JP4015136B2 - Deoxyribose derivatives having a phenol skeleton and photoresponsive nucleotides - Google Patents
Deoxyribose derivatives having a phenol skeleton and photoresponsive nucleotides Download PDFInfo
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Description
この出願の発明は、光応答性ヌクレオチドに関する。さらに詳しくは、この出願の発明は、アンチセンスＤＮＡ、アンチジーンＤＮＡ、光遺伝子診断治療、ＤＮＡ−蛋白質相互作用の解析等への応用に有用で、光照射により核酸に対し選択的にクロスリンクし、核酸を選択的にラベル化することが可能な光応答性ヌクレオチドに関する。 The invention of this application relates to photoresponsive nucleotides. More specifically, the invention of this application is useful for applications such as antisense DNA, antigene DNA, photogene diagnostic treatment, and analysis of DNA-protein interactions, and selectively crosslinks to nucleic acids by light irradiation. , Relates to a light-responsive nucleotide capable of selectively labeling a nucleic acid.
ＤＮＡ、ＲＮＡ、ＰＮＡ等の核酸の相互における核酸同士のクロスリンク（架橋反応）を特異的に制御することは、遺伝子機能の解析、各種の生理活性物質や生体分子の作用機構の解明など分子遺伝学分野において重要である。さらに、新規な生理活性物質の探索や創製、具体的にはアンチセンスＤＮＡ、アンチジーンＤＮＡ、遺伝子診断治療、すなわち遺伝子発現の制御にかかる分子医薬分野おいても極めて重要である。
現在までのところ、核酸に対するクロスリンクを行う手段としては、ソラレン誘導体（Chang, E. et al. Biochemistry 1991, 30, 8283）やアミノプリン誘導体（特開2001-206896）が知られている。しかしながら、前者は５´−ＡＴ−３´である塩基配列のチミン特異的に反応し、後者は配列依存性はないもののシチジン特異的であるなど、適用範囲に一定の制約があった。
Specific control of the cross-linking (cross-linking reaction) between nucleic acids such as DNA, RNA, PNA, etc. means that molecular inheritance such as analysis of gene function, elucidation of action mechanism of various physiologically active substances and biomolecules Important in academic fields. Furthermore, it is extremely important in the field of molecular medicine related to the search and creation of new physiologically active substances, specifically, antisense DNA, antigene DNA, gene diagnostic therapy, that is, control of gene expression.
To date, psoralen derivatives (Chang, E. et al. Biochemistry 1991, 30, 8283) and aminopurine derivatives (Japanese Patent Laid-Open No. 2001-206896) are known as means for cross-linking nucleic acids. However, there are certain restrictions on the application range, such as the former reacts specifically with thymine of the base sequence 5′-AT-3 ′, and the latter is cytidine-specific although it has no sequence dependency.
この出願の発明は、上記の従来技術にかかる核酸架橋反応試薬における問題点を鑑み、塩基配列依存性を持たず、かつ、従来技術においては不可能であったアデニン特異的なクロスリンク反応を生成することが可能なデオキシリボース誘導体および、それを利用した光応答性ヌクレオチドを提供することを課題とする。 The invention of this application generates an adenine-specific cross-linking reaction that has no base sequence dependency and has been impossible in the prior art in view of the problems in the nucleic acid crosslinking reaction reagent according to the above-described prior art. It is an object of the present invention to provide a deoxyribose derivative that can be produced and a photoresponsive nucleotide utilizing the same.
この出願の発明は上記の課題を解決するために下記の手段を提供する。すなわち、この出願の発明は、第一に、一般式（１） The invention of this application provides the following means in order to solve the above problems. That is, the invention of this application is firstly a general formula (1)
〔Ｘは水素原子、ジメトキシトリチル基、リン酸基または１塩基以上のヌクレオチド鎖を示し、Ｙは水素原子、次式（２）のフォスフォロアミダイト基 [X represents a hydrogen atom, a dimethoxytrityl group, a phosphate group or a nucleotide chain of one or more bases, Y represents a hydrogen atom, a phosphoramidite group of the following formula (2)
（Ａは結合部位を示す。）、リン酸基または１塩基以上のヌクレオチド鎖を示し、Ｒは−ＣＯＯＨまたは、−ＣＯＯＣＨ 3 を示す。〕であるフェノール骨格を有するデオキシリボース誘導体を提供する。
(A represents a binding site), a phosphate group or a nucleotide chain of one or more bases, and R represents —COOH or —COOCH 3 . And a deoxyribose derivative having a phenol skeleton.
さらに、この出願の発明は、第２には前記Ｙが式２に示すフォスフォロアミダイト基である前記デオキシリボース誘導体を含むことを特徴とする核酸合成用アミダイト試薬を、第３には、前記Ｘおよび前記Ｙそれぞれに１塩基以上のヌクレオチド鎖が結合されていることを特徴とする前記デオキシリボース誘導体からなる光応答性ヌクレオチドを提供する。そして第４には、前記Ｘまたは前記Ｙのいずれか一方に１塩基以上のヌクレオチド鎖が結合されていることを特徴とする前記デオキシリボース誘導体からなる光応答性ヌクレオチドを提供する。 Furthermore, the invention of this application is characterized in that secondly, the amidite reagent for nucleic acid synthesis is characterized in that it contains the deoxyribose derivative, wherein Y is a phosphoramidite group represented by Formula 2, and thirdly, the X And a photoresponsive nucleotide comprising the deoxyribose derivative, wherein a nucleotide chain of one base or more is bound to each Y. Fourthly, a photoresponsive nucleotide comprising the deoxyribose derivative, wherein one or more nucleotide chains are bound to either X or Y, is provided.
加えて、この出願の発明は、第５には、前記核酸合成用アミダイト試薬を使用することを特徴とする前記光応答性ヌクレオチドの合成方法、ならびに第６には、前記光応答性ヌクレオチドを含む核酸のクロスリンク試薬を提供する。 In addition, the invention of this application includes, in the fifth aspect, the method for synthesizing the photoresponsive nucleotide, wherein the amidite reagent for nucleic acid synthesis is used, and sixth, the photoresponsive nucleotide. Nucleic acid cross-linking reagents are provided.
