JP2006248975A - Nucleoside phosphoroamidite compound - Google Patents
Nucleoside phosphoroamidite compound Download PDFInfo
- Publication number
- JP2006248975A JP2006248975A JP2005067121A JP2005067121A JP2006248975A JP 2006248975 A JP2006248975 A JP 2006248975A JP 2005067121 A JP2005067121 A JP 2005067121A JP 2005067121 A JP2005067121 A JP 2005067121A JP 2006248975 A JP2006248975 A JP 2006248975A
- Authority
- JP
- Japan
- Prior art keywords
- group
- nucleoside phosphoramidite
- phosphoramidite compound
- nucleoside
- carbon atoms
- 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.)
- Pending
Links
- 0 CCCO*OC([C@@](CO)O)=C([C@@](C)*(C=C(CC)C(*1)=O)C1=O)O Chemical compound CCCO*OC([C@@](CO)O)=C([C@@](C)*(C=C(CC)C(*1)=O)C1=O)O 0.000 description 1
- UHUUMBQOTZWCRQ-CYBMUJFWSA-N NCCCCCNCC(C(N1)=O)=CN([C@@H](C2)OC(CO)=C2O)C1=O Chemical compound NCCCCCNCC(C(N1)=O)=CN([C@@H](C2)OC(CO)=C2O)C1=O UHUUMBQOTZWCRQ-CYBMUJFWSA-N 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
本発明は、ヌクレオシドホスホロアミダイト化合物に関する。更に詳細には、熱的安定性を有するＡ型ＲＮＡ二重鎖を形成し得る中間体として用い得る、ヌクレオシドホスホロアミダイト化合物に関する。 The present invention relates to nucleoside phosphoramidite compounds. More particularly, the present invention relates to a nucleoside phosphoramidite compound that can be used as an intermediate capable of forming a type A RNA duplex having thermal stability.
オリゴヌクレオチドは、単鎖ＲＮＡ又は単鎖ＤＮＡにハイブリダイズすることが知られている。ハイブリダイズする（ハイブリダイゼーション）とは、標的ＲＮＡ又はＤＮＡの塩基に対し、オリゴヌクレオチドの塩基が配列特異的な塩基対で水素結合することであり、そのように水素結合する場合、そのような塩基対は相補的であると言われる。 Oligonucleotides are known to hybridize to single stranded RNA or single stranded DNA. Hybridization (hybridization) means that a base of an oligonucleotide is hydrogen-bonded to a base of a target RNA or DNA by a sequence-specific base pair. The pair is said to be complementary.
相補的な核酸に対するオリゴヌクレオチドのハイブリダイゼーションの程度を決定する場合、相補的な核酸に結合するオリゴヌクレオチドの相対能力は、特定のハイブリダイゼーション複合体の融解温度を測定することによって確認することができる。融解温度（Ｔｍ）は、二重らせんに特徴的な物理特性であり、５０％のらせん形（ハイブリダイズした）対コイル形（ハイブリダイズしていない）が存在する温度を意味する。 When determining the degree of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of the oligonucleotide to bind to the complementary nucleic acid can be confirmed by measuring the melting temperature of a particular hybridization complex. . Melting temperature (Tm) is a physical property characteristic of a double helix and refers to the temperature at which 50% helical (hybridized) vs. coiled (unhybridized) is present.
Ａ型構造と呼ばれているＲＮＡが存在しているが、ＤＮＡは、通常はＢ型構造で存在しており、一般に、ＲＮＡ−ＲＮＡ二重鎖はＤＮＡ−ＤＮＡ二重鎖よりも安定であるか、又はより高い融解温度（Ｔｍ）を有している。しかし、ＤＮＡ−ＲＮＡハイブリッド二本鎖はＲＮＡ：ＲＮＡ二重鎖よりも安定性が低く、それらの配列に依存して、ＤＮＡのＤＮＡ二本鎖よりも安定性が高かったり低かったりする。 Although there is RNA called A-type structure, DNA usually exists in B-type structure, and in general, RNA-RNA duplex is more stable than DNA-DNA duplex Or have a higher melting temperature (Tm). However, DNA-RNA hybrid duplexes are less stable than RNA: RNA duplexes and are more or less stable than DNA DNA duplexes depending on their sequence.
一方、ｍＲＮＡやｓｉＲＮＡから生じるとされるｍｉＲＮＡ等の機能性のＲＮＡの細胞内における発現状態や、機能、塩基配列等を網羅的に解析するための技術として、ＤＮＡチップの利用が知られている。しかし、現状のＤＮＡチップでは、特に短鎖であるｓｉＲＮＡやｍｉＲＮＡによって形成されるＤＮＡ−ＲＮＡ二重鎖の熱的安定性が低いことから、これらの検出には不適当である。さらに、ターゲットとするＲＮＡはＤＮＡと比較して高次構造を形成しやすいため、あまり熱安定性の期待できないプローブでは、その高次構造を解きほぐすことができず、検出感度が低下するという問題がある。 On the other hand, the use of a DNA chip is known as a technique for exhaustively analyzing the expression state, function, base sequence, etc. of intracellular functional RNA such as miRNA that is supposed to be generated from mRNA or siRNA. . However, the current DNA chip is not suitable for the detection of these because the thermal stability of the DNA-RNA duplex formed by siRNA or miRNA which are short strands is particularly low. Furthermore, the target RNA is more likely to form a higher order structure than DNA, so that a probe that is not expected to have a very high thermal stability cannot unravel the higher order structure, resulting in a decrease in detection sensitivity. is there.
ＤＮＡ二重鎖については、修飾核酸を用いることによる核酸二重鎖の安定化方法が開発されている。修飾核酸を用いた核酸二重鎖を安定化させるために有効な方法として、核酸の塩基部分にカチオン性側鎖を導入することにより、リン酸基上に生じる負電荷同士の静電的反発を減少させ、核酸二重鎖を安定化する技術が知られている（非特許文献１）。Ｂ型ＤＮＡ二重鎖中にカチオン性側鎖を有する様々な修飾核酸が組み込まれ、その挙動が研究されてきているが、二重鎖安定化能を獲得したカチオン性側鎖を有する修飾核酸が存在するが、反対に二重鎖を不安定化するものも報告されている（非特許文献２）。 For DNA duplexes, methods for stabilizing nucleic acid duplexes by using modified nucleic acids have been developed. As an effective method for stabilizing nucleic acid duplexes using modified nucleic acids, the introduction of a cationic side chain into the base part of the nucleic acid prevents electrostatic repulsion between negative charges generated on the phosphate group. A technique for reducing and stabilizing nucleic acid duplexes is known (Non-patent Document 1). Various modified nucleic acids having cationic side chains have been incorporated into B-type DNA duplexes, and their behavior has been studied. Modified nucleic acids having cationic side chains that have acquired the ability to stabilize double strands On the other hand, the one that destabilizes the double chain has also been reported (Non-patent Document 2).
