%).
1 H NMR (500 MHz, DMSO-d 6 ) δ 11.39 (s, 1H), 7.89 (d, J = 8.1 Hz, 1H), 6.81 (s, 1H), 6.62 (s, 1H), 5.95 (d, J = 6.3 Hz, 1H), 5.69 (d, J = 8.1 Hz, 1H), 5.26 (t, J = 4.9 Hz, 1H), 5.04 (dd, J = 6.3, 5.1 Hz, 1H), 4.38 (dd, J = 5.1, 3.3 Hz, 1H), 3.87 (q, J = 3.3 Hz, 1H), 3.65 (dt, J = 12.0, 4.1 Hz, 1H), 3.54 (dt, J = 12.0, 4.1 Hz, 1H), 0.88 (s, 9H), 0.08 (s, 6H).
MS m / z calculated for C 16 H 27 N 3 NaO 7 Si + [M + H] + : 424.1510, found: 424.1511.
（２）2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)ウリジン 5'-トリホスフェートの合成 (2) Synthesis of 2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) uridine 5'-triphosphate
2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)ウリジン(181mg, 0.45 mmol)を脱水ピリジン (1ml) により5回共沸した。アルゴン存在下で脱水ピリジン : 脱水1,4-ジオキサン(2 : 3, v/v, 1 ml)に溶かし、1, 4-ジオキサン(600 μl) に溶かした2-クロロ-4H-1,3,2-ベンゾジオキサホスホリン-4-オン (100 mg, 0.50 mmol) を加えた。10分間反応させたのち、トリブチルアミン(400 μl) とDMF (1.3 ml) に溶かしたピロホスフェートトリブチルアンモニウム塩 (372 mg, 0.50 mmol) を加え、10分間反応させた。つづいて 1% ヨウ素 ピリジン/水 溶液 (98 : 2, v/v, 8ml)を加え、15分間反応させた。水 (10 ml) 加え、5% 亜硫酸水素ナトリウム水溶液 (300μl) を加え、30分間撹拌した。反応終了後、溶媒を減圧下で留去し、残渣を逆相カラムクロマトグラフィ(0.1 M酢酸トリエチルアミン緩衝液 : アセトニトリル, 70 : 30 → 50 : 50) を行い精製したのち、溶媒を凍結乾燥し、留去した。そうして得た残渣をメタノール (1 ml) で溶かし、そこに0.6 M 過塩素酸ナトリウム アセトン溶液 (10 ml) を加えた。この反応系を遠心分離 (7000 rpm, 4分) し、上澄みをデキャンタで取り除いた。さらに、アセトン (10 ml)を加え、遠心分離 (7000 rpm, 4分) をし、上澄みを取り除く作業を3回行った。こうして得た残渣を凍結乾燥することにより、表題の化合物(182 mg, 61%) を得た。
1H NMR (500 MHz, DMSO-d6) δ 11.36 (br s, 1H), 7.86 (br s, 1H), 6.78 (br s, 1H), 6.70-6.41 (br s, 1H), 5.94 (br s, 1H), 5.67 (br s, 1H), 5.23 (br s, 1H), 5.02 (br s, 1H), 4.37 (br s, 1H), 3.86 (br s, 1H), 3.58-3.70 (m, 1H), 3.48-3.60 (m, 1H), 0.86 (s, 9H), 0.25-0.08 (br s, 6H).
MS m/z calculated for C16H29N3O16P3Si- [M-H]-: 640.0535, found: 640.527.
2′-O-carbamoyl-3′-O- (tert-butyldimethylsilyl) uridine (181 mg, 0.45 mmol) was azeotroped 5 times with dehydrated pyridine (1 ml). Dehydrated pyridine in the presence of argon: 2-chloro-4H-1,3, dissolved in dehydrated 1,4-dioxane (2: 3, v / v, 1 ml) and 1,4-dioxane (600 μl) 2-Benzodioxaphosphorin-4-one (100 mg, 0.50 mmol) was added. After reacting for 10 minutes, pyrophosphate tributylammonium salt (372 mg, 0.50 mmol) dissolved in tributylamine (400 μl) and DMF (1.3 ml) was added and reacted for 10 minutes. Subsequently, 1% iodine pyridine / water solution (98: 2, v / v, 8 ml) was added and allowed to react for 15 minutes. Water (10 ml) was added, 5% aqueous sodium bisulfite solution (300 μl) was added, and the mixture was stirred for 30 min. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase column chromatography (0.1 M triethylamine acetate buffer: acetonitrile, 70: 30 → 50: 50), and then the solvent was lyophilized and distilled. Left. The residue thus obtained was dissolved in methanol (1 ml), and 0.6 M sodium perchlorate acetone solution (10 ml) was added thereto. The reaction system was centrifuged (7000 rpm, 4 minutes), and the supernatant was removed with a decanter. Furthermore, acetone (10 ml) was added, and centrifugation (7000 rpm, 4 minutes) was performed, and the operation of removing the supernatant was performed three times. The residue thus obtained was lyophilized to give the title compound (182 mg, 61%).
1 H NMR (500 MHz, DMSO-d 6 ) δ 11.36 (br s, 1H), 7.86 (br s, 1H), 6.78 (br s, 1H), 6.70-6.41 (br s, 1H), 5.94 (br s, 1H), 5.67 (br s, 1H), 5.23 (br s, 1H), 5.02 (br s, 1H), 4.37 (br s, 1H), 3.86 (br s, 1H), 3.58-3.70 (m , 1H), 3.48-3.60 (m, 1H), 0.86 (s, 9H), 0.25-0.08 (br s, 6H).
MS m / z calculated for C 16 H 29 N 3 O 16 P 3 Si - [MH] -: 640.0535, found: 640.527.