以上詳しく説明したとおり、この出願の発明によって、塩基配列依存性を持たず、かつ、従来技術においては不可能であったアデニン特異的なクロスリンク反応を生成することが可能なデオキシリボース誘導体および、それを利用した光応答性ヌクレオチドを提供する。この出願の発明は、各種の生理活性物質や生体分子の作用機構の解明、新規な生理活性物質の探索や創製、遺伝子の機能解析や発現制御おいての研究開発や実施利用への応用によるそれらの発展に寄与するものとなる。 As described above in detail, according to the invention of this application, a deoxyribose derivative that has no base sequence dependency and can generate an adenine-specific cross-linking reaction that was impossible in the prior art, and A photoresponsive nucleotide utilizing the same is provided. The invention of this application is based on elucidation of the mechanism of action of various physiologically active substances and biomolecules, search and creation of novel physiologically active substances, gene function analysis and application to research and development and practical use in expression control. It will contribute to the development of.
この出願の発明は上記のとおりの特徴をもつものであるが、以下にその実施の形態について説明する。 The invention of this application has the features as described above, and an embodiment thereof will be described below.
前記式（１）に示すこの出願の発明の提供するデオキシリボース誘導体は、２−デオキシリボースの１位Ｃに、フェノキシ基がα位で結合しており、このフェノキシ基の３位Ｃにはフェノール骨格に対しＥ配置で置換基Ｒを有するビニル基が付加されている特徴のある構造を有している。Ｒとしては、−ＣＯＯＨまたは、−ＣＯＯＣＨ 3 を用いることができる。この構造における２−ビニルフェノキシ基および置換基Ｒの存在が、核酸との架橋反応を光制御可能とせしめている。 The deoxyribose derivative provided by the invention of this application represented by the above formula (1) has a phenoxy group bonded to the 1-position C of 2-deoxyribose at the α-position, and the 3-position C of this phenoxy group has a phenol. It has a characteristic structure in which a vinyl group having a substituent R in E configuration is added to the skeleton. The R, -COOH or Ru can be used -COOCH 3. The presence of 2-vinyl phenoxy groups and substituents R in the structure of this has a cross-linking reaction with a nucleic acid made to allow the light control.
この出願の発明の提供するデオキシリボース誘導体（式１）は、２−デオキシリボースの５位Ｃに結合したＯと結合するＸが、水素原子であってもよく、アセチル基、４，４'，４''−トリス（４−ベンゾイルオキシ）トリチル基、ジメトキシトリチル基などの適当な保護基を導入してもよく、保護基を導入する場合、好ましくは容易で効率的に除去可能であるジメトキシトリチル基を用いる。また、３位Ｃに結合したＯと結合するＹは、水素原子あるいは式（２）のフォスフォロアミダイト基である。 In the deoxyribose derivative (formula 1) provided by the invention of this application, X bonded to O bonded to 5-position C of 2-deoxyribose may be a hydrogen atom, an acetyl group, 4, 4 ′, A suitable protecting group such as a 4 ″ -tris (4-benzoyloxy) trityl group or a dimethoxytrityl group may be introduced. When a protecting group is introduced, dimethoxytrityl is preferably easily and efficiently removable. Use groups. Y bonded to O bonded to the 3-position C is a hydrogen atom or a phosphoramidite group of the formula (2).
この出願の発明の提供する光応答性ヌクレオチドは、前記式（１）のデオキシリボース誘導体のＹを式（２）のフォスフォロアミダイト基としたデオキシリボース誘導体を含むアミダイド試薬により、フォスフォロアミダイト法によるＤＮＡ合成により提供され、前記式（１）のデオキシリボース誘導体のＸおよびＹのいずれか一方、あるいは双方に１塩基以上のヌクレオチド鎖、すなわちＤＮＡ、ＲＮＡあるいはＰＮＡのヌクレオチド鎖を結合したものである。また、前記デオキシリボース誘導体のＸおよびＹは、これらヌクレオチド鎖とのフォスフォジエステル結合形成を目的としてリン酸基によって修飾されていてもよい。 The photoresponsive nucleotide provided by the invention of this application is obtained by a phosphoramidite method using an amidide reagent containing a deoxyribose derivative in which Y of the deoxyribose derivative of formula (1) is a phosphoramidite group of formula (2). Provided by DNA synthesis and having one or more nucleotide chains, that is, DNA, RNA or PNA nucleotide chains bound to either or both of X and Y of the deoxyribose derivative of formula (1). Further, X and Y of the deoxyribose derivative may be modified with a phosphate group for the purpose of forming a phosphodiester bond with these nucleotide chains.
また、この出願の発明の提供する光応答性ヌクレオチドは、前記デオキシリボース誘導体を含む機能性核酸と呼ぶこともできる。ベンゼン環やヌクレオシド部分に許容される他の置換基を有していてもよい。 The photoresponsive nucleotide provided by the invention of this application can also be called a functional nucleic acid containing the deoxyribose derivative. It may have other substituents allowed in the benzene ring or nucleoside moiety.
以下、この出願の発明の提供するデオキシリボース誘導体の作成方法を説明する。 Hereinafter, a method for producing a deoxyribose derivative provided by the invention of this application will be described.
この出願の発明の提供する式（１）に示すデオキシリボース誘導体の一態様であって、Ｘ、Ｙともに水素原子、すなわち２−デオキシリボースの３位および５位がともにヒドロキシル基あって、Ｒがメチルカルボニル基である式（７）に示す（Ｅ）−１−α−（３´−（メトキシカルボニルビニル）フェノキシ）−２−デオキシリボースは、化５に示す経路によって、式（３）に示すm-iodophenol、および式（４）に示すChlorosugarより、式（４）、式（５）および式（６）に示す反応中間体を経由することによって合成することができる。 In one embodiment of the deoxyribose derivative represented by the formula (1) provided by the invention of this application, both X and Y are hydrogen atoms, that is, hydroxyl groups are present at both the 3-position and 5-position of 2-deoxyribose, and R is (E) -1-α- (3 ′-(methoxycarbonylvinyl) phenoxy) -2-deoxyribose represented by the formula (7), which is a methylcarbonyl group, is represented by the formula (3) according to the route represented by Chemical Formula 5. From m-iodophenol and Chlorosugar shown in Formula (4), it can synthesize | combine via the reaction intermediate shown in Formula (4), Formula (5), and Formula (6).