例えば、下記式（Ｉ）で表わされる化合物（デオキシウリジンのウラシル環の５位にアミノヘプチル基を有する化合物）をＢ型ＤＮＡ二重鎖中に組み込んだ場合、デオキシウリジンが組み込まれたＢ型ＤＮＡ二重鎖との融解温度の差（ΔＴｍ値）は−０．１℃とほぼ同等である（非特許文献３）。また、下記式（II）で表わされる化合物（式（Ｉ）で表わされる化合物のウラシル環の５位の置換基のウラシル環に近い部位にアミノ基を導入した化合物）をＢ型ＤＮＡ二重鎖中に組み込んだ場合、デオキシウリジンが組み込まれたＢ型ＤＮＡ二重鎖との融解温度の差（ΔＴｍ値）は−３．９℃となり、二重鎖は不安定化される（非特許文献２）。また、下記式（III）で表わされる化合物（デオキシウリジンのウラシル環の５位にアミノプロピル基を有する化合物）をＢ型ＤＮＡ二重鎖中に組み込んだ場合、デオキシウリジンが組み込まれたＢ型ＤＮＡ二重鎖との融解温度の差（ΔＴｍ値）は−０．６℃となり、二重鎖は若干不安定化される（非特許文献４）。また、下記式（IV）で表わされる化合物（化合物（III）に比べ、ウラシル環の５位に結合した置換基が不飽和結合を有する化合物）をＢ型ＤＮＡ二重鎖中に組み込んだ場合、デオキシウリジンが組み込まれたＢ型ＤＮＡ二重鎖との融解温度の差（ΔＴｍ値）は＋２〜４℃であり、二重鎖形成が大幅に安定化される（非特許文献４）。 For example, when a compound represented by the following formula (I) (a compound having an aminoheptyl group at the 5-position of the uracil ring of deoxyuridine) is incorporated into a B-type DNA duplex, B-type DNA into which deoxyuridine is incorporated The difference in melting temperature from the duplex (ΔTm value) is almost equivalent to −0.1 ° C. (Non-patent Document 3). Further, a compound represented by the following formula (II) (a compound in which an amino group is introduced at a position close to the uracil ring of the substituent at the 5-position of the uracil ring of the compound represented by formula (I)) is converted into a B-type DNA duplex. When incorporated in, the difference in melting temperature (ΔTm value) from the B-type DNA duplex in which deoxyuridine is incorporated is −3.9 ° C., and the duplex is destabilized (Non-patent Document 2) ). In addition, when a compound represented by the following formula (III) (a compound having an aminopropyl group at the 5-position of the uracil ring of deoxyuridine) is incorporated into a B-type DNA duplex, B-type DNA into which deoxyuridine is incorporated The difference in melting temperature from the duplex (ΔTm value) is −0.6 ° C., and the duplex is slightly destabilized (Non-patent Document 4). In addition, when a compound represented by the following formula (IV) (a compound in which the substituent bonded to the 5-position of the uracil ring has an unsaturated bond as compared with the compound (III)) is incorporated into the B-type DNA duplex, The difference in melting temperature (ΔTm value) from the B-type DNA duplex incorporating deoxyuridine is +2 to 4 ° C., and the duplex formation is greatly stabilized (Non-patent Document 4).
上述したように、Ｂ型ＤＮＡ二重鎖の場合は、二重鎖を安定化させるための側鎖等の修飾がある程度明らかになっているが、Ｂ型ＤＮＡ二重鎖とは構造が異なる、Ａ型ＲＮＡ二重鎖についての報告はあまりないのが現状であり、Ａ型ＲＮＡ二重鎖の安定化を向上することのできる最適な修飾については未だに明らかとはなっていない。 As described above, in the case of a B-type DNA duplex, modification of side chains and the like for stabilizing the duplex has been clarified to some extent, but the structure is different from that of a B-type DNA duplex. There are currently few reports on the A-type RNA duplex, and the optimal modification that can improve the stabilization of the A-type RNA duplex has not yet been clarified.
一方、修飾核酸を用いることによる、Ｂ型ＤＮＡの安定な二重鎖を形成させる他の方法としては、２’水酸基が２’−Ｏ−アルキル化（メチル化、メトキシエチル化等）された修飾核酸や、核酸の２’位の酸素原子と４’位の炭素原子とを架橋して糖部コンフォメーションを化学的に固定化した修飾核酸等（ＢＮＡ、ＥＮＡ等）を用いることにより、糖部がＡ型ＲＮＡ二重鎖を組み込みやすいコンホメーションををとり、安定な二重鎖を形成されることが知られている（非特許文献５、６及び７）。しかし、Ａ型ＲＮＡ二重鎖の安定化を狙った報告例は未だにあまりないのが現状である。 On the other hand, another method for forming a stable duplex of B-type DNA by using a modified nucleic acid is modification in which the 2 ′ hydroxyl group is 2′-O-alkylated (methylated, methoxyethylated, etc.) By using nucleic acids or modified nucleic acids (BNA, ENA, etc.) in which the sugar moiety conformation is chemically immobilized by crosslinking the 2′-position oxygen atom and the 4′-position carbon atom of the nucleic acid. Is known to adopt a conformation that facilitates incorporation of an A-type RNA duplex and to form a stable duplex (Non-Patent Documents 5, 6 and 7). However, there are currently few reports on the stabilization of A-type RNA duplexes.
従って、本発明の目的は、熱的安定性を有するＡ型ＲＮＡ二重鎖を形成し得る中間体として用い得る、ヌクレオシドホスホロアミダイト化合物を提供することにある。 Accordingly, an object of the present invention is to provide a nucleoside phosphoramidite compound that can be used as an intermediate capable of forming an A-type RNA duplex having thermal stability.
上記目的を達成するため、本発明者らは鋭意検討した結果、特定のヌクレオシドホスホロアミダイト化合物が、上記目的を達成し得るという知見を得た。
本発明は、上記知見に基づいてなされたものであり、下記一般式（１）で表わされるヌクレオシドホスホロアミダイト化合物を提供するものである。
In order to achieve the above object, the present inventors have intensively studied, and as a result, have found that a specific nucleoside phosphoramidite compound can achieve the above object.
The present invention has been made based on the above findings, and provides a nucleoside phosphoramidite compound represented by the following general formula (1).
（上記式中、Ｒ１はリン酸保護基を表し、Ｒ２は窒素原子上に炭素数１〜６個の同一又は異なるアルキル基が２個結合したジアルキルアミノ基を表し、Ｒ３はアルコキシ基であるか、又はリボースの４’位炭素と結合して環を形成しており、Ｒ４は、炭素数３個以上のアルキル基、炭素数３個以上のアルケニル基又は炭素数３個以上のアルキニル基を表わし、アルキル基、アルケニル基又はアルキニル基はトリフルオロアセチルアミノ基と結合していてもよく、Ｒ５は水酸基の保護基を表し、Ｘは酸素原子又は硫黄原子を表す。）
また、本発明は、上記ヌクレオシドホスホロアミダイト化合物を用いることで、修飾されたヌクレオシドを構成成分として含有するオリゴ核酸を提供する。
(In the above formula, R 1 represents a phosphate protecting group, R 2 represents a dialkylamino group in which two identical or different alkyl groups having 1 to 6 carbon atoms are bonded on a nitrogen atom, and R 3 represents an alkoxy group. Or bonded to the 4′-position carbon of ribose to form a ring, and R 4 is an alkyl group having 3 or more carbon atoms, an alkenyl group having 3 or more carbon atoms, or a group having 3 or more carbon atoms. Represents an alkynyl group, the alkyl group, alkenyl group or alkynyl group may be bonded to a trifluoroacetylamino group, R 5 represents a protecting group for a hydroxyl group, and X represents an oxygen atom or a sulfur atom.)
The present invention also provides an oligonucleic acid containing a modified nucleoside as a constituent component by using the nucleoside phosphoramidite compound.
本発明のヌクレオシドホスホロアミダイト化合物は、熱的安定性を有するＡ型ＲＮＡ二重鎖を形成し得る中間体として用い得る。
本発明のオリゴ核酸は、熱的安定性を有しており、アンチセンス医薬、アンチジーン医薬、特定の遺伝子を検出するためのプローブ、又は増幅開始のためのプライマー等として用い得る。
The nucleoside phosphoramidite compound of the present invention can be used as an intermediate capable of forming an A-type RNA duplex having thermal stability.
The oligonucleic acid of the present invention has thermal stability and can be used as an antisense drug, an antigene drug, a probe for detecting a specific gene, a primer for starting amplification, or the like.
以下、本発明のヌクレオシドホスホロアミダイト化合物について説明する。
本発明のヌクレオシドホスホロアミダイト化合物は下記一般式（１）で表わされる。
Hereinafter, the nucleoside phosphoramidite compound of the present invention will be described.
The nucleoside phosphoramidite compound of the present invention is represented by the following general formula (1).