（３）2'-O-カルバモイルウリジン 5'-トリホスフェートの合成 (3) Synthesis of 2'-O-carbamoyluridine 5'-triphosphate
2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)ウリジン 5'-トリホスフェート (182 mg, 0.28 mmol) に８％酢酸水溶液 (5 ml)を加え、37 ℃で、20時間反応させた。反応終了後、反応溶媒を凍結乾燥により、留去した。そうして得た残渣をメタノール (1 ml) で溶かし、そこに0.6 M過塩素酸ナトリウム アセトン溶液 (10 ml) を加えた。この反応系を遠心分離 (7000 rpm, 4分) し、上澄みをデキャンタで取り除いた。さらに、アセトン (10 ml)を加え、遠心分離 (7000 rpm, 4分) をし、上澄みを取り除く作業を3回行った。そうして得た残渣を凍結乾燥することにより、表題の化合物 (139 mg, 92%) を得た。
1H NMR (500 MHz, D2O) δ 7.94 (d, J = 8.2 Hz, 1H), 6.09 (d, J = 4.1 Hz, 1H), 5.99 (d, J = 8.2 Hz, 1H), 5.23 (t, J = 4.9 Hz, 1H), 4.60 (t, J = 5.7 Hz, 1H), 4.24-4.39 (m, 3H).
31P NMR (D2O) δ -8.26, -10.00, -21.00.
MS m/z calculated for C10H13N3Na2O16P3- [M-Na]-: 569.9310, found: 570.1221.
2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) uridine 5'-triphosphate (182 mg, 0.28 mmol) was added with 8% acetic acid aqueous solution (5 ml), and the mixture was incubated at 37 ° C for 20 hours. Reacted. After completion of the reaction, the reaction solvent was distilled off by lyophilization. The residue thus obtained was dissolved in methanol (1 ml), and 0.6 M sodium perchlorate acetone solution (10 ml) was added thereto. The reaction system was centrifuged (7000 rpm, 4 minutes), and the supernatant was removed with a decanter. Furthermore, acetone (10 ml) was added, and centrifugation (7000 rpm, 4 minutes) was performed, and the operation of removing the supernatant was performed three times. The residue thus obtained was lyophilized to give the title compound (139 mg, 92%).
1 H NMR (500 MHz, D 2 O) δ 7.94 (d, J = 8.2 Hz, 1H), 6.09 (d, J = 4.1 Hz, 1H), 5.99 (d, J = 8.2 Hz, 1H), 5.23 ( t, J = 4.9 Hz, 1H), 4.60 (t, J = 5.7 Hz, 1H), 4.24-4.39 (m, 3H).
31 P NMR (D 2 O) δ -8.26, -10.00, -21.00.
MS m / z calculated for C 10 H 13 N 3 Na 2 O 16 P 3- [M-Na] - : 569.9310, found: 570.1221.
〔実施例２〕 2'-O-カルバモイルアデノシン三リン酸の合成
（１） 2'-O-カルバモイル-3',5'-O-(1,1,3,3-テトライソプロピイルジシロキサン-1,3,-ジイル)-6-N-(4,4'-ジメトキシトリチル)アデノシンの合成
[Example 2] Synthesis of 2'-O-carbamoyladenosine triphosphate (1) 2'-O-carbamoyl-3 ', 5'-O- (1,1,3,3-tetraisopropiyldisiloxane-1 Synthesis of (, 3, -diyl) -6-N- (4,4'-dimethoxytrityl) adenosine
3',5'-O-(1,1,3,3-テトライソプロピイルジシロキサン-1,3,-ジイル)-6-N-(4,4'-ジメトキシトリチル)アデノシン (6.19 g, 7.60 mmol) を脱水ピリジン(2 ml, ×3)で共沸し、脱水ピリジン(76 ml)に溶解させた。クロロギ酸フェニル (1.15 ml, 9.12 mmol) を加え、室温で4時間、アルゴン存在下で反応させた。反応終了後、反応系に、酢酸エチル (20 ml) を加え、飽和炭酸水素ナトリウム水溶液で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下で留去し、残渣を得た。そうして得られた残渣を、精製作業を行わずに次の反応に使用した。得られた残渣に2 M NH3/ EtOH(25 ml)を加え、室温で1時間反応させた。反応終了後、飽和炭酸水素ナトリウム水溶液で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下で留去し、残渣を得た。そうして得られた残渣をシリカゲルカラムクロマトグラフィー(C-200、ジクロロメタン：メタノール=100:1)によって精製し、表題の化合物(4.74 g, 72%)を得た。
1H NMR (500 MHz, CDCl3) δ 8.00 (s, 1H), 7.84 (s, 1H), 7.38-7.13 (m, 9H), 6.85 (s, 1H), 6.79 (d, J = 8.6 Hz, 4H), 5.96 (s, 1H), 5.66 (d, J = 5.5 Hz, 1H), 5.20 (dd, J = 8.6, 5.5 Hz, 1H), 4.74 (s, 2H), 4.13 (dd, J = 12.8, 3.1 Hz, 1H), 4.08-3.95 (m, 2H), 3.78 (s, 6 H), 1.25-0.81 (m, 28H).
3 ', 5'-O- (1,1,3,3-Tetraisopropiyldisiloxane-1,3, -diyl) -6-N- (4,4'-dimethoxytrityl) adenosine (6.19 g, 7.60 mmol ) Was azeotroped with dehydrated pyridine (2 ml, x3) and dissolved in dehydrated pyridine (76 ml). Phenyl chloroformate (1.15 ml, 9.12 mmol) was added and reacted at room temperature for 4 hours in the presence of argon. After completion of the reaction, ethyl acetate (20 ml) was added to the reaction system, and extraction was performed three times with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried using sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue. The residue thus obtained was used for the next reaction without any purification work. 2 M NH 3 / EtOH (25 ml) was added to the obtained residue, and the mixture was reacted at room temperature for 1 hour. After completion of the reaction, extraction was performed 3 times with a saturated aqueous solution of sodium bicarbonate. The organic layer was collected and dried using sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue. The residue thus obtained was purified by silica gel column chromatography (C-200, dichloromethane: methanol = 100: 1) to obtain the title compound (4.74 g, 72%).
1 H NMR (500 MHz, CDCl 3 ) δ 8.00 (s, 1H), 7.84 (s, 1H), 7.38-7.13 (m, 9H), 6.85 (s, 1H), 6.79 (d, J = 8.6 Hz, 4H), 5.96 (s, 1H), 5.66 (d, J = 5.5 Hz, 1H), 5.20 (dd, J = 8.6, 5.5 Hz, 1H), 4.74 (s, 2H), 4.13 (dd, J = 12.8 , 3.1 Hz, 1H), 4.08-3.95 (m, 2H), 3.78 (s, 6 H), 1.25-0.81 (m, 28H).