また、この出願の発明の提供する式（１）のデオキシリボース誘導体の一態様であって、Ｘがジメトキシトリチル基、Ｙが水素原子である式（８）に示す（Ｅ）−１−α−（３´−（メトキシカルボニルビニル）フェノキシ）−２−デオキシ―５−Ｏ−（４，４´−ジメトキシトリチル）―リボース、および別の態様である、Ｘがジメトキシトリチル基、Ｙが式（２）のフォスフォロアミダイトである式（９）に示す（Ｅ）−１−α−（３´−（メトキシカルボニルビニル）フェノキシ）−２−デオキシ―５−Ｏ−ジメトキシトリチル―リボシド−３−Ｏ−（シアノエトキシ−Ｎ，Ｎ−ジイソプロピル）フォスフォロアミダイトは、それぞれ化５に示す経路によって、式（７）で示す（Ｅ）−１−α−（３´−（メトキシカルボニルビニル）フェノキシ）−２−デオキシリボースより合成することができる。 Further, an embodiment of the deoxyribose derivative of the formula (1) provided by the invention of this application, wherein X is a dimethoxytrityl group and Y is a hydrogen atom (E) -1-α- (3 '-(methoxycarbonylvinyl) phenoxy) -2-deoxy-5-O- (4,4'-dimethoxytrityl) -ribose, and another embodiment, X is a dimethoxytrityl group, Y is a compound of formula (2 (E) -1-α- (3 ′-(methoxycarbonylvinyl) phenoxy) -2-deoxy-5-O-dimethoxytrityl-riboside-3-O— represented by formula (9) which is a phosphoramidite of (Cyanoethoxy-N, N-diisopropyl) phosphoramidite is converted into (E) -1-α- (3 ′-(methoxycarbonylvinyl) phenol represented by the formula (7) by the route shown in Chemical Formula 5 respectively. Alkoxy) can be synthesized from 2-deoxyribose.
前記式（９）のフォスフォロアミダイトは核酸合成用アミダイト試薬としてＤＮＡ合成およびＲＮＡ合成に用いることができる。 The phosphoramidite of the formula (9) can be used as amidite reagent for nucleic acid synthesis in DNA synthesis and RNA synthesis.
この出願の発明の提供する光応答性ヌクレオチドは、２塩基以上１００塩基未満のオリゴヌクレオチドあるいは１００塩基以上のポリヌクレオチドからなるＤＮＡあるいはＲＮＡであり、これより誘導されるＰＮＡも含まれる。この光応答性ヌクレオチドは、前記式（９）のフォスフォロアミダイトを用いフォスフォロアミダイト法によって合成することが可能であり、この合成核酸をもとに公知の方法あるいはこの出願の発明の提供する方法によって切断、あるいはさらに他のＤＮＡ、ＲＮＡ、タンパク質等の生体分子と結合、またはこれらの結合・切断方法を組み合わせて作成されたＤＮＡ、ＲＮＡあるいはＰＮＡであってもよい。この出願の発明の提供する光応答性ヌクレオチドは、各種の分析や診断を目的として公知の方法により放射性同位体、蛍光物質の結合などによって化学修飾されたヌクレオチドであってもよい。 The photoresponsive nucleotide provided by the invention of this application is DNA or RNA comprising an oligonucleotide of 2 to 100 bases or a polynucleotide of 100 or more bases, and includes PNA derived therefrom. This photoresponsive nucleotide can be synthesized by the phosphoramidite method using the phosphoramidite of the formula (9), and a known method based on this synthetic nucleic acid or a method provided by the invention of this application Alternatively, it may be DNA, RNA, or PNA that is prepared by cleaving or binding to other biomolecules such as DNA, RNA, or protein, or by combining these cleaving and cleaving methods. The photoresponsive nucleotide provided by the invention of this application may be a nucleotide chemically modified by binding of a radioisotope or a fluorescent substance by known methods for the purpose of various analyzes and diagnosis.
この出願の発明の提供する光応答性ヌクレオチドをもちいた光架橋反応は、架橋形成対象の核酸の共存下に光照射することで可能とされる。具体的には、たとえば３６６ｎｍの光励起により光架橋反応を生じさせることができる。これら光の照射の方法としては各種の手段から選択され、たとえばトランスイルミネータを用いることができる。 The photocrosslinking reaction using the photoresponsive nucleotide provided by the invention of this application is made possible by irradiating with light in the presence of the nucleic acid to be crosslinked. Specifically, for example, a photocrosslinking reaction can be caused by photoexcitation at 366 nm. These light irradiation methods are selected from various means. For example, a transilluminator can be used.
以下に実施例を用いてこの出願の発明をさらに詳細に説明するが、下記実施例はこの出願の発明の一態様にすぎず、この出願の発明の実施が下記実施例に制限されるものではない。 The invention of this application will be described in more detail below using examples, but the following examples are only one aspect of the invention of this application, and the implementation of the invention of this application is not limited to the following examples. Absent.
〔実施例１〕＜反応中間体の合成１＞
式（３）に示すm-iodophenol (1.04 g, 4.74 mmol)のTHF溶液 (80 mL)に0 °CでNaH (0.20 g, 5.00 mmol)と式（４）に示すChlorosugar（2-deoxy-3,5-di-O-(p-toluoyl)-α- D -ribofuranosyl chloride） (2.14 g, 5.00 mmol)を順に加えて10分間撹拌した。反応溶液を室温で19時間撹拌し、TLC (CHCl3)で原料の消失を確認した。溶媒を除去した後、カラムクロマトグラフィー (CHCl3)で精製し、無色油状の生成物１(1.97 g, 73%)を得た。この生成物１は下記の質量分析値およびNMR分析結果より、式（５）に示す反応中間体、すなわち1-α-(m-iodo)phenoxy-2-deoxy-3,5-di-O-(p-toluoyl)-riboseと同定された。
[Example 1] <Synthesis of reaction intermediate 1>
A THF solution (80 mL) of m-iodophenol (1.04 g, 4.74 mmol) represented by formula (3) and NaH (0.20 g, 5.00 mmol) and Chlorosugar (2-deoxy-3) represented by formula (4) at 0 ° C. , 5-di-O- (p-toluoyl) -α-D-ribofuranosyl chloride) (2.14 g, 5.00 mmol) was sequentially added and stirred for 10 minutes. The reaction solution was stirred at room temperature for 19 hours, and disappearance of raw materials was confirmed by TLC (CHCl 3 ). After removing the solvent, the residue was purified by column chromatography (CHCl 3 ) to obtain a colorless oily product 1 (1.97 g, 73%). This product 1 was obtained from the following mass spectrometric value and NMR analysis result, and the reaction intermediate represented by the formula (5), that is, 1-α- (m-iodo) phenoxy-2-deoxy-3,5-di-O— It was identified as (p-toluoyl) -ribose.