上記一般式（１）において、Ｒ１はリン酸保護基を表す。リン酸保護基としては、ホスホロアミダイト法に用いられるものであれば、特に制限なく用いることができ、例えば、メチル基、２−シアノエチル基、２−トリメチルシリルエチル基等が挙げられる。 In the above general formula (1), R 1 represents a phosphate protecting group. Any phosphoric acid protecting group can be used without particular limitation as long as it is used in the phosphoramidite method, and examples thereof include a methyl group, a 2-cyanoethyl group, and a 2-trimethylsilylethyl group.
Ｒ２は窒素原子上に炭素数１〜６個の同一又は異なるアルキル基が２個結合したジアルキルアミノ基を表す。なお、２個のアルキル基が互いに結合して環を形成していてもよい。このようなジアルキルアミノ基としては、例えば、ジエチルアミノ基、ジイソプロピルアミノ基、ジメチルアミノ基等が挙げられる。 R 2 represents a dialkylamino group in which two identical or different alkyl groups having 1 to 6 carbon atoms are bonded on a nitrogen atom. Two alkyl groups may be bonded to each other to form a ring. Examples of such a dialkylamino group include a diethylamino group, a diisopropylamino group, and a dimethylamino group.
Ｒ３はアルコキシ基であるか、又はリボースの４’位炭素と結合して環を形成していてもよい。アルコキシ基としては、炭素数が１〜６個のアルコキシ基が好ましく、このようなアルコキシ基としては、例えば、メトキシ基、エトキシ基、プロポキシ基、１−ブチルオキシ基、１−ペンチルオキシ基、１−ヘキシルオキシ基等が挙げられ、また、２−プロピルオキシ基、イソブチルオキシ基等のように分枝したアルコキシ基、シクロプロピルオキシ基、シクロブチルオキシ基、シクロペンチルオキシ基、シクロヘキシルオキシ基、シクロプロピルメチルオキシ基等の、側鎖の一部もしくは全部が環化したアルコキシ基も含む。 R 3 is an alkoxy group, or may be bonded to the 4′-position carbon of ribose to form a ring. The alkoxy group is preferably an alkoxy group having 1 to 6 carbon atoms. Examples of such an alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a 1-butyloxy group, a 1-pentyloxy group, 1- Hexyloxy group and the like, and branched alkoxy groups such as 2-propyloxy group, isobutyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group, cyclopropylmethyl Also included are alkoxy groups in which part or all of the side chain is cyclized, such as oxy groups.
Ｒ４は、炭素数３個以上のアルキル基、炭素数３個以上のアルケニル基又は炭素数３個以上のアルキニル基を表わし、アルキル基、アルケニル基又はアルキニル基はトリフルオロアセチルアミノ基を結合していてもよい。
ここで、アルキル基とは、炭素数が３〜６個のアルキル基が好ましく、このようなアルキル基としては、例えば、プロピル基、ｎ−ブチル基、イソブチル基、ペンチル基、ヘキシル基などのアルキル基を示し、イソプロピル基、ｓｅｃ−ブチル基、ｔｅｒｔ−ブチル基、ネオペンチル基などのように分枝したアルキル基、シクロプロピル基、シクロブチル基、シクロペンチル基、シクロヘキシル基等のシクロアルキル基も含む。アルケニル基としては、プロペニル基、ブチニル基等が挙げられる。また、アルキニル基としては、プロピニル基等が挙げられる。
R 4 represents an alkyl group having 3 or more carbon atoms, an alkenyl group having 3 or more carbon atoms, or an alkynyl group having 3 or more carbon atoms, and the alkyl group, alkenyl group or alkynyl group binds a trifluoroacetylamino group. It may be.
Here, the alkyl group is preferably an alkyl group having 3 to 6 carbon atoms. Examples of such an alkyl group include alkyl groups such as a propyl group, an n-butyl group, an isobutyl group, a pentyl group, and a hexyl group. A cycloalkyl group such as an isopropyl group, a sec-butyl group, a tert-butyl group, a neopentyl group, a cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group. Examples of the alkenyl group include a propenyl group and a butynyl group. Examples of the alkynyl group include a propynyl group.
また、Ｒ５は水酸基の保護基を表す。水酸基の保護基としては、ホスホロアミダイト法で用いられるものであれば特に制限はなく、例えば、ジメトキシトリチル基、モノメトキシトリチル基等が挙げられる。Ｘは酸素原子又は硫黄原子であり、Ｘが酸素原子の場合は、本発明のヌクレオシドホスホロアミダイト化合物はウラシル誘導体となり、硫黄原子の場合はチオウラシル誘導体となる。 R 5 represents a hydroxyl-protecting group. The hydroxyl protecting group is not particularly limited as long as it is used in the phosphoramidite method, and examples thereof include a dimethoxytrityl group and a monomethoxytrityl group. X is an oxygen atom or a sulfur atom. When X is an oxygen atom, the nucleoside phosphoramidite compound of the present invention is a uracil derivative, and when it is a sulfur atom, it is a thiouracil derivative.
本発明のヌクレオシドホスホロアミダイト化合物の具体例としては、下記式（２）で表わされる化合物（5’−Ｏ−(4,4’−ジメトキシトリチル)−5−[3−（Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジン 3’−Ｏ− (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）又は（３）で表わされる化合物（5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピル]−2’−Ｏ−メチルウリジン 3‘−Ｏ− (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が挙げられる。 Specific examples of the nucleoside phosphoramidite compound of the present invention include compounds represented by the following formula (2) (5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl). ) -Aminopropyn-1-yl] -2'-O-methyluridine 3'-O- (2-cyanoethyl) -N, N-diisopropylphosphoramidite) or a compound represented by (3) (5'- O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl] -2'-O-methyluridine 3'-O- (2-cyanoethyl) -N, N -Diisopropyl phosphoramidite.
本発明のヌクレオシドホスホロアミダイト化合物は、当業者に公知の方法を用いて合成することができる。例えば、本明細書の実施例に記載されている方法に従って合成することができる。 The nucleoside phosphoramidite compounds of the present invention can be synthesized using methods known to those skilled in the art. For example, it is compoundable according to the method described in the Example of this specification.
次に、本発明のオリゴ核酸について説明する。本発明のオリゴ核酸は、本発明のヌクレオシドホスホロアミダイト化合物を用いることで、修飾されたヌクレオシドを構成成分として含有してなる。本発明のオリゴ核酸に含まれる、本発明のヌクレオシドホスホロアミダイト化合物の種類、数、組み合わせ、位置等も、使用目的及び用途に応じて、当業者が適宜選択することができる。
本発明のオリゴ核酸は、本発明のヌクレオシドホスホロアミダイト化合物を用いて、当業者に公知の任意の方法で合成することができる。本発明のオリゴ核酸は、その用途によっても異なるが、ヌクレオチド単位が、通常は１０〜３０個程度からなり、その構成単位として、本発明のヌクレオシドホスホロアミダイト化合物を少なくとも１個含有してなる。本発明のオリゴ核酸は、本発明のヌクレオシドホスホロアミダイト化合物を含有してなり、熱的安定性に優れたものとなり、以下に説明する種々の用途に用いることができる。
Next, the oligonucleic acid of the present invention will be described. The oligonucleic acid of the present invention contains a modified nucleoside as a constituent by using the nucleoside phosphoramidite compound of the present invention. The type, number, combination, position, etc. of the nucleoside phosphoramidite compound of the present invention contained in the oligonucleic acid of the present invention can also be appropriately selected by those skilled in the art according to the intended purpose and use.
The oligonucleic acid of the present invention can be synthesized by any method known to those skilled in the art using the nucleoside phosphoramidite compound of the present invention. Although the oligonucleic acid of the present invention varies depending on its use, it usually comprises about 10 to 30 nucleotide units, and contains at least one nucleoside phosphoramidite compound of the present invention as its structural unit. The oligonucleic acid of the present invention contains the nucleoside phosphoramidite compound of the present invention, has excellent thermal stability, and can be used for various applications described below.