（２）2'-O-カルバモイル-6-N-(4,4'-ジメトキシトリチル)アデノシンの合成 (2) Synthesis of 2'-O-carbamoyl-6-N- (4,4'-dimethoxytrityl) adenosine
2'-O-カルバモイル-3',5'-O-(1,1,3,3-テトライソプロピイルジシロキサン-1,3,-ジイル)-6-N-(4,4'-ジメトキシトリチル)アデノシン(4.27 g, 5.00 mmol) をテトラヒドロフラン(50 ml)に溶解させた。トリエチルアミン (1.25 ml, 9.00 mmol) を加えたのち、トリエチルアミン3フッ化水素(2.85 ml, 17.5 ml)を加え、室温で3時間、反応させた。反応終了後、反応系に、2-(トリメチルシリル)エタノール (5.98 ml, 40 mmol) を加え、室温で8時間、反応させた。反応終了後、飽和炭酸水素ナトリウム水溶液で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下で留去し、残渣を得た。そうして得られた残渣を、シリカゲルカラムクロマトグラフィー(C-200, ジクロロメタン：メタノール=100:5)によって精製し、表題の化合物(3.13 g, quant)を得た。
1H NMR (500 MHz, CDCl3) δ 8.00 (s, 1H), 7.78 (s, 1H), 7.37-7.13 (m, 9H), 7.06 (s, 1H), 6.86 -6.74 (m, 4H), 5.93 (d, J = 7.2 Hz, 1H), 5.60 (dd, J = 7.2, 4.9 Hz, 1H), 5.00 (s, 2H), 4.76 (d, J = 4.9, 1H), 4.24 (s, 1H), 3.90 (d, J = 12.3 Hz, 1H), 3.77 (s, 6H), 3.71 (d, J = 12.3 Hz, 1H).
2'-O-carbamoyl-3 ', 5'-O- (1,1,3,3-tetraisopropyldisiloxane-1,3, -diyl) -6-N- (4,4'-dimethoxytrityl) Adenosine (4.27 g, 5.00 mmol) was dissolved in tetrahydrofuran (50 ml). Triethylamine (1.25 ml, 9.00 mmol) was added, then triethylamine trifluoride (2.85 ml, 17.5 ml) was added, and the mixture was allowed to react at room temperature for 3 hours. After completion of the reaction, 2- (trimethylsilyl) ethanol (5.98 ml, 40 mmol) was added to the reaction system and reacted at room temperature for 8 hours. After completion of the reaction, extraction was performed 3 times with a saturated aqueous solution of sodium bicarbonate. The organic layer was collected and dried using sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue. The residue thus obtained was purified by silica gel column chromatography (C-200, dichloromethane: methanol = 100: 5) to obtain the title compound (3.13 g, quant).
1 H NMR (500 MHz, CDCl 3 ) δ 8.00 (s, 1H), 7.78 (s, 1H), 7.37-7.13 (m, 9H), 7.06 (s, 1H), 6.86 -6.74 (m, 4H), 5.93 (d, J = 7.2 Hz, 1H), 5.60 (dd, J = 7.2, 4.9 Hz, 1H), 5.00 (s, 2H), 4.76 (d, J = 4.9, 1H), 4.24 (s, 1H) , 3.90 (d, J = 12.3 Hz, 1H), 3.77 (s, 6H), 3.71 (d, J = 12.3 Hz, 1H).
（３）2'-O-カルバモイル-5'-O-6-N-ビス-(4,4'-ジメトキシトリチル)アデノシンの合成 (3) Synthesis of 2'-O-carbamoyl-5'-O-6-N-bis- (4,4'-dimethoxytrityl) adenosine
2'-O-カルバモイル-6-N-(4,4'-ジメトキシトリチル)アデノシン(3.00 g, 4.90 mmol) を脱水ピリジン(2 ml, ×3)で共沸し、脱水ピリジン (49 ml)に溶解させた。DMTrCl(2.00 g, 5.90 mmol) を加え、室温で6時間、アルゴン存在下で反応させた。反応終了後、反応系に、メタノール(20 ml) を加え、飽和炭酸水素ナトリウム水溶液で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下で留去し、残渣を得た。そうして得られた残渣をシリカゲルカラムクロマトグラフィー(C-200, ジクロロメタン：メタノール=100:2)によって精製し、表題の化合物(4.17 g, 93%)を得た。
1H NMR (500 MHz, CDCl3) δ 8.01 (s, 1H), 7.94 (s, 1H), 7.50-7.13 (m, 18H), 6.90 (s, 1H), 6.87-6.72 (m, 8H), 6.21 (d, J = 4.9 Hz, 1H), 5.66 (t, J = 4.9 Hz, 1H), 4.77 (q, J = 4.9 Hz, 1H), 4.19 (q, J = 4.1 Hz, 1H), 3.77 (s, 6H), 3.76 (s, 6H). 3.47 (dd, J = 10.6, 3.3 Hz, 1H), 3.39 (dd, J = 10.6, 4.2 Hz, 1H), 2.95 (d, J = 4.9 Hz, 1H).
2'-O-carbamoyl-6-N- (4,4'-dimethoxytrityl) adenosine (3.00 g, 4.90 mmol) was azeotroped with dehydrated pyridine (2 ml, × 3) to give dehydrated pyridine (49 ml). Dissolved. DMTrCl (2.00 g, 5.90 mmol) was added and reacted at room temperature for 6 hours in the presence of argon. After completion of the reaction, methanol (20 ml) was added to the reaction system, and extraction was performed three times with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried using sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue. The residue thus obtained was purified by silica gel column chromatography (C-200, dichloromethane: methanol = 100: 2) to obtain the title compound (4.17 g, 93%).