1H NMR (CDCl3) d 7.91 (d, 2H, J = 8.1 Hz), 7.85 (d, 2H, J = 8.1 Hz), 7.34 (t, 1H, J = 2.4 Hz), 7.28 (dt, 1H, J = 6.9, 1.8 Hz), 7.23 (d, 2H, J = 8.1 Hz), 7.18 (d, 2H, J = 8.1 Hz), 6.90-6.98 (m, 2H), 5.92 (dd, 1H, J = 5.9, 2.7 Hz), 5.67-5.72 (m, 1H), 4.59-4.63 (m, 1H), 4.54-4.59 (m, 1H), 4.40 (dd, 1H, J = 11.6, 3.4 Hz), 2.82 (ddd, 1H, J = 14.5, 6.9, 2.4 Hz), 2.53 (ddd, 1H, J = 14.5, 5.3, 5.1 Hz), 2.40 (s, 3H), 2.38 (s, 3H);
HRMS (ESI) cald. for C27H25IO6Na (MNa+): 595.0588, found: 595.0531.
〔実施例２〕＜反応中間体の合成２＞
前記実施例１の生成物１(1.96 g, 3.43 mmol；式（５）)のdichloromethane溶液(100 mL) に0.5 M methanolic NaOMe (20 mL, 10 mmol)を加えて、反応溶液を45 °Cで12時間撹拌した。TLC (CHCl3 : MeOH = 98 : 2)で原料の消失を確認した。溶媒を除去した後、カラムクロマトグラフィー(CHCl3 : MeOH = 98 : 2)で精製し、白色粉末として生成物２ (0.77 g, 67%)を得た。この生成物２は下記の質量分析値およびNMR分析結果より、式（６）に示す反応中間体、すなわち1-α-(m-iodo)phenoxy-2-deoxyriboseと同定された。
1H NMR (CDCl3) d 7.37-7.38 (m, 1H), 7.31-7.34 (m, 1H), 6.96-6.98 (m, 2H), 5.83 (dd, 1H, J = 5.6, 1.8 Hz), 4.60-4.66 (m, 1H), 4.09 (dd, 1H, J = 8.7, 4.5 Hz), 3.60-3.76 (m, 2H), 2.52 (ddd, 1H, J = 13.8, 6.9, 1.8 Hz), 2.30 (dt, 1H, J = 13.8, 6.0 Hz), 1.89-1.95 (m, 2H);
HRMS (ESI) cald. for C11H13IO4Na (MNa+): 358.9751, found: 358.9729.
〔実施例３〕＜デオキシリボース誘導体の合成＞
triphenylphosphine (87 mg, 0.3 μmol)のジオキサン溶液(15 mL)にpalladium acetate (25 mg, 0.1 μmol)とtriethylamine (0.37μL, 2+.64 mmol)を順に加えて、75°Cで5分間撹拌した。前記実施例２の生成物２(0.74 g, 2.20 mmol)とmethyl acrylate (0.40 μL, 4.40 mmol)を加えて、反応溶液を2時間環流した。TLC (CHCl3 : MeOH = 9 : 1)で生成物を確認した後、綿ろ過でpalladium粉末を取り除いた。カラムクロマトグラフィー(CHCl3 : MeOH = 95 : 5)で精製し、黄色油状物質として生成物３ (0.6 g, 93%)を得た。生成物３はNOESY実験（図１）によってα体と同定され、また下記の質量および紫外吸光度分析値およびNMR分析結果より、式（７）に示すデオキシリボース誘導体、すなわち(E)-1-α-(m-metoxycarbonylvinyl)phenoxy-2-deoxyriboseと同定された。
1 H NMR (CDCl 3 ) d 7.91 (d, 2H, J = 8.1 Hz), 7.85 (d, 2H, J = 8.1 Hz), 7.34 (t, 1H, J = 2.4 Hz), 7.28 (dt, 1H, J = 6.9, 1.8 Hz), 7.23 (d, 2H, J = 8.1 Hz), 7.18 (d, 2H, J = 8.1 Hz), 6.90-6.98 (m, 2H), 5.92 (dd, 1H, J = 5.9 , 2.7 Hz), 5.67-5.72 (m, 1H), 4.59-4.63 (m, 1H), 4.54-4.59 (m, 1H), 4.40 (dd, 1H, J = 11.6, 3.4 Hz), 2.82 (ddd, 1H, J = 14.5, 6.9, 2.4 Hz), 2.53 (ddd, 1H, J = 14.5, 5.3, 5.1 Hz), 2.40 (s, 3H), 2.38 (s, 3H);
HRMS (ESI) cald.for C 27 H 25 IO 6 Na (MNa + ): 595.0588, found: 595.0531.
[Example 2] <Synthesis 2 of reaction intermediate>
0.5 M methanolic NaOMe (20 mL, 10 mmol) was added to a dichloromethane solution (100 mL) of the product 1 of Example 1 (1.96 g, 3.43 mmol; formula (5)), and the reaction solution was heated at 45 ° C. Stir for 12 hours. The disappearance of the raw materials was confirmed by TLC (CHCl 3 : MeOH = 98: 2). After removing the solvent, the residue was purified by column chromatography (CHCl 3 : MeOH = 98: 2) to obtain the product 2 (0.77 g, 67%) as a white powder. This product 2 was identified as a reaction intermediate represented by the formula (6), that is, 1-α- (m-iodo) phenoxy-2-deoxyribose, from the following mass spectrometry and NMR analysis results.