本発明のオリゴ核酸は熱安定性に優れるという特性を有しており、この熱的安定性に優れるという特性を利用し、遺伝子解析用プローブ、核酸間のハイブリダイゼーション法に使用することができる。このようなハイブリダイゼーション法は、ＰＣＲ法及びＤＮＡチップ等の分野で当業者に公知の様々な測定手段において利用されている。
本発明のオリゴ核酸は、ＰＣＲ用のＤＮＡプライマー（特定遺伝子の増幅開始用プライマー）、ＤＮＡ医薬品素材（アンチセンスＤＮＡ、デコイＤＮＡ、オリゴＤＮＡを利用した遺伝子修復用素材）、遺伝子解析用ＲＮＡプローブ、ＲＮＡ医薬品素材（アンチセンスＲＮＡ、アンチジーンＲＮＡ、リボザイム、ＲＮＡｉを利用した遺伝子発現制御）、ＤＮＡチップ又はＲＮＡチップの材料等としても用いることができる。
The oligonucleic acid of the present invention has the property of being excellent in thermal stability, and can be used for a hybridization method between a probe for gene analysis and a nucleic acid by utilizing the property of being excellent in thermal stability. Such a hybridization method is used in various measuring means known to those skilled in the art in the fields of PCR method and DNA chip.
The oligonucleic acid of the present invention comprises a DNA primer for PCR (primer for starting amplification of a specific gene), a DNA pharmaceutical material (antisense DNA, decoy DNA, a material for gene repair using oligo DNA), an RNA probe for gene analysis, It can also be used as RNA drug material (antisense RNA, antigene RNA, ribozyme, gene expression control using RNAi), DNA chip or RNA chip material.
本発明のオリゴ核酸を医薬素材として投与する場合の投与形態としては、例えば、錠剤、カプセル剤、顆粒剤、散剤若しくはシロップ剤等による経口投与又は注射剤若しくは坐剤等による非経口投与が挙げられる。これらの製剤は、従来公知の、滑沢剤、結合剤、崩壊剤、安定剤、矯味矯臭剤、希釈剤等の添加剤を用いて周知の方法で製造される。
医薬素材として用いる場合、その使用量は症状、年齢、投与方法等により異なり、適宜選択することができる。
Examples of the administration form when the oligonucleic acid of the present invention is administered as a pharmaceutical material include oral administration using tablets, capsules, granules, powders or syrups, or parenteral administration using injections or suppositories. . These preparations are produced by known methods using conventionally known additives such as lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.
When used as a pharmaceutical material, the amount used varies depending on symptoms, age, administration method, etc., and can be appropriately selected.
以下、本発明を実施例により更に詳細に説明する。なお、本発明の範囲は、かかる実施例に限定されないことはいうまでもない。
比較製造例１
5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[Ｎ− (トリフルオロアセチル)−メチルアミノメチル]−2’−Ｏ−メチルウリジン 3’−Ｏ− (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
2’−Ｏ−メチルウリジン(9.80 g, 40.1 mmol)を0.5 M KOH水溶液(80 mL)に溶解し、パラホルムアルデヒド(20.0 g, 667 mmol)を加えて、65 ℃の温度で1週間撹拌した。溶媒を減圧下留去した後、残留物を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=96：4）で精製し、更にメタノールで再結晶させ、5−ヒドロキシメチル−2’−Ｏ−メチルウリジンを得た（4.40 g、収率；40%)を得た。
mp 183.0-185.0 ℃; １H NMR (270 MHz, DMSO) δ3.33 (3H, s, 2’−OCH3), 3.52−3.64 (2H, m, 5’−H), 3.78−3.84 (2H, m, 3’−H, 4’−H), 4.11 (3H, bs, CH２, OH), 4.88 (1H, bs, OH), 5.08 (1H, bs, OH), 5.14−5.16 (1H, m, 2’−H), 5.89 (1H, d, 1’−H,Ｊ１’、２’ =5.6 Hz), 7.81 (1H, s, 6−H), 11.35 (1H, s, 3−NH);１３C NMR (67.8 MHz, DMSO) δ56.30, 57.46, 60.83, 68.45, 82.42, 85.25, 85.69, 114.39, 136.88, 150.51, 162.64; ESI−mass m/z Calcd for C１１H１７N２O７289.1036; Observed [M + H] 289.1034
Hereinafter, the present invention will be described in more detail with reference to examples. Needless to say, the scope of the present invention is not limited to such examples.
Comparative production example 1
5'-O- (4,4'-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl] -2'-O-methyluridine 3'-O- (2-cyanoethyl) -N Of N, N-diisopropyl phosphoramidite
2′-O-methyluridine (9.80 g, 40.1 mmol) was dissolved in 0.5 M aqueous KOH solution (80 mL), paraformaldehyde (20.0 g, 667 mmol) was added, and the mixture was stirred at a temperature of 65 ° C. for 1 week. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography (chloroform: methanol = 96: 4) and further recrystallized with methanol to obtain 5-hydroxymethyl-2′-O-methyluridine. (4.40 g, yield; 40%) was obtained.
mp 183.0-185.0 ° C; 1 H NMR (270 MHz, DMSO) δ3.33 (3H, s, 2'-OCH 3 ), 3.52-3.64 (2H, m, 5'-H), 3.78-3.84 (2H, m, 3'-H, 4'- H), 4.11 (3H, bs, CH 2, OH), 4.88 (1H, bs, OH), 5.08 (1H, bs, OH), 5.14-5.16 (1H, m , 2'-H), 5.89 (1H, d, 1'-H, J 1 ', 2' = 5.6 Hz), 7.81 (1H, s, 6-H), 11.35 (1H, s, 3-NH) 13 C NMR (67.8 MHz, DMSO) δ56.30, 57.46, 60.83, 68.45, 82.42, 85.25, 85.69, 114.39, 136.88, 150.51, 162.64; ESI-mass m / z Calcd for C 11 H 17 N 2 O 7 289.1036; Observed [M + H] 289.1034
上述のようにして得られた5−ヒドロキシメチル−2’−Ｏ−メチルウリジン (1.0 g, 3.65 mmol)を4.0 M HCl ジオキサン溶液(10 mL)に溶解し、0℃の温度で2.5時間撹拌した。溶媒を減圧下留去した後、無水ジオキサンでHClを共沸した。残留物を無水ジオキサン(10 mL)に溶解し、40% CH３NH２ （メタノール溶液）(20 mL)を加えて、室温で10時間撹拌した。溶媒を減圧下留去し、残留物を無水ピリジンで3回共沸脱水した後、無水ピリジン(30 mL)に溶解し、トリフルオロ酢酸無水物(772 μL, 5.47 mmol)を加えて、室温で3時間撹拌した。１ｍｌのメタノールを加えて反応を停止した後、溶媒を減圧下留去した。残留物を無水ピリジンで3回共沸脱水した後、無水ピリジン(30 mL)に溶解し、4,4’-ジメトキシトリチルクロリド(1.36 g, 4.02 mmol)を加えて、アルゴン雰囲気下、室温で3時間撹拌した。メタノールを加えて反応を停止した後、反応溶液を２００ｍｌのクロロホルムで希釈して水で2回洗浄した。さらに5%炭酸水素ナトリウム水溶液で3回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をシリカゲルカラムクロマトグラフィー（ヘキサン：酢酸エチル=50：50）で精製し、5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[Ｎ−(トリフルオロアセチル)−メチルアミノメチル]−2’−Ｏ−メチルウリジンを得た (900 mg、収率： 36%)。
１H NMR (270 MHz, CDCl3) δ3.18 (3H, s, CH３−N), 3.46−3.85 (14H, m, 2’−OCH３, OCH３of DMTr, CH２, 4’−H, 5’−H), 4.06−4.11 (1H, m, 3’−H), 4.23−4.26 (1H, m, 2’−H), 5.92 (1H, d, 1’−H,Ｊ1’,2’=2.1 Hz), 6.79−7.45 (13H, m, ArH of DMTr), 7.87(1H, s, 6−H);１３C NMR (67.8 MHz, CDCl３) δ36.80, 46.84, 55.17, 58.77, 62.65, 68.96, 83.28, 83.48, 86.66, 87.56, 108.41, 113.20, 114.05, 118.28, 126.89, 127.91, 128.24, 130.26, 135.56, 135.68, 141.61, 144.63, 149.77, 156.63, 157.16, 158.57, 163.21; ESI−mass m/z Calcd for C３５H３７F３N３O９722.2301; Observed [M + H] 722.2301
5-Hydroxymethyl-2′-O-methyluridine (1.0 g, 3.65 mmol) obtained as described above was dissolved in 4.0 M HCl dioxane solution (10 mL) and stirred at a temperature of 0 ° C. for 2.5 hours. . After the solvent was distilled off under reduced pressure, HCl was azeotroped with anhydrous dioxane. The residue was dissolved in anhydrous dioxane (10 mL), 40% CH 3 NH 2 (methanol solution) (20 mL) was added, and the mixture was stirred at room temperature for 10 hr. The solvent was distilled off under reduced pressure, the residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (30 mL), trifluoroacetic anhydride (772 μL, 5.47 mmol) was added, and Stir for 3 hours. The reaction was stopped by adding 1 ml of methanol, and then the solvent was distilled off under reduced pressure. The residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (30 mL), 4,4'-dimethoxytrityl chloride (1.36 g, 4.02 mmol) was added, and the mixture was added at room temperature under argon atmosphere at room temperature. Stir for hours. After stopping the reaction by adding methanol, the reaction solution was diluted with 200 ml of chloroform and washed twice with water. Furthermore, the organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 50: 50), and 5′-O- (4,4′-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl. ] -2′-O-methyluridine was obtained (900 mg, yield: 36%).