1 H NMR (500 MHz, CDCl 3 ) δ 8.01 (s, 1H), 7.94 (s, 1H), 7.50-7.13 (m, 18H), 6.90 (s, 1H), 6.87-6.72 (m, 8H), 6.21 (d, J = 4.9 Hz, 1H), 5.66 (t, J = 4.9 Hz, 1H), 4.77 (q, J = 4.9 Hz, 1H), 4.19 (q, J = 4.1 Hz, 1H), 3.77 ( s, 6H), 3.76 (s, 6H). 3.47 (dd, J = 10.6, 3.3 Hz, 1H), 3.39 (dd, J = 10.6, 4.2 Hz, 1H), 2.95 (d, J = 4.9 Hz, 1H ).
（４）2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)-5'-O-6-N-ビス-(4,4'-ジメトキシトリチル)アデノシンの合成 (4) Synthesis of 2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) -5'-O-6-N-bis- (4,4'-dimethoxytrityl) adenosine
2'-O-カルバモイル-5'-O-6-N-ビス-(4,4'-ジメトキシトリチル)アデノシン(4.39 g, 4.80 mmol) を脱水ピリジン(2 ml, ×3), 脱水トルエン (2 ml, ×3), 脱水アセトニトリル (2 ml, ×3) で共沸し、脱水DMF (12 ml)に溶解させた。imidazole (0.980 g, 14.4 mmol) を加えたのち、tert-ブチルジメチルクロロシラン(0.868 g, 5.76 mmol) を加え、室温で16時間、アルゴン存在下で反応させた。反応終了後、反応系に、酢酸エチル (30 ml) を加え、飽和炭酸水素ナトリウム水溶液で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下で留去し、残渣を得た。そうして得られた残渣をシリカゲルカラムクロマトグラフィー(C-200, ジクロロメタン：メタノール=100:1)によって精製し、表題の化合物(4.48 g, 90%)を得た。
1H NMR (500 MHz, CDCl3) δ 8.04 (s, 1H), 7.94 (s, 1H), 7.51-7.10 (m, 18H), 6.85 (s, 1H), 6.82-6.74 (m, 8H), 6.18 (d, J = 5.4 Hz, 1H), 5.70 (t, J = 5.4 Hz, 1H), 4.92 - 4.70 (m, 3H), 4.15 (q, J = 4.0 Hz, 1H), 3.76 (s, 12H), 3.46 (dd, J = 10.6, 3.5 Hz, 1H), 3.26 (dd, J = 10.6, 4.2 Hz, 1H), 0.83 (s, 9H), 0.04 (s, 3H), -0.03 (s, 3H).
2'-O-carbamoyl-5'-O-6-N-bis- (4,4'-dimethoxytrityl) adenosine (4.39 g, 4.80 mmol) was added to dehydrated pyridine (2 ml, × 3), dehydrated toluene (2 ml, x3), dehydrated acetonitrile (2 ml, x3), and dissolved in dehydrated DMF (12 ml). After adding imidazole (0.980 g, 14.4 mmol), tert-butyldimethylchlorosilane (0.868 g, 5.76 mmol) was added, and the mixture was allowed to react at room temperature for 16 hours in the presence of argon. After completion of the reaction, ethyl acetate (30 ml) was added to the reaction system, and extraction was performed three times with a saturated aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried using sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue. The residue thus obtained was purified by silica gel column chromatography (C-200, dichloromethane: methanol = 100: 1) to obtain the title compound (4.48 g, 90%).
1 H NMR (500 MHz, CDCl 3 ) δ 8.04 (s, 1H), 7.94 (s, 1H), 7.51-7.10 (m, 18H), 6.85 (s, 1H), 6.82-6.74 (m, 8H), 6.18 (d, J = 5.4 Hz, 1H), 5.70 (t, J = 5.4 Hz, 1H), 4.92-4.70 (m, 3H), 4.15 (q, J = 4.0 Hz, 1H), 3.76 (s, 12H ), 3.46 (dd, J = 10.6, 3.5 Hz, 1H), 3.26 (dd, J = 10.6, 4.2 Hz, 1H), 0.83 (s, 9H), 0.04 (s, 3H), -0.03 (s, 3H ).
（５）2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)アデノシンの合成 (5) Synthesis of 2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) adenosine
2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)-5'-O-6-N-ビス-(4,4'-ジメトキシトリチル)アデノシン (0.515 g, 0.500 mmol) を3% トリフルオロ酢酸/ジクロロメタン (6ml)を加え、室温で20分間反応させた。その後、反応系にピリジン：メタノール=1:1(10 ml) を加え、室温で20分間反応させた。反応終了後、飽和炭酸水素ナトリウム水溶液で、抽出操作を3回行った。有機層を回収し、硫酸ナトリウムを用いて乾燥させ溶媒を減圧下で留去し、残渣を得た。そうして得られた残渣をシリカゲルカラムクロマトグラフィー(C-200, ジクロロメタン：メタノール=100:8)によって精製し、表題の化合物 (0.195 mg, 92%)を得た。
1H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H), 8.14 (s, 1H), 7.37 (s, 2H), 6.80 (s, 1H), 6.60 (s, 1H), 6.10 (d, J = 6.7 Hz, 1H), 5.58 (dd, J = 6.7, 4.9 Hz, 1H), 5.42 (s, 1H), 4.63 (dd, J = 4.9, 2.7 Hz, 1H), 3.98 (d, J = 3.4 Hz, 1H), 3.70 (d, J = 11.9 Hz, 1H), 3.55 (d, J = 11.9 Hz, 1H), 0.91 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H).
2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) -5'-O-6-N-bis- (4,4'-dimethoxytrityl) adenosine (0.515 g, 0.500 mmol) % Trifluoroacetic acid / dichloromethane (6 ml) was added and reacted at room temperature for 20 minutes. Thereafter, pyridine: methanol = 1: 1 (10 ml) was added to the reaction system and reacted at room temperature for 20 minutes. After completion of the reaction, extraction was performed 3 times with a saturated aqueous solution of sodium bicarbonate. The organic layer was collected and dried using sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue. The residue thus obtained was purified by silica gel column chromatography (C-200, dichloromethane: methanol = 100: 8) to obtain the title compound (0.195 mg, 92%).