1 H NMR (CDCl 3 ) d 7.37-7.38 (m, 1H), 7.31-7.34 (m, 1H), 6.96-6.98 (m, 2H), 5.83 (dd, 1H, J = 5.6, 1.8 Hz), 4.60 -4.66 (m, 1H), 4.09 (dd, 1H, J = 8.7, 4.5 Hz), 3.60-3.76 (m, 2H), 2.52 (ddd, 1H, J = 13.8, 6.9, 1.8 Hz), 2.30 (dt , 1H, J = 13.8, 6.0 Hz), 1.89-1.95 (m, 2H);
HRMS (ESI) cald.for C 11 H 13 IO 4 Na (MNa + ): 358.9751, found: 358.9729.
[Example 3] <Synthesis of deoxyribose derivative>
Palladium acetate (25 mg, 0.1 μmol) and triethylamine (0.37 μL, 2 + .64 mmol) were sequentially added to a dioxane solution (15 mL) of triphenylphosphine (87 mg, 0.3 μmol), and the mixture was stirred at 75 ° C for 5 minutes. . The product 2 of Example 2 (0.74 g, 2.20 mmol) and methyl acrylate (0.40 μL, 4.40 mmol) were added, and the reaction solution was refluxed for 2 hours. After confirming the product by TLC (CHCl 3 : MeOH = 9: 1), palladium powder was removed by cotton filtration. Purification by column chromatography (CHCl 3 : MeOH = 95: 5) gave the product 3 (0.6 g, 93%) as a yellow oil. The product 3 was identified as α-form by the NOESY experiment (FIG. 1), and from the following mass and ultraviolet absorbance analysis values and NMR analysis results, the deoxyribose derivative represented by the formula (7), that is, (E) -1-α It was identified as-(m-metoxycarbonylvinyl) phenoxy-2-deoxyribose.
1H NMR (CDCl3) d 7.62 (d, 1H, J = 15.9 Hz), 7.25-7.30 (m, 1H), 7.14-7.17 (m, 2H), 6.99-7.05 (m, 1H), 6.41 (d, 1H, J = 15.9 Hz), 5.89 (dd, 1H, J = 5.6, 2.4 Hz), 4.63-4.69 (m, 1H), 4.10 (dd, 1H, J = 8.4, 4.2 Hz), 3.79 (s, 3H), 3.62-3.76 (m, 2H), 2.55 (ddd, 1H, J = 13.8, 6.6, 1.8 Hz), 2.33 (dt, 1H, J = 13.8, 5.7 Hz), 1.89-1.93 (m, 2H);
HRMS (ESI) cald. for C15H18O6Na (MNa+): 317.0996, found: 317.0981;
UV (H2O : CH3OH = 1 : 1) lmax (e) 278 nm (1.2×104 M-1cm-1), 366 nm (68 M-1cm-1).
〔実施例４〕＜ジメチルトリチル基を導入したデオキシリボース誘導体の合成＞
式（７）に示すデオキシリボース誘導体の５位のヒドロキシル基に保護基（ジメチルトリチル基）の導入を試みた。pyridine (2.0 mL×2)で共沸した前記デオキシリボース誘導体である実施例３得た生成物３(0.20 g, 0.68 mmol)にpyridine (1.0 mL)を加えた。反応溶液に4,4'-dimethoxytritylchloride (0.23 g, 0.82 mmol)と4-(dimethylamino)pyridine (17.0 mg, 0.14 mmol)のピリジン溶液(2.0 mL)を加えて、反応溶液を室温で14時間撹拌した。TLC (CHCl3 : MeOH = 97 : 3)で生成物を確認した後、pyridineを除去した。カラムクロマトグラフィー(CHCl3 : MeOH = 98 : 2)で精製し、黄色粉末として生成物４(0.30 g, 75%)を得た。生成物４は下記の質量分析値およびNMR分析結果より、式（８）に示すデオキシリボース誘導体と同定された。
1 H NMR (CDCl 3 ) d 7.62 (d, 1H, J = 15.9 Hz), 7.25-7.30 (m, 1H), 7.14-7.17 (m, 2H), 6.99-7.05 (m, 1H), 6.41 (d , 1H, J = 15.9 Hz), 5.89 (dd, 1H, J = 5.6, 2.4 Hz), 4.63-4.69 (m, 1H), 4.10 (dd, 1H, J = 8.4, 4.2 Hz), 3.79 (s, 3H), 3.62-3.76 (m, 2H), 2.55 (ddd, 1H, J = 13.8, 6.6, 1.8 Hz), 2.33 (dt, 1H, J = 13.8, 5.7 Hz), 1.89-1.93 (m, 2H) ;
HRMS (ESI) cald.for C 15 H 18 O 6 Na (MNa + ): 317.0996, found: 317.0981;
UV (H 2 O: CH 3 OH = 1: 1) l max (e) 278 nm (1.2 × 10 4 M -1 cm -1 ), 366 nm (68 M -1 cm -1 ).
[Example 4] <Synthesis of deoxyribose derivative having a dimethyltrityl group introduced>
An attempt was made to introduce a protecting group (dimethyltrityl group) into the hydroxyl group at the 5-position of the deoxyribose derivative represented by the formula (7). Pyridine (1.0 mL) was added to the product 3 (0.20 g, 0.68 mmol) obtained in Example 3, which was the deoxyribose derivative azeotroped with pyridine (2.0 mL × 2). A pyridine solution (2.0 mL) of 4,4'-dimethoxytritylchloride (0.23 g, 0.82 mmol) and 4- (dimethylamino) pyridine (17.0 mg, 0.14 mmol) was added to the reaction solution, and the reaction solution was stirred at room temperature for 14 hours. . After confirming the product by TLC (CHCl 3 : MeOH = 97: 3), pyridine was removed. Purification by column chromatography (CHCl 3 : MeOH = 98: 2) gave the product 4 (0.30 g, 75%) as a yellow powder. The product 4 was identified as a deoxyribose derivative represented by the formula (8) from the following mass spectrometry value and NMR analysis result.