1 H NMR (270 MHz, CDCl 3 ) δ 3.18 (3H, s, CH 3 -N), 3.46-3.85 (14H, m, 2'-OCH 3 , OCH 3 of DMTr, CH 2 , 4'-H , 5'−H), 4.06−4.11 (1H, m, 3′−H), 4.23−4.26 (1H, m, 2′−H), 5.92 (1H, d, 1′−H, J 1 ′, 2 ' = 2.1 Hz), 6.79-7.45 (13H, m, ArH of DMTr), 7.87 (1H, s, 6-H); 13 C NMR (67.8 MHz, CDCl 3 ) δ36.80, 46.84, 55.17, 58.77 , 62.65, 68.96, 83.28, 83.48, 86.66, 87.56, 108.41, 113.20, 114.05, 118.28, 126.89, 127.91, 128.24, 130.26, 135.56, 135.68, 141.61, 144.63, 149.77, 156.63, 157.16, 158.57, 163.2s; E m / z Calcd for C 35 H 37 F 3 N 3 O 9 722.2301; Observed [M + H] 722.2301
上述のようにして得られた5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[Ｎ−(トリフルオロアセチル)−メチルアミノメチル]−2’−Ｏ−メチルウリジン (300 mg, 0.43 mmol)を無水アセトニトリルで3回、無水ジクロロメタンで1回共沸脱水した後、無水ジクロロメタン(4.3 mL)に溶解し、1−H テトラゾール(30.1 mg, 0.43 mmol)とジイソプロピルアミン(60 μL, 0.43 mmol)を加えて、アルゴン雰囲気下、室温で10分撹拌した。さらに、(2-シアノエトキシ)−ビス−(Ｎ，Ｎ−ジイソプロピルアミノ)ホスフィン(273 μL, 0.86 mmol)を加えて、アルゴン雰囲気下、室温で3時間撹拌した。１ｍｌの水を加えて反応を停止した後、３０ｍｌのクロロホルムで希釈して飽和食塩水で3回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をジエチルエーテル：ジイソプロピルエーテル=1：1の混合溶媒で希釈して0.1 M水酸化ナトリウム水溶液で5回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をリサイクル分取HPLC（アセトニトリル）で精製し、5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[Ｎ− (トリフルオロアセチル)−メチルアミノメチル]−2’−Ｏ−メチルウリジン 3’−Ｏ− (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（下記式（４）で表わされる化合物）を得た (210 mg, 収率：55%)。
1H NMR (270 MHz, CDCl3) δ0.95-0.97 (4H, m), 1.10-1.15(12H, m), 2.35-2.39(1H, m),2.60-2.70 (1H, m), 3.17 (3H, s), 3.30-3.50 (7H,m), 3.71-3.83 (7H,m), 3.90-3.98 (1H, m), 4.18-4.25 (1H, m),5.90-5.99(1H, m), 6.75-6.90 (4H, m),7.20-7.46 (13 H, m), 7.83, 7.90 (1H, 2s); 13C NMR (67.8 MHz, CDCl3) δ20.10, 20.27, 22.85, 24.42, 24.54, 36.59, 43.08, 43.24, 43.42, 46.84, 55.16, 55.20, 58.36, 58.51, 58.78, 62.92, 68.34, 70.01, 82.32, 82.57, 86.69, 88.04, 108.24, 108.34, 112.95, 113.17, 126.98, 127.89, 128.36, 128.42, 130.32, 130.38, 135.65, 141.59, 144.57, 149.51, 158.60, 158.79, 162.68; 31P NMR (109.4 MHz, CDCL3) δ151.13, 151.15; ESI-mass m/z Calcd for C44H54F3N5O10P 900.3560; Observed [M + H] 900.3580
5′-O- (4,4′-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl] -2′-O-methyluridine (300 mg, 0.43 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile and once with anhydrous dichloromethane, and then dissolved in anhydrous dichloromethane (4.3 mL), and 1-H tetrazole (30.1 mg, 0.43 mmol) and diisopropylamine (60 μL, 0.43 mmol) was added, and the mixture was stirred at room temperature for 10 minutes under an argon atmosphere. Further, (2-cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (273 μL, 0.86 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon atmosphere. The reaction was stopped by adding 1 ml of water, diluted with 30 ml of chloroform and washed 3 times with saturated saline. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was diluted with a mixed solvent of diethyl ether: diisopropyl ether = 1: 1, washed 5 times with 0.1 M aqueous sodium hydroxide solution, the organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. . The residue was purified by recycle preparative HPLC (acetonitrile) and 5'-O- (4,4'-dimethoxytrityl) -5- [N- (trifluoroacetyl) -methylaminomethyl] -2'-O- Methyluridine 3′-O- (2-cyanoethyl) -N, N-diisopropyl phosphoramidite (a compound represented by the following formula (4)) was obtained (210 mg, yield: 55%).
1 H NMR (270 MHz, CDCl 3 ) δ0.95-0.97 (4H, m), 1.10-1.15 (12H, m), 2.35-2.39 (1H, m), 2.60-2.70 (1H, m), 3.17 ( 3H, s), 3.30-3.50 (7H, m), 3.71-3.83 (7H, m), 3.90-3.98 (1H, m), 4.18-4.25 (1H, m), 5.90-5.99 (1H, m), 6.75-6.90 (4H, m), 7.20-7.46 (13 H, m), 7.83, 7.90 (1H, 2s); 13 C NMR (67.8 MHz, CDCl 3 ) δ20.10, 20.27, 22.85, 24.42, 24.54, 36.59, 43.08, 43.24, 43.42, 46.84, 55.16, 55.20, 58.36, 58.51, 58.78, 62.92, 68.34, 70.01, 82.32, 82.57, 86.69, 88.04, 108.24, 108.34, 112.95, 113.17, 126.98, 127.89, 128.36, 128.42 130.32, 130.38, 135.65, 141.59, 144.57, 149.51, 158.60, 158.79, 162.68; 31 P NMR (109.4 MHz, CDCL 3 ) δ151.13, 151.15; ESI-mass m / z Calcd for C 44 H 54 F 3 N 5 O 10 P 900.3560; Observed [M + H] 900.3580
比較製造例１の合成反応を式で表わすと以下の通りである。 The synthesis reaction of Comparative Production Example 1 is represented by the following formula.