1 H NMR (500 MHz, DMSO-d 6 ) δ 8.39 (s, 1H), 8.14 (s, 1H), 7.37 (s, 2H), 6.80 (s, 1H), 6.60 (s, 1H), 6.10 ( d, J = 6.7 Hz, 1H), 5.58 (dd, J = 6.7, 4.9 Hz, 1H), 5.42 (s, 1H), 4.63 (dd, J = 4.9, 2.7 Hz, 1H), 3.98 (d, J = 3.4 Hz, 1H), 3.70 (d, J = 11.9 Hz, 1H), 3.55 (d, J = 11.9 Hz, 1H), 0.91 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H ).
（６）2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)アデノシン 5'-トリホスフェートの合成 (6) Synthesis of 2'-O-carbamoyl-3'-O- (tert-butyldimethylsilyl) adenosine 5'-triphosphate
2'-O-カルバモイル-3'-O-(tert-ブチルジメチルシリル)アデノシン(127 mg, 0.300 mmol)を脱水ピリジン (1ml) により5回共沸した。アルゴン存在下で脱水ピリジン : 脱水1,4-ジオキサン(1 : 3, v/v, 1 ml)に溶かし、1, 4-ジオキサン(360μl) に溶かした2-クロロ-4H-1,3,2-ベンゾジオキサホスホリン-4-オン (67 mg, 0.330 mmol) を加えた。10分間反応させたのち、トリブチルアミン(300 μl) とDMF (0.900 ml) に溶かしたトリブチルアンモニウムピロホスフェート(250 mg, 0.330 mmol) を加え、10分間反応させた。 1% ヨウ素 ピリジン/水 溶液 (98 : 2, v/v, 6ml)を加え、15分間反応させた。水 (10 ml) 加え、5% 亜硫酸水素ナトリウム水溶液 (450μl) を加え、30分間撹拌した。反応終了後、溶媒を減圧下で留去し、残渣を逆相カラムクロマトグラフィ(0.1 M酢酸トリエチルアミン緩衝液 : アセトニトリル, 100 : 0 → 70 : 30) を行い精製したのち、溶媒を凍結乾燥し、留去し表題の化合物を得た。
1H NMR (500 MHz, D2O) δ 8.63 (s, 1H), 8.42 (s, 1H), 6.55-6.40 (m, 1H), 5.61 (s, 1H), 4.55-4.45 (m, 1H), 4.45-4.30 (m, 1H), 1.09 (s, 9H), 0.36 (s, 3H), 0.34 (s, 3H).
2′-O-carbamoyl-3′-O- (tert-butyldimethylsilyl) adenosine (127 mg, 0.300 mmol) was azeotroped 5 times with dehydrated pyridine (1 ml). Dehydrated pyridine in the presence of argon: 2-chloro-4H-1,3,2 dissolved in dehydrated 1,4-dioxane (1: 3, v / v, 1 ml) and dissolved in 1,4-dioxane (360 μl) -Benzodioxaphosphorin-4-one (67 mg, 0.330 mmol) was added. After reacting for 10 minutes, tributylammonium pyrophosphate (250 mg, 0.330 mmol) dissolved in tributylamine (300 μl) and DMF (0.900 ml) was added and reacted for 10 minutes. 1% iodine pyridine / water solution (98: 2, v / v, 6 ml) was added and allowed to react for 15 minutes. Water (10 ml) was added, 5% aqueous sodium hydrogen sulfite solution (450 μl) was added, and the mixture was stirred for 30 min. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by reverse-phase column chromatography (0.1 M triethylamine acetate buffer: acetonitrile, 100: 0 → 70: 30), and then the solvent was lyophilized and distilled. The title compound was obtained.
1 H NMR (500 MHz, D 2 O) δ 8.63 (s, 1H), 8.42 (s, 1H), 6.55-6.40 (m, 1H), 5.61 (s, 1H), 4.55-4.45 (m, 1H) , 4.45-4.30 (m, 1H), 1.09 (s, 9H), 0.36 (s, 3H), 0.34 (s, 3H).
〔実施例３〕 2'-O-カルバモイルウリジン-5'-三リン酸を用いた転写実験
各テンプレート-DNA (2 μl, 5 μM), 10×T7 RNA Polymerase Buffer (2 μl, 400 mM Tris-HCl (pH 8.0), 80 mM MgCl2, 20 mM スペルミジン), ATP (2 μL, 4 mM), GTP (2 μl, 4 mM)、UcmTP (2 μL, 4 mM), CTP (2 μl, 3.2 mM), FAM-CTP (2 μl, 0.8 mM), 50 mM DTT (2 μl), T7 RNA Polymerase または T7 RNA Polymerase TM(100 Unit)の順にチューブに入れ、37℃で1 時間転写反応を行った。75 ℃で５分間チューブを加熱し、酵素を失活させたのち、氷浴で1分間冷やし、反応停止剤 (20 μl, 95% formamide, 0.05% BPB)を加えた。95 ℃で3分加熱したのち、7 M 尿素 20%ポリアクリルアミドゲルを用いて電気泳動 (チャージ量10 μl, 160 min, 300 V, 30 mA)を行った。フルオロイメージアナライザー (FUJIFILM FLA-7000)を用いて、FAMの蛍光を検出した。Lane 1 のマーカーは標品のRNA (20 μl, 2 μM, 5'-FAM, 30mer)に、反応停止剤 (20 μl, 95% formamide, 0.05% BPB)を加えたものを用いた。
[Example 3] Transcription experiment using 2'-O-carbamoyluridine-5'-triphosphate Each template-DNA (2 μl, 5 μM), 10 × T7 RNA Polymerase Buffer (2 μl, 400 mM Tris- HCl (pH 8.0), 80 mM MgCl 2 , 20 mM spermidine), ATP (2 μL, 4 mM), GTP (2 μl, 4 mM), U cm TP (2 μL, 4 mM), CTP (2 μl, 3.2 mM), FAM-CTP (2 μl, 0.8 mM), 50 mM DTT (2 μl), T7 RNA Polymerase or T7 RNA Polymerase TM (100 Unit) in this order, and perform the transcription reaction at 37 ° C for 1 hour. It was. The tube was heated at 75 ° C. for 5 minutes to inactivate the enzyme, cooled in an ice bath for 1 minute, and a reaction terminator (20 μl, 95% formamide, 0.05% BPB) was added. After heating at 95 ° C. for 3 minutes, electrophoresis (charge amount 10 μl, 160 min, 300 V, 30 mA) was performed using 7 M urea 20% polyacrylamide gel. FAM fluorescence was detected using a fluoro image analyzer (FUJIFILM FLA-7000). The Lane 1 marker used was a standard RNA (20 μl, 2 μM, 5′-FAM, 30mer) plus a reaction terminator (20 μl, 95% formamide, 0.05% BPB).