1H NMR (CDCl3) d 7.62 (d, 1H, J = 15.9 Hz), 7.35-7.38 (m, 1H), 7.23-7.28 (m, 6H), 7.10-7.17 (m, 5H), 6.99-7.02 (m, 1H), 6.69 (dd, 4H, J = 9.0, 2.4 Hz), 6.40 (d, 1H, J = 15.9 Hz), 5.87 (dd, 1H, J = 5.3, 2.1 Hz), 4.56-4.62 (m, 1H), 4.05 (dd, 1H, J = 10.3, 5.7 Hz), 3.78 (s, 3H), 3.73 (s, 3H), 3.72 (s, 3H), 3.24 (dd, 1H, J = 9.2, 5.1 Hz), 3.16 (dd, 1H, J = 9.8, 5.4 Hz), 2.50 (ddd, 1H, J = 13.3, 6.8, 2.4 Hz), 2.27 (ddd, 1H, J = 14.2, 6.6, 5.4 Hz), 1.81-1.83 (m, 1H);
HRMS (ESI) cald. for C36H36O8Na (MNa+): 619.2302, found: 619.2268.
〔実施例５〕＜フォスフォロアミダイト誘導体の合成＞
式（８）に示す５位をジメチルトリチル基で保護したデオキシリボース誘導体の３位のヒドロキシル基にフォスフォロアミダイトの導入を試みた。acetonitrile (1.0 mL)で共沸した前記実施例４で得た生成物４ (0.22 g, 0.36 mmol)にacetonitrile (3.0 mL)を加えた。反応溶液に2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoro-diamidite (0.12 μL, 0.36 mmol)と0.45 M tetrazoleのacetonitrile溶液 (0.80 mL, 0.36 mmol)を加えて、反応溶液を室温で1.5時間撹拌した。反応溶液を脱酢酸処理した酢酸エチルで2回抽出し、Sat. NaHCO3 aq.とH2Oで洗浄した。有機相をMgSO4で乾燥し、溶媒を除去した。白色粉末の粗生成物である生成物５ (0.33 g)を得た。この生成物５は下記の質量分析値およびNMR分析結果より、式（９）に示すフォスフォロアミダイト誘導体と同定された。
1 H NMR (CDCl 3 ) d 7.62 (d, 1H, J = 15.9 Hz), 7.35-7.38 (m, 1H), 7.23-7.28 (m, 6H), 7.10-7.17 (m, 5H), 6.99-7.02 (m, 1H), 6.69 (dd, 4H, J = 9.0, 2.4 Hz), 6.40 (d, 1H, J = 15.9 Hz), 5.87 (dd, 1H, J = 5.3, 2.1 Hz), 4.56-4.62 ( m, 1H), 4.05 (dd, 1H, J = 10.3, 5.7 Hz), 3.78 (s, 3H), 3.73 (s, 3H), 3.72 (s, 3H), 3.24 (dd, 1H, J = 9.2, 5.1 Hz), 3.16 (dd, 1H, J = 9.8, 5.4 Hz), 2.50 (ddd, 1H, J = 13.3, 6.8, 2.4 Hz), 2.27 (ddd, 1H, J = 14.2, 6.6, 5.4 Hz), 1.81-1.83 (m, 1H);
HRMS (ESI) cald.for C 36 H 36 O 8 Na (MNa + ): 619.2302, found: 619.2268.
[Example 5] <Synthesis of phosphoramidite derivative>
An attempt was made to introduce a phosphoramidite into the hydroxyl group at the 3-position of the deoxyribose derivative in which the 5-position shown in Formula (8) was protected with a dimethyltrityl group. Acetonitrile (3.0 mL) was added to the product 4 (0.22 g, 0.36 mmol) obtained in Example 4 azeotropically with acetonitrile (1.0 mL). To the reaction solution was added 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphoro-diamidite (0.12 μL, 0.36 mmol) and 0.45 M tetrazole acetonitrile solution (0.80 mL, 0.36 mmol). Stir for 1.5 hours. The reaction solution was extracted twice with deacetic acid-treated ethyl acetate and washed with Sat. NaHCO 3 aq. And H 2 O. The organic phase was dried over MgSO 4 and the solvent was removed. Product 5 (0.33 g) was obtained as a white powder crude product. This product 5 was identified as a phosphoramidite derivative represented by the formula (9) from the following mass spectrometry and NMR analysis results.
1H NMR (CDCl3) d 7.63 (d, 1H, J = 16.2 Hz), 7.35-7.38 (m, 1H), 7.22-7.27 (m, 6H), 7.10-7.14 (m, 5H), 7.02-7.08 (m, 1H), 6.65 (d, 4H, J = 8.8 Hz), 6.40 (d, 1H,J = 16.2 Hz), 5.90 (m, 1H), 4.60-4.70 (m, 1H), 4.18-4.22 (m, 1H), 4.10-4.14 (m,1H), 3.78 (s, 3H), 3.71 (s, 3H), 3.70 (s, 3H), 3.42-3.60 (m, 1H), 3.08-3.26 (m,1H), 2.74 (t, 1H, J = 6.0 Hz), 2.50-2.56 (m, 1H), 2.28-2.34 (m, 1H), 1.11-1.29 (m, 12H);HRMS (MALDI) cald. for C45H53N2O9Na (MNa+): 819.3387, found: 819.8709.
〔実施例６〕＜光応答性ヌクレオチドの合成＞
ABI 3400 DNA合成機を用いてb-シアノエチルホスフォアミダイト法により以下のオリゴデオキシヌクレオチド（ＯＤＮ）をそれぞれ合成した。前記実施例５で得たフォスフォロアミダイト誘導体をゴムシールボトルにアセトニトリルで移し3回共沸した後、さらなる精製をせずにアミダイト試薬として使用した。ＯＤＮは得られた反応混合物のアンモニア水溶液を65 °Cで4時間置き、脱保護した。アンモニア水溶液を除去した後、日本分光PU-980、UV-970、HG-980-31による逆相高速液体クロマトグラフィーを通じて精製した。それぞれのＯＤＮはApplide Biosystems Voyager-DE-PRO-SFによる質量分析とＰ１ヌクレアーゼ、アルカリフォスファターゼを用いて酵素分解を行いＡ、Ｇ、Ｃ、Ｔ、Ｕの各ヌクレオシドの組成比から下記のとおり塩基配列と分子量が同定された。
ODN1（配列番号１）: HRMS (MALDI) calcd for 5'-TGTGCT: [(M-H)-] 1796.3, found 1796.6.
ODN2（配列番号２）: HRMS (MALDI) calcd for 5'-cvPhGCGTG: [(M-H)-] 1858.4, found 1858.4.