製造例２
3’， 5’−ビス−アセチル−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピル]−2’−Ｏ−メチルウリジン(400 mg, 0.80 mmol)を、ピリジン、28%アンモニア水(v/v=1:1)の混合溶媒(8 mL)に溶解し、50℃の温度で8時間撹拌した。溶媒を減圧下留去し、残留物をエタノール(8 mL)に溶解し、トリフルオロ酢酸エチルエステル(191μL, 1.60 mmol)及びトリエチルアミン(455μL, 2.40 mmol)を加えて、室温で2時間撹拌した。溶媒を減圧下留去し、残留物を無水ピリジンで3回共沸脱水した後、無水ピリジン(8 mL)に溶解し、4,4’−ジメトキシトリチルクロリド(325 mg, 0.96 mmol)を加えて、アルゴン雰囲気下、室温で3時間撹拌した。１ｍｌのメタノールを加えて反応を停止した後、反応溶液を５０ｍｌのクロロホルムで希釈して5%炭酸水素ナトリウム水溶液で3回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル=50：50）で精製し、5’−Ｏ−(4,4’−ジメトキシトリチル)−5−[3−（Ｎ−トリフルオロアセチル)−アミノプロピル]−2’−Ｏ−メチルウリジンを得た(450 mg,収率：80%)。
１H NMR (270 MHz, CDCl３)δ1.69−1.74 (2H, m, CH２), 2.14−2.19 (2H, m, CH２), 2.32−2.40 (2H, m, CH２), 3.53 (3H, s, 2’−Ｏ−Me), 3.70−3.85 (8H, m, OCH３ of DMTr, 5’−H), 3.91−3.99 (2H, m, 3’−H, 4’−H), 4.23−4.31 (1H, m, 2’−H), 5.76 (1H, d, 1’−H, Ｊ1’,2’=3.3 Hz), 6.77−6.80 (4H, d, ArH of DMTr, Ｊ1’,2’=8.9 Hz), 7.15−7.39 (10H, m, ArH of DMTr, NH), 7.59 (1H, s, 6−H); １３C NMR (67.8 MHz, CDCl３)δ24.22, 28.18, 42.70, 55.19, 58.63, 60.91, 68.43, 69.99, 82.89, 84.80, 89.19, 113.09, 114.40, 126.24, 127.82, 128.42, 129.72, 136.68, 138.33, 146.52, 149.80, 157.86, 162.82; ESI−mass m/z Calcd for C３６H３８F３N３NaO９736.2458; Observed [M + Na] 736.2736
Production Example 2
3 ′, 5′-bis-acetyl-5- [3- (N-trifluoroacetyl) -aminopropyl] -2′-O-methyluridine (400 mg, 0.80 mmol) was added to pyridine, 28% aqueous ammonia ( It was dissolved in a mixed solvent (8 mL) of v / v = 1: 1) and stirred at a temperature of 50 ° C. for 8 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in ethanol (8 mL), trifluoroacetic acid ethyl ester (191 μL, 1.60 mmol) and triethylamine (455 μL, 2.40 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure, the residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (8 mL), and 4,4'-dimethoxytrityl chloride (325 mg, 0.96 mmol) was added. The mixture was stirred at room temperature for 3 hours under an argon atmosphere. After stopping the reaction by adding 1 ml of methanol, the reaction solution was diluted with 50 ml of chloroform and washed 3 times with 5% aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Distilled off. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 50: 50), and 5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl. ] -2′-O-methyluridine was obtained (450 mg, yield: 80%).
1 H NMR (270 MHz, CDCl 3 ) δ 1.69−1.74 (2H, m, CH 2 ), 2.14−2.19 (2H, m, CH 2 ), 2.32−2.40 (2H, m, CH 2 ), 3.53 ( 3H, s, 2'-O- Me), 3.70-3.85 (8H, m, OCH 3 of DMTr, 5'-H), 3.91-3.99 (2H, m, 3'-H, 4'-H), 4.23−4.31 (1H, m, 2′−H), 5.76 (1H, d, 1′−H, J 1 ′, 2 ′ = 3.3 Hz), 6.77−6.80 (4H, d, ArH of DMTr, J 1 ', 2' = 8.9 Hz), 7.15-7.39 (10H, m, ArH of DMTr, NH), 7.59 (1H, s, 6-H); 13 C NMR (67.8 MHz, CDCl 3 ) δ24.22, 28.18 , 42.70, 55.19, 58.63, 60.91, 68.43, 69.99, 82.89, 84.80, 89.19, 113.09, 114.40, 126.24, 127.82, 128.42, 129.72, 136.68, 138.33, 146.52, 149.80, 157.86, 162.82; ESI-mass m / z Calcd for C 36 H 38 F 3 N 3 NaO 9 736.2458; Observed [M + Na] 736.2736
上述のようにして得られた5’−Ｏ−(4,4’−ジメトキシトリチル)−5−[3−（Ｎ−トリフルオロアセチル)−アミノプロピル]−2’−Ｏ−メチルウリジン (250 mg, 0.35 mmol) を無水アセトニトリルで3回、無水ジクロロメタンで1回共沸脱水した後、無水ジクロロメタン(3.5 mL)に溶解し、1−H テトラゾール(14.7 mg, 0.21 mmol)及びジイソプロピルアミン(29.5 μL, 0.21 mmol)を加えて、アルゴン雰囲気下、室温で10分撹拌した。さらに、(2−シアノエトキシ)−ビス−(Ｎ，Ｎ−ジイソプロピルアミノ)ホスフィン(133μL, 0.42 mmol)を加えて、アルゴン雰囲気下、室温で5時間撹拌した。１ｍｌの水を加えて反応を停止した後、２０ｍｌのクロロホルムで希釈して飽和食塩水で3回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をジエチルエーテル：ジイソプロピルエーテル=1：1の混合溶媒で希釈し、0.1 M水酸化ナトリウム水溶液で5回洗浄した。有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をリサイクル分取HPLC（アセトニトリル）で精製し、5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピル]−2’−Ｏ−メチルウリジン 3’−Ｏ−(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（式（３）で表わされる化合物）を得た(140 mg, 収率：44%)。
１H NMR (270 MHz, CDCl３)δ0.91−1.10 (14H, m), 1.19−1.28 (2H, m), 1.50−1.88 (2H, m), 2.26−2.31 (1H, m), 2.52−2.57 (1H, m), 2.81−3.23 (2H, m), 3.43 (3H, s), 3.43−3.57 (2H, m), 3.68, 3.69 (6H, 2s), 3.80−3.91 (1H, m), 4.05−4.19 (1H, m), 4.32−4.48 (1H, m), 5.90−6.06 (1H, m), 7.17−7.35 (10H, m), 7.44, 7.50 (1H, 2s);１３C NMR (67.8 MHz, CDCl３)δ20.05, 20.16, 20.24, 20.32, 23.05, 24.39, 24.50, 24.59, 28.28, 38.27, 42.95, 43.13, 43.31, 55.17, 57.70, 57.99, 58.18, 58.50, 58.74, 61.64, 62.40, 70.11, 70.33, 82.39, 82.82, 83.52, 86.70, 86.84, 87.32, 113.12, 113.79, 114.02, 117.47, 117.99, 127.24, 127.86, 128.22, 128.39, 128.94, 130.18, 130.30, 135.05, 135.13, 136.38, 144.05, 156.69, 157.23, 158.71;３１P NMR (109.4 MHz, CDCL３)δ151.17, 151.23; ESI−mass m/z Calcd for C４５H５６F３N５O１０P 914.3717; Observed [M ＋ H] 914.3711
5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl] -2'-O-methyluridine (250 mg) obtained as described above. , 0.35 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile and once with anhydrous dichloromethane, then dissolved in anhydrous dichloromethane (3.5 mL), and 1-H tetrazole (14.7 mg, 0.21 mmol) and diisopropylamine (29.5 μL, 0.21 mmol) was added, and the mixture was stirred at room temperature for 10 minutes under an argon atmosphere. Furthermore, (2-cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (133 μL, 0.42 mmol) was added, and the mixture was stirred at room temperature for 5 hours under an argon atmosphere. The reaction was stopped by adding 1 ml of water, diluted with 20 ml of chloroform and washed 3 times with saturated saline. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was diluted with a mixed solvent of diethyl ether: diisopropyl ether = 1: 1 and washed 5 times with 0.1 M aqueous sodium hydroxide solution. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by recycle preparative HPLC (acetonitrile) and 5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyl] -2'-O. -Methyluridine 3'-O- (2-cyanoethyl) -N, N-diisopropyl phosphoramidite (compound represented by formula (3)) was obtained (140 mg, yield: 44%).