電気移動の結果を図１に示す。また、実験に用いた鋳型DNA及び生成するRNAの配列も図１に示す。この図に示すように、2'-O-カルバモイルウリジン-5'-三リン酸（UcmTP）を用いた場合も、ウリジン-5'-三リン酸（UTP）を用いた場合と同様に、全長転写産物が検出できた。このことから、UcmTPは、T7 RNA Polymeraseの基質として認識されることが確認された。 The result of electromigration is shown in FIG. In addition, the template DNA used in the experiment and the sequence of the generated RNA are also shown in FIG. As shown in this figure, when 2'-O-carbamoyluridine-5'-triphosphate (U cm TP) is used, it is the same as when uridine-5'-triphosphate (UTP) is used. A full-length transcript was detected. From this, it was confirmed that U cm TP is recognized as a substrate of T7 RNA Polymerase.
また、全長転写産物を示すバンドの蛍光強度を数値化したところ、UcmTPはUTPの74％の値を示した（図２）。 Further, when the fluorescence intensity of the band representing the full-length transcript was quantified, U cm TP showed a value of 74% of UTP (FIG. 2).
〔実施例４〕 オリゴヌクレオチドを用いた一塩基伸張逆転写実験
テンプレート RNA (2 μl, 1 μM)、cDNA primer (2 μl, 1 μM)、5×RT Buffer (2 μl)、dNTP (ATP, GTP, CTP) (それぞれ1 μl, 10 mM)、0.1M DTT (0.5 μl)、 SuperScriptIII (0.5 μl, 300 Unit)を42 ℃で30 分間反応させた。そののち、チューブを75 ℃で10分加熱した。氷冷したのち、RNase (0.5U)を加え、37℃で15分間反応させた。そののち、75 ℃で5分間加熱し、氷冷した。反応停止剤 (10 μl, 95% formamide, 0.05% BPB) を加え、95 ℃で3分間加熱し、氷冷し、7 M Urea 20%ポリアクリルアミドゲルを用いて電気泳動 (チャージ量5 μl, 180 min, 300 V, 30 mA)を行った。フルオロイメージアナライザー (FUJIFILM FLA-7000)を用いて、FAMの蛍光を検出した。
[Example 4] Single-base extension reverse transcription experiment using oligonucleotide Template RNA (2 μl, 1 μM), cDNA primer (2 μl, 1 μM), 5 × RT Buffer (2 μl), dNTP (ATP, GTP) , CTP) (1 μl, 10 mM, respectively), 0.1 M DTT (0.5 μl), and SuperScript III (0.5 μl, 300 Unit) at 42 ° C. for 30 minutes. The tube was then heated at 75 ° C. for 10 minutes. After cooling with ice, RNase (0.5 U) was added and reacted at 37 ° C. for 15 minutes. Thereafter, the mixture was heated at 75 ° C. for 5 minutes and cooled on ice. Add a reaction terminator (10 μl, 95% formamide, 0.05% BPB), heat at 95 ° C for 3 minutes, cool on ice, and perform electrophoresis using 7 M Urea 20% polyacrylamide gel (charge amount 5 μl, 180% min, 300 V, 30 mA). FAM fluorescence was detected using a fluoro image analyzer (FUJIFILM FLA-7000).
電気移動の結果を図３に示す。この図に示すように、鋳型とするRNAが2'-O-カルバモイルウリジンを含む場合（X=Ucm）も、ウリジンを含む場合（X=U）と同様に、一塩基鎖伸長バンドは、dGTPを加えたときは検出されず、dATPを加えたときにのみ検出された。このことから、dGTPは取り込まれず、dATPのみが特異的にcDNAに取り込まれることが確認された。 The result of electromigration is shown in FIG. As shown in this figure, when the RNA used as the template contains 2'-O-carbamoyluridine (X = U cm ), as in the case of containing uridine (X = U), the single-base chain extension band is It was not detected when dGTP was added, but only when dATP was added. From this, it was confirmed that dGTP was not incorporated and only dATP was specifically incorporated into cDNA.
〔実施例５〕 オリゴヌクレオチドを用いた完全長逆転写実験
テンプレート RNA (2 μl, 1 μM)、cDNA primer (2 μl, 1 μM)、5×RT Buffer (2 μl)、dNTPs (1 μl, 10 mM)、滅菌水 (2 μl)、0.1M DTT (0.5 μl)、SuperScriptIII (0.5 μl, 300 Unit)を45 ℃でそれぞれ上記の時間反応させた。そののち、チューブを75 ℃で10分加熱した。氷冷したのち、RNase (0.5U)を加え、37℃で15分間反応させた。そののち、75 ℃で5分間加熱し、氷冷した。反応停止剤 (20 μl, 95% formamide, 0.05% BPB)を加え、95 ℃で3分間加熱し、氷冷し、7 M Urea 20%ポリアクリルアミドゲルを用いて電気泳動 (チャージ量5 μl, 180 min, 300 V, 30 mA)を行った。フルオロイメージアナライザー (FUJIFILM FLA-7000)を用いて、FAMの蛍光を検出した。
[Example 5] Full-length reverse transcription experiment using oligonucleotide Template RNA (2 μl, 1 μM), cDNA primer (2 μl, 1 μM), 5 × RT Buffer (2 μl), dNTPs (1 μl, 10 mM), sterilized water (2 μl), 0.1M DTT (0.5 μl), and SuperScript III (0.5 μl, 300 Unit) were each reacted at 45 ° C. for the above times. The tube was then heated at 75 ° C. for 10 minutes. After cooling with ice, RNase (0.5 U) was added and reacted at 37 ° C. for 15 minutes. Thereafter, the mixture was heated at 75 ° C. for 5 minutes and cooled on ice. Add a reaction terminator (20 μl, 95% formamide, 0.05% BPB), heat at 95 ° C for 3 minutes, cool on ice, and perform electrophoresis using 7 M Urea 20% polyacrylamide gel (charge amount 5 μl, 180% min, 300 V, 30 mA). FAM fluorescence was detected using a fluoro image analyzer (FUJIFILM FLA-7000).