ODN3（配列番号３）: HRMS (MALDI) calcd for 3'-ACACGAACGCAC: [(M+H)+] 3607.4, found 3607.7.
ODN5（配列番号４）: HRMS (MALDI) calcd for 3'-ACACGGACGCAC: [(M+H)+] 3625.4, found 3625.7.
なお、ヌクレオチド配列中のcvPhはこの出願の発明かかるデオキシリボース誘導体を示す。
1 H NMR (CDCl 3 ) d 7.63 (d, 1H, J = 16.2 Hz), 7.35-7.38 (m, 1H), 7.22-7.27 (m, 6H), 7.10-7.14 (m, 5H), 7.02-7.08 (m, 1H), 6.65 (d, 4H, J = 8.8 Hz), 6.40 (d, 1H, J = 16.2 Hz), 5.90 (m, 1H), 4.60-4.70 (m, 1H), 4.18-4.22 ( m, 1H), 4.10-4.14 (m, 1H), 3.78 (s, 3H), 3.71 (s, 3H), 3.70 (s, 3H), 3.42-3.60 (m, 1H), 3.08-3.26 (m, 1H), 2.74 (t, 1H, J = 6.0 Hz), 2.50-2.56 (m, 1H), 2.28-2.34 (m, 1H), 1.11-1.29 (m, 12H); HRMS (MALDI) cald. For C 45 H 53 N 2 O 9 Na (MNa + ): 819.3387, found: 819.8709.
[Example 6] <Synthesis of photoresponsive nucleotide>
The following oligodeoxynucleotides (ODN) were respectively synthesized by the b-cyanoethyl phosphoramidite method using an ABI 3400 DNA synthesizer. The phosphoramidite derivative obtained in Example 5 was transferred to a rubber seal bottle with acetonitrile and azeotroped three times, and then used as an amidite reagent without further purification. ODN was deprotected by placing an aqueous ammonia solution of the obtained reaction mixture at 65 ° C. for 4 hours. After removing the aqueous ammonia solution, it was purified through reverse phase high performance liquid chromatography using JASCO PU-980, UV-970, HG-980-31. Each ODN is subjected to mass spectrometry using Applide Biosystems Voyager-DE-PRO-SF and enzymatic degradation using P1 nuclease and alkaline phosphatase. From the composition ratio of each nucleoside of A, G, C, T, U, the base sequence is as follows: And the molecular weight was identified.
ODN1 (SEQ ID NO : 1) : HRMS (MALDI) calcd for 5'-TGTGCT: [(MH)-] 1796.3, found 1796.6.
ODN2 (SEQ ID NO : 2) : HRMS (MALDI) calcd for 5'- cv PhGCGTG: [(MH)-] 1858.4, found 1858.4.
ODN3 (SEQ ID NO : 3) : HRMS (MALDI) calcd for 3'-ACACGAACGCAC: [(M + H) + ] 3607.4, found 3607.7.
ODN 5 (SEQ ID NO : 4) : HRMS (MALDI) calcd for 3'-ACACGGACGCAC: [(M + H) + ] 3625.4, found 3625.7.
Note that cv Ph in the nucleotide sequence represents the deoxyribose derivative according to the invention of this application.
〔実施例７〕＜光応答性ヌクレオチドによる架橋反応１＞
ＯＤＮ１(10 mM)、ＯＤＮ２(20 mM)およびＯＤＮ３(10 mM)をカコジル酸ナトリウム緩衝溶液 (30 ml)に溶解し、トランスイルミネーターを用いて0℃で5時間、366 nmの光照射を行った。光反応生成物をChemcobond 5-ODS-Hカラムを用いＨＰＬＣ分析した（図２）。溶離液は、30分間にギ酸アンモニウム : アセトニトリル = 97 :3から80 : 20へと移動相の組成を変化させたものを用いた。
[Example 7] <Crosslinking reaction 1 with photoresponsive nucleotide>
ODN1 (10 mM), ODN2 (20 mM) and ODN3 (10 mM) are dissolved in sodium cacodylate buffer solution (30 ml), and irradiated with light at 366 nm for 5 hours at 0 ° C. using a transilluminator. It was. The photoreaction product was analyzed by HPLC using a Chemcobond 5-ODS-H column (FIG. 2). The eluent used was one in which the composition of the mobile phase was changed from ammonium formate: acetonitrile = 97: 3 to 80:20 for 30 minutes.
図２に示すＨＰＬＣ分析結果においては、光反応前後でＯＤＮ１のピーク面積がほとんど変化が認められなかった。また、天然の塩基（Ａ、Ｇ、Ｃ、Ｔ）のみで構成されるのＯＤＮの場合、上記反応条件では相補鎖が形成されないことより、この出願の発明かかるデオキシリボース誘導体を含むＯＤＮ２のみがＯＤＮ３に366 nmの光照射によって特異的にクロスリンクしたことが確認された。図３に上記反応のスキームを示す。クロスリンクした光連結体ＯＤＮ４の分子量は実施例６と同様に下記のとおり同定した。 In the HPLC analysis results shown in FIG. 2, there was almost no change in the peak area of ODN1 before and after the photoreaction. Further, in the case of an ODN composed only of natural bases (A, G, C, T), since a complementary strand is not formed under the above reaction conditions, only ODN2 containing the deoxyribose derivative according to the invention of this application is ODN3. Were confirmed to be specifically cross-linked by light irradiation at 366 nm. FIG. 3 shows a scheme of the above reaction. The molecular weight of the cross-linked photocoupler ODN4 was identified as follows in the same manner as in Example 6.
HRMS (MALDI) calcd for ODN: [(M+H)+] 5469.7, found 5470.3.
次に、光連結体ＯＤＮ４をＰ１ヌクレアーゼとアルカリフォスファターゼとSnake Venomを用いて酵素分解を行った。この分解産物の分子量をMALDI-TOF MSで測定した結果、式（１０）に示すcvPh-Ａ付加体が検出され、これよりこの出願の発明かかるデオキシリボース誘導体はアデニンとクロスリンクすることが確認された。
HRMS (MALDI) calcd for ODN: [(M + H) + ] 5469.7, found 5470.3.