1 H NMR (270 MHz, CDCl 3 ) δ0.91-1.10 (14H, m), 1.19-1.28 (2H, m), 1.50-1.88 (2H, m), 2.26-2.31 (1H, m), 2.52− 2.57 (1H, m), 2.81−3.23 (2H, m), 3.43 (3H, s), 3.43−3.57 (2H, m), 3.68, 3.69 (6H, 2s), 3.80−3.91 (1H, m), 4.05−4.19 (1H, m), 4.32−4.48 (1H, m), 5.90−6.06 (1H, m), 7.17−7.35 (10H, m), 7.44, 7.50 (1H, 2s); 13 C NMR (67.8 MHz, CDCl 3 ) δ20.05, 20.16, 20.24, 20.32, 23.05, 24.39, 24.50, 24.59, 28.28, 38.27, 42.95, 43.13, 43.31, 55.17, 57.70, 57.99, 58.18, 58.50, 58.74, 61.64, 62.40, 70.11 , 70.33, 82.39, 82.82, 83.52, 86.70, 86.84, 87.32, 113.12, 113.79, 114.02, 117.47, 117.99, 127.24, 127.86, 128.22, 128.39, 128.94, 130.18, 130.30, 135.05, 135.13, 136.38, 144.05, 156.69, 15 , 158.71; 31 P NMR (109.4 MHz, CDCL 3 ) δ 151.17, 151.23; ESI-mass m / z Calcd for C 45 H 56 F 3 N 5 O 10 P 914.3717; Observed [M + H] 914.3711
製造例１の合成反応を式で表わすと以下の通りである。 The synthesis reaction of Production Example 1 is represented by the following formula.
製造例２
5’−Ｏ− (4,4’−ジメトキシトリチル)−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジン 3’−Ｏ− (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
既知の化合物である、3’， 5’−ビス-アセチル−5−ヨード−2’−Ｏ−メチルウリジン(2.0 g, 4.3 mmol)を、３ｍｌの無水アセトニトリルで3回、３ｍｌの無水ジメチルホルムアミドで1回共沸脱水した後、無水ジメチルホルムアミド(23 mL)に溶解し、減圧しながら超音波脱気を5回行い、アルゴン置換した。トリエチルアミン(1.63 mL, 8.6 mmol)、テトラキス（トリフェニルホスフィン）パラジウム（０）(248 mg, 0.215 mmol)、ヨウ化銅(81.9 mg, 0.43 mmol)を加えて、室温で10分間撹拌した。Ｎ−トリフルオロアセチル-アミノプロピン(730 μL, 6.4 mmol)を脱気済みの無水ジメチルホルムアミド(20 mL)に溶解させ、滴下ロートを用いて反応系に滴下させ、アルゴン雰囲気下、室温で10時間撹拌した。反応溶液を１５０ｍｌの酢酸エチルで希釈し、5%炭酸水素ナトリウム水溶液で3回洗浄し、1%アンモニア水で2回洗浄した。有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した後、残留物をシリカゲルカラムクロマトグラフィー（ヘキサン：酢酸エチル=60：40, ヘキサン：クロロホルム=20:80）で精製し、3’, 5’−ビス-アセチル−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジンを得た(1.41 g,収率： 67%)。
１H NMR (270 MHz, CDCl３)δ2.13, 2.17 (6H, 2s, Ac), 3.48 (3H, s, 2’−Ｏ−Me), 4.04−4.07 (1H, m, 5’−H), 4.29−4.42 (5H, m, 3’−H, 4’−H, 5’−H, CH２−NH), 4.89−4.94 (1H, m, 2’-H), 5.90 (1H, d, 1’-H, Ｊ1’,2’=2.3 Hz), 7.69 (1H, bs, NH), 7.92 (1H, s, 6−H), 9.77 (1H, s, 3−NH);１３C NMR (67.8 MHz, CDCl３) δ20.56, 20.68, 30.26, 59.00, 61.77, 69.37, 75.23, 79.35, 81.77, 87.87, 88.63, 99.02, 113.55, 117.78, 142.96, 148.98, 156.75, 157.31, 162.02, 1770.23, 170.32; ESI−mass m/z Calcd for C１９H２１F３N３O９492.1230; Observed [M + H] 492.1290
Production Example 2
5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2'-O-methyluridine 3'-O- ( Synthesis of 2-cyanoethyl) -N, N-diisopropyl phosphoramidite 3 ′, 5′-bis-acetyl-5-iodo-2′-O-methyluridine (2.0 g, 4.3 mmol), a known compound After azeotropic dehydration 3 times with 3 ml of anhydrous acetonitrile and once with 3 ml of anhydrous dimethylformamide, the product was dissolved in anhydrous dimethylformamide (23 mL), and ultrasonic deaeration was performed 5 times while reducing the pressure, followed by substitution with argon. Triethylamine (1.63 mL, 8.6 mmol), tetrakis (triphenylphosphine) palladium (0) (248 mg, 0.215 mmol) and copper iodide (81.9 mg, 0.43 mmol) were added, and the mixture was stirred at room temperature for 10 minutes. N-trifluoroacetyl-aminopropyne (730 μL, 6.4 mmol) is dissolved in degassed anhydrous dimethylformamide (20 mL) and added dropwise to the reaction system using a dropping funnel. Stir for hours. The reaction solution was diluted with 150 ml of ethyl acetate, washed 3 times with 5% aqueous sodium hydrogen carbonate solution and twice with 1% aqueous ammonia. The organic layer was collected and dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 60: 40, hexane: chloroform = 20: 80) 3 ′, 5′-bis-acetyl-5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2′-O-methyluridine was obtained (1.41 g, yield: 67 %).