電気移動の結果を図４に示す。この図に示すように、鋳型とするRNAが2'-O-カルバモイルウリジンを含む場合（X=Ucm）も、ウリジンを含む場合（X=U）と同様に、完全鎖伸長バンドが検出された。このことから、Ucmを含む鋳型鎖は逆転写酵素により精度よく逆転写されることが確認された。 The result of electromigration is shown in FIG. As shown in this figure, when the template RNA contains 2'-O-carbamoyluridine (X = U cm ), the full-strand extension band is detected as in the case of uridine (X = U). It was. From this, it was confirmed that the template strand containing U cm is accurately reverse transcribed by reverse transcriptase.
〔実施例６〕 酵素耐性試験
蛇毒ホスホジエステラーゼ (SVPDE) (40 μU) を、オリゴヌクレオチド (8 nmol) を溶かした50 mMトリス-塩酸緩衝液 (72 mM塩化ナトリウム、14 mM塩化マグネシウム、pH 7.0) に対し加えた。加水分解反応は37 ℃で行った。それぞれの時間において反応系から20 μL混合液を取り出し、100 ℃で3分間加熱して反応を停止させた後、CENTRICUTを用いて限外ろ過した。ろ液を逆相HPLCにより解析し、得られたピークの面積比から原料であるオリゴヌクレオチドの存在比を算出した。
[Example 6] Enzyme resistance test Snake venom phosphodiesterase (SVPDE) (40 μU) was added to 50 mM Tris-HCl buffer solution (72 mM sodium chloride, 14 mM magnesium chloride, pH 7.0) in which oligonucleotide (8 nmol) was dissolved. Added. The hydrolysis reaction was carried out at 37 ° C. At each time, 20 μL of the mixed solution was taken out from the reaction system, heated at 100 ° C. for 3 minutes to stop the reaction, and then ultrafiltered using CENTRICUT. The filtrate was analyzed by reverse-phase HPLC, and the abundance ratio of the oligonucleotide as a raw material was calculated from the area ratio of the obtained peaks.
この結果を図５に示す。この図に示すように、2'-O-カルバモイル修飾オリゴヌクレオチド（5'-UcmUcm-T）は、2'-O-メチル修飾オリゴヌクレオチド（5'-UOMeUOMe-T）と同等の蛇毒ホスホジエステラーゼ耐性を有していた。 The result is shown in FIG. As shown in this figure, 2'-O-carbamoyl modified oligonucleotide (5'-U cm U cm -T) and 2'-O-methyl modified oligonucleotide (5'-U OMe U OMe -T) Has equivalent snake venom phosphodiesterase resistance.
Claims (11)
で表されるヌクレオシド三リン酸誘導体。 Formula (I):
A nucleoside triphosphate derivative represented by:
で表されるヌクレオシド三リン酸誘導体から弱酸性条件下でＲ３を脱離させる工程を含む請求項１乃至４のいずれか一項に記載のヌクレオシド三リン酸誘導体の製造方法。 General formula (II):
Method for producing a nucleoside triphosphate derivative according to any one of claims 1 to 4 comprising the step of nucleoside three to phosphoric acid derivative under mildly acidic conditions keep an R 3 de expressed in.
で示される基を表し、Ｙ２はｎの繰り返しにおいて異なっていてもよく、ｎは１以上の整数を表す。但し、Ｙ１、Ｙ２、及びＹ３の少なくとも一つは一般式（VI）で示される基を表す。〕
で表されるRNAアプタマー。 Formula (V):
Y 2 may be different in the repetition of n, and n represents an integer of 1 or more. However, at least one of Y 1 , Y 2 , and Y 3 represents a group represented by the general formula (VI). ]
RNA aptamer represented by
（１）逆転写酵素をRNAの集団又はRNA誘導体の集団（このRNA誘導体の集団は、工程（４）で選択されたRNA誘導体の集団であってもよい。）に作用させ、DNAの集団を合成する工程、
（２）工程（１）で合成されたDNAの集団を、ポリメラーゼ連鎖反応により増幅させる工程、
（３）請求項１乃至４のいずれか一項に記載のヌクレオシド三リン酸誘導体の存在下で、工程（２）で増幅されたDNAの集団にDNA依存RNAポリメラーゼを作用させ、前記ヌクレオシド三リン酸誘導体を残基として含むRNA誘導体の集団を合成する工程、
（４）工程（３）で合成したRNA誘導体の集団を標的物質と接触させ、標的物質に親和性を示すRNA誘導体の集団を選択する工程。 A method for producing an RNA derivative, characterized by comprising the following steps:
(1) A reverse transcriptase is allowed to act on a population of RNA or a population of RNA derivatives (this population of RNA derivatives may be a population of RNA derivatives selected in step (4)). The step of synthesizing,
(2) Amplifying the population of DNA synthesized in step (1) by polymerase chain reaction;
(3) In the presence of the nucleoside triphosphate derivative according to any one of claims 1 to 4, a DNA-dependent RNA polymerase is allowed to act on the population of DNA amplified in step (2), whereby the nucleoside triphosphate Synthesizing a population of RNA derivatives containing acid derivatives as residues,
(4) A step of contacting the population of RNA derivatives synthesized in step (3) with a target substance and selecting a population of RNA derivatives showing affinity for the target substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013152578A JP6261027B2 (en) | 2013-07-23 | 2013-07-23 | 2'-O-carbamoyl modified nucleoside triphosphates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013152578A JP6261027B2 (en) | 2013-07-23 | 2013-07-23 | 2'-O-carbamoyl modified nucleoside triphosphates |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015020994A true JP2015020994A (en) | 2015-02-02 |
| JP6261027B2 JP6261027B2 (en) | 2018-01-17 |
Family
ID=52485747
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013152578A Expired - Fee Related JP6261027B2 (en) | 2013-07-23 | 2013-07-23 | 2'-O-carbamoyl modified nucleoside triphosphates |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6261027B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019044974A1 (en) | 2017-08-31 | 2019-03-07 | 株式会社Ｖｅｒｉｔａｓ Ｉｎ Ｓｉｌｉｃｏ | Small guide antisense nucleic acid and use thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5643889A (en) * | 1984-07-11 | 1997-07-01 | Temple University-Of The Commonwealth System Of Pennsylvania | Cholesterol conjugates of 2'5'-oligoadenylate derivatives and antiviral uses thereof |
| JP2002322192A (en) * | 2000-08-10 | 2002-11-08 | Sankyo Co Ltd | 2'-o,4'-crosslinked nucleoside triphosphate |
| JP2008526877A (en) * | 2005-01-05 | 2008-07-24 | エージェンコート パーソナル ジェノミクス | Reversible nucleotide terminator and use thereof |