Next, the photoligand ODN4 was subjected to enzymatic degradation using P1 nuclease, alkaline phosphatase and Snake Venom. As a result of measuring the molecular weight of this degradation product by MALDI-TOF MS, a cv Ph-A adduct represented by the formula (10) was detected. From this, it was confirmed that the deoxyribose derivative according to the present invention cross-links with adenine. It was done.
〔実施例８〕＜光応答性ヌクレオチドによる架橋反応２＞
ＯＤＮ２(20 mM)とＯＤＮ５(10 mM)をカコジル酸ナトリウム緩衝溶液 (30 ml)に溶解し、トランスイルミネーターを用いて0℃で5時間、366 nmの光照射を行った。光反応生成物を実施例７と同様にＨＰＬＣ分析した（図４）。
[Example 8] <Crosslinking reaction 2 with photoresponsive nucleotide>
ODN2 (20 mM) and ODN5 (10 mM) were dissolved in a sodium cacodylate buffer solution (30 ml), and irradiated with light at 366 nm for 5 hours at 0 ° C. using a transilluminator. The photoreaction product was analyzed by HPLC as in Example 7 (FIG. 4).
図３に示すＨＰＬＣ分析結果より、この出願の発明かかるデオキシリボース誘導体を含むＯＤＮ２とＯＤＮ５のクロスリンク生成が確認された。図５に上記反応のスキームを示す。クロスリンクした光連結体ＯＤＮ６の分子量は実施例６と同様に下記のとおり同定した。 From the results of the HPLC analysis shown in FIG. 3, it was confirmed that ODN2 and ODN5 containing the deoxyribose derivative according to the invention of this application were cross-linked. FIG. 5 shows a scheme of the above reaction. The molecular weight of the cross-linked photocoupler ODN6 was identified as follows in the same manner as in Example 6.
HRMS (MALDI) calcd for ODN 6: [(M+H)+] 5485.7, found 5486.2.
次に、ＯＤＮ２／ＯＤＮ５のTm値、および光連結体ＯＤＮ６のTm値をJASCO V-550 UV/Vis spectrophotometerで測定した。標準プロトコールに従いそれぞれのＯＤＮ試料を1.0 °C/minの速度で昇温させ、紫外領域260 nmの吸光度を測定した（図６）。図６に示す吸光度曲線（ａ）は前者のＯＤＮ２／ＯＤＮ５、（ｂ）はＯＤＮ６をそれぞれ示す。これより前者のTm値は19.0 °Cであったが、後者のTm値は65.3 °Cと、この出願の発明かかるデオキシリボース誘導体によるクロスリンクは核酸の熱的安定性を向上させることを確認した。
HRMS (MALDI) calcd for ODN 6: [(M + H) + ] 5485.7, found 5486.2.
Next, the Tm value of ODN2 / ODN5 and the Tm value of photocoupler ODN6 were measured with JASCO V-550 UV / Vis spectrophotometer. Each ODN sample was heated at a rate of 1.0 ° C./min according to a standard protocol, and the absorbance in the ultraviolet region 260 nm was measured (FIG. 6). The absorbance curve (a) shown in FIG. 6 shows the former ODN2 / ODN5, and (b) shows ODN6. The former Tm value was 19.0 ° C, but the latter Tm value was 65.3 ° C. It was confirmed that the crosslink by the deoxyribose derivative according to the invention of this application improves the thermal stability of nucleic acids. .
〔実施例９〕＜光応答性ヌクレオチドによる架橋反応における塩基特異性、配列依存性の検討＞
実施例６と同様にＯＤＮ３Ｘ (3'-ACACGAXCGCAC-5'、塩基XはA,G,C,Tのいずれかを示す；配列番号５)を作成した。ＯＤＮ３Ｘ(20 mM)とＯＤＮ２(30 mM)を実施例７と同様に366 nm光照射によりクロスリンクさせた。各ＯＤＮ３Ｘの塩基Xの種類とクロスリンク形成効率であるＯＤＮの減少率を表１に示す。表1よりこの出願の発明かかるデオキシリボース誘導体はアデニンとのクロスリンク形成において高い反応性を示し、シトシン、チミン、グアニンとは反応性が低いことが確認された。
[Example 9] <Examination of base specificity and sequence dependency in crosslinking reaction with photoresponsive nucleotide>
In the same manner as in Example 6, ODN3X (3′-ACACGAXCGCAC-5 ′, base X represents one of A, G, C, and T; SEQ ID NO: 5 ) was prepared. ODN3X (20 mM) and ODN2 (30 mM) were cross-linked by 366 nm light irradiation in the same manner as in Example 7. Table 1 shows the type of base X of each ODN3X and the decrease rate of ODN, which is crosslink formation efficiency. From Table 1, it was confirmed that the deoxyribose derivative according to the invention of this application showed high reactivity in cross-linking with adenine and low reactivity with cytosine, thymine and guanine.
次に、ＯＤＮ２(20 mM)とＯＤＮ３(20 mM)、およびＯＤＮ２(20 mM)とＯＤＮ５(20 mM)それぞれについて実施例７と同様に366 nm光照射によりクロスリンク反応を行った。それぞれのODNの減少率を表２に示す。なお、ＯＤＮ３およびＯＤＮ５は、それぞれ3'-ACACGYACGCAC-5'の塩基配列を有し、かかる塩基Yは前者においてはA、後者においてはGである。また、ＯＤＮ２のcvPhはかかる塩基Yの５´に隣接するAと特異的に反応することが上記実施例において確認されている。
表２よりこの出願の発明かかるデオキシリボース誘導体におけるアデニンとのクロスリンク形成においては周辺の塩基配列依存性は認められなかった。
Next, ODN2 (20 mM) and ODN3 (20 mM), and ODN2 (20 mM) and ODN5 (20 mM) were each subjected to a cross-link reaction by 366 nm light irradiation in the same manner as in Example 7. Table 2 shows the reduction rate of each ODN. ODN3 and ODN5 each have a base sequence of 3′-ACACGYACGCAC-5 ′, and such base Y is A in the former and G in the latter. In addition, it was confirmed in the above Examples that cv Ph of ODN2 reacts specifically with A adjacent to 5 ′ of such base Y.
From Table 2, the dependency of the surrounding base sequence on the cross-linking with adenine in the deoxyribose derivative according to the invention of this application was not recognized.
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