1 H NMR (270 MHz, CDCl3) δ 2.13, 2.17 (6H, 2s, Ac), 3.48 (3H, s, 2'-O-Me), 4.04-4.07 (1H, m, 5'-H), 4.29-4.42 (5H, m, 3'- H, 4'-H, 5'-H, CH 2 -NH), 4.89-4.94 (1H, m, 2'-H), 5.90 (1H, d, 1 '-H, J 1', 2 ' = 2.3 Hz), 7.69 (1H, bs, NH), 7.92 (1H, s, 6-H), 9.77 (1H, s, 3-NH); 13 C NMR ( 67.8 MHz, CDCl 3 ) δ20.56, 20.68, 30.26, 59.00, 61.77, 69.37, 75.23, 79.35, 81.77, 87.87, 88.63, 99.02, 113.55, 117.78, 142.96, 148.98, 156.75, 157.31, 162.02, 1770.23, 170.32; ESI-mass m / z Calcd for C 19 H 21 F 3 N 3 O 9 492.1230; Observed [M + H] 492.1290
上述のようにして得られた3’, 5’−ビス-アセチル−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジン(870 mg, 1.77 mmol)をピリジン、28%アンモニア水(v/v=1:1)の混合溶媒(18 mL)に溶解し、50℃の温度で8時間撹拌した。溶媒を減圧下留去した後、残留物をエタノール(18 mL)に溶解し、トリフルオロ酢酸エチルエステル(844μL, 7.08 mmol)とトリエチルアミン(1.68 mL, 8.85 mmol)を加えて、室温で2時間撹拌した。溶媒を減圧下留去し、残留物を無水ピリジンで3回共沸脱水した後、無水ピリジン(18 mL)に溶解し、4,4’−ジメトキシトリチルクロリド(718.3 mg, 2.12 mmol)を加えて、アルゴン雰囲気下、室温で3時間撹拌した。メタノールを加えて反応を停止した後、反応溶液を５０ｍｌのクロロホルムで希釈して5%炭酸水素ナトリウム水溶液で3回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル=50：50）で精製し、5’−Ｏ−(4,4’−ジメトキシトリチル)−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジンを得た (800 mg,収率：50%)。
１H NMR (270 MHz, CDCl３)δ3.47 (2H, s, CH２), 3.63 (3H, s, 2’−Ｏ−CH３), 3.77−3.81 (8H, m, OCH３of DMTr, 5’−H), 3.92−3.97 (1H, m, 4’−H), 4.08−4.11(1H, m, 3’−H), 4.52−4.55 (1H, m, 2’−H), 5.93 (1H, s, 1’−H), 6.82−6.86 (4H, m, ArH of DMTr), 7.21−7.54 (10H, m, ArH of DMTr, NH), 8.25 (1H, s, 6−H);１３C NMR (67.8 MHz, CDCl３)δ30.17, 55.12, 58.74, 61.64, 68.45, 75.02, 83.72, 86.84, 87.51, 98.95, 113.31, 117.63, 121.87, 126.87, 127.81, 128.00, 129.88, 129.94, 135.40, 135.43, 143.20, 144.52, 149.31, 155.66, 156.22, 156.77, 157.33, 158.51, 158.55, 162.46; ESI−mass m/z Calcd for C３６H３４F３N３NaO９732.2145; Observed [M + Na] 732.2127
3 ′, 5′-bis-acetyl-5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2′-O-methyluridine (870 mg) obtained as described above. , 1.77 mmol) was dissolved in a mixed solvent (18 mL) of pyridine and 28% aqueous ammonia (v / v = 1: 1) and stirred at a temperature of 50 ° C. for 8 hours. After the solvent was distilled off under reduced pressure, the residue was dissolved in ethanol (18 mL), ethyl trifluoroacetate (844 μL, 7.08 mmol) and triethylamine (1.68 mL, 8.85 mmol) were added, and the mixture was stirred at room temperature for 2 hours. did. The solvent was distilled off under reduced pressure, the residue was azeotropically dehydrated three times with anhydrous pyridine, dissolved in anhydrous pyridine (18 mL), and 4,4'-dimethoxytrityl chloride (718.3 mg, 2.12 mmol) was added. The mixture was stirred at room temperature for 3 hours under an argon atmosphere. After stopping the reaction by adding methanol, the reaction solution was diluted with 50 ml of chloroform and washed 3 times with 5% aqueous sodium hydrogen carbonate solution. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. did. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 50: 50), and 5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopro Pin-1-yl] -2′-O-methyluridine was obtained (800 mg, yield: 50%).
1 H NMR (270 MHz, CDCl 3 ) δ 3.47 (2H, s, CH 2 ), 3.63 (3H, s, 2′-O—CH 3 ), 3.77-3.81 (8H, m, OCH 3 of DMTr, 5'−H), 3.92−3.97 (1H, m, 4′−H), 4.08−4.11 (1H, m, 3′−H), 4.52−4.55 (1H, m, 2′−H), 5.93 ( 1H, s, 1'-H), 6.82-6.86 (4H, m, ArH of DMTr), 7.21-7.54 (10H, m, ArH of DMTr, NH), 8.25 (1H, s, 6-H); 13 C NMR (67.8 MHz, CDCl 3 ) δ30.17, 55.12, 58.74, 61.64, 68.45, 75.02, 83.72, 86.84, 87.51, 98.95, 113.31, 117.63, 121.87, 126.87, 127.81, 128.00, 129.88, 129.94, 135.40, 135.43 , 143.20, 144.52, 149.31, 155.66, 156.22, 156.77, 157.33, 158.51, 158.55, 162.46; ESI-mass m / z Calcd for C 36 H 34 F 3 N 3 NaO 9 732.2145; Observed [M + Na] 732.2127
上述のようにして得られた5’−Ｏ−(4,4’−ジメトキシトリチル)−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジン (400 mg, 0.56 mmol)を無水アセトニトリルで3回、無水ジクロロメタンで1回共沸脱水した後、無水ジクロロメタン(5.6 mL)に溶解し、1−H テトラゾール(23.8 mg, 0.34 mmol)とジイソプロピルアミン(47.8 μL, 0.34 mmol)を加えて、アルゴン雰囲気下、室温で10分撹拌した。さらに、(2のシアノエトキシ)−ビス−(Ｎ，Ｎ−ジイソプロピルアミノ)ホスフィン(212 μL, 0.67 mmol)を加えて、アルゴン雰囲気下、室温で3時間撹拌した。１ｍｌの水を加えて反応を停止した後、３０ｍｌのクロロホルムで希釈して飽和食塩水で3回洗浄し、有機層を集めて無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残留物をジエチルエーテル：ジイソプロピルエーテル=1：1の混合溶媒で希釈し、0.1 M水酸化ナトリウム水溶液で5回洗浄した。この際に析出した粉体を、無水テトラヒドロフラン(5.6 mL)に溶解し、トリフルオロ酢酸エチルエステル(267μL, 2.24 mmol)とトリエチルアミン(850 mL, 4.48 mmol)を加えて、室温で1時間撹拌した。溶媒を減圧下留去し、残留物をリサイクル分取HPLC（アセトニトリル）で精製し、5’−Ｏ−(4,4’−ジメトキシトリチル)−5−[3−(Ｎ−トリフルオロアセチル)−アミノプロピン−1−イル]−2’−Ｏ−メチルウリジン 3−Ｏ−(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（式（２）で表わされる化合物）を得た(200 mg,収率：40%)。
1H NMR (270 MHz, CDCl３)δ0.96−0.98 (4H, m), 1.07−1.11(10H, m), 2.29−2.31(1H, m),2.51−2.55 (1H, m), 3.31−3.52 (6H, m), 3.52−3.83 (7H, m), 3.98−4.05 (1H, m), 4.13−4.22 (1H, m), 4.40−4.59(1H, m), 5.57−5.86 (1H, m), 6.75−6.78 (4H, m), 7.04 (1H, bs), 7.07−7.41 (9H, m), 8.15, 8.19 (1H, 2s,); １３C NMR (67.8 MHz, CDCl３)δ7.47, 20.27, 20.31, 20.37, 24.36, 24.43, 24.47, 24.54, 24.64, 30.12, 43.04, 43.23, 55.13, 58.18, 58.37, 58.40, 58.46, 58.83, 58.87, 75.34, 75.51, 76.53, 82.74, 86.64, 86.80, 86.84, 86.86, 88.14, 88.60, 98.61, 98.91, 113.22, 113.33, 117.56, 117.59, 117.75, 126.81, 126.85, 127.90, 127.93, 128.10, 130.03, 135.32, 135.39, 135.44, 135.46, 143.40, 144.49, 144.63, 149.32, 149.40, 156.10, 156.65, 158.51, 158.55, 162.13, 162.27; ３１P NMR (109.4 MHz, CDCL３) δ150.58, 150.97; ESI−mass m/z Calcd for C４５H５１F３N５NaO１０P 932.3223; Observed [M ＋ Na] 932.3892
製造例２の合成反応を式で表わすと以下の通りである。
5'-O- (4,4'-dimethoxytrityl) -5- [3- (N-trifluoroacetyl) -aminopropyn-1-yl] -2'-O- obtained as described above. Methyluridine (400 mg, 0.56 mmol) was azeotropically dehydrated three times with anhydrous acetonitrile and once with anhydrous dichloromethane, then dissolved in anhydrous dichloromethane (5.6 mL), and 1-H tetrazole (23.8 mg, 0.34 mmol) and diisopropyl Amine (47.8 μL, 0.34 mmol) was added, and the mixture was stirred at room temperature for 10 minutes under an argon atmosphere. Further, (2 cyanoethoxy) -bis- (N, N-diisopropylamino) phosphine (212 μL, 0.67 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon atmosphere. The reaction was stopped by adding 1 ml of water, diluted with 30 ml of chloroform and washed 3 times with saturated saline. The organic