-
2013
- 2013-07-23 JP JP2013152578A patent/JP6261027B2/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5643889A (en) * | 1984-07-11 | 1997-07-01 | Temple University-Of The Commonwealth System Of Pennsylvania | Cholesterol conjugates of 2'5'-oligoadenylate derivatives and antiviral uses thereof |
| JP2002322192A (en) * | 2000-08-10 | 2002-11-08 | Sankyo Co Ltd | 2'-o,4'-crosslinked nucleoside triphosphate |
| JP2008526877A (en) * | 2005-01-05 | 2008-07-24 | エージェンコート パーソナル ジェノミクス | Reversible nucleotide terminator and use thereof |
Non-Patent Citations (9)
| Title |
|---|
| BIOCHEMISTRY, vol. 31, JPN6017009648, 1992, pages 9636 - 9641, ISSN: 0003523025 * |
| BIOORGANIC & MEDICINAL CHEMISTRY, vol. 17, JPN6017009655, 2009, pages 7275 - 7280, ISSN: 0003523030 * |
| CSJ CURRENT REVIEW, JPN6017009649, 2011, pages 78 - 85, ISSN: 0003523026 * |
| DISSERTATION ABSTRACTS INTERNATIONAL B, vol. 50, no. 4, JPN6017009644, 1989, pages 1211, ISSN: 0003523023 * |
| NUCLEIC ACIDS RESEARCH, vol. 27, no. 6, JPN6017009646, 1999, pages 1561 - 1563, ISSN: 0003523024 * |
| 日本化学会講演予稿集, vol. 85, no. 2, JPN6017009654, 2005, pages 1393 - 1, ISSN: 0003523029 * |
| 日本化学会講演予稿集, vol. 86, no. 2, JPN6017009651, 2006, pages 860 - 2, ISSN: 0003523027 * |
| 日本化学会講演予稿集, vol. 88, no. 2, JPN6017009652, 2008, pages 1531 - 3, ISSN: 0003523028 * |
| 社団法人日本化学会編, 化学便覧 応用化学編 第６版, JPN6017009657, 2003, pages 1543 - 1545, ISSN: 0003523031 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019044974A1 (en) | 2017-08-31 | 2019-03-07 | 株式会社Ｖｅｒｉｔａｓ Ｉｎ Ｓｉｌｉｃｏ | Small guide antisense nucleic acid and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6261027B2 (en) | 2018-01-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5689054B2 (en) | RNA chemical synthesis method | |
| JP5157168B2 (en) | Method for producing phosphoramidite compound and oligo RNA | |
| JPH10501809A (en) | Novel method for producing known and novel 2'-nucleosides by intramolecular nucleophilic substitution | |
| WO2010110775A1 (en) | Reagents for reversibly terminating primer extension | |
| JP3893057B2 (en) | Novel nucleic acid base pair | |
| EP1995253B1 (en) | Method for detaching protecting group on nucleic acid | |
| WO2019021945A1 (en) | Photoresponsive nucleotide analog capable of photocrosslinking in visible light region | |
| WO2021020562A1 (en) | Primer, device for producing double-stranded dna using same, and method for producing double-stranded dna | |
| JP6261027B2 (en) | 2'-O-carbamoyl modified nucleoside triphosphates | |
| EP2017282A1 (en) | Method of capping oligonucleic acid | |
| EP1995252A1 (en) | Method for removal of nucleic acid-protecting group | |
| JP4802712B2 (en) | Solution phase synthesis of ribonucleic acid compounds and oligonucleic acid compounds | |
| JP5076504B2 (en) | Amidite for nucleic acid synthesis and nucleic acid synthesis method | |
| Saneyoshi et al. | Chemical synthesis of RNA via 2′-O-cyanoethylated intermediates | |
| JP2009221168A (en) | Nucleoside or nucleotide derivative, nucleic acid derivative, target substance-capturing material, method for capturing and/or releasing target substance, and hemin aptamer | |
| JP4665758B2 (en) | Thionucleoside-S-nitrosyl derivative | |
| EP2053054A1 (en) | Method for introducing nucleic-acid-protecting group | |
| WO2004018494A1 (en) | 4'-thionucleotide | |
| WO2021020561A1 (en) | Primer, apparatus for producing double-stranded dna using same, and method for producing double-stranded dna | |
| WO2023282245A1 (en) | Method for purifying nucleotides, device for purifying nucleotides, hydrophobic reagent, and hydrophobic substrate | |
| RU2415862C2 (en) | Phosphoramidite derivative and method of producing rna oligo | |
| WO2019150564A1 (en) | Dna replication method using oligonucleotide having sulfonamide skeleton as template | |
| Mori et al. | Synthesis and hybridization property of a boat-shaped pyranosyl nucleic acid containing an exocyclic methylene group in the sugar moiety | |
| JP5425780B2 (en) | Photoresponsive nucleotide molecule and method for producing photoresponsive modified nucleic acids using the photoresponsive nucleotide molecule | |
| WO1994001445A1 (en) | Base-protected nucleotide analogs with protected thiol groups |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20160223 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20160223 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20170328 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170519 |
|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20170519 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20170525 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170519 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170721 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170721 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20171205 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20171207 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6261027 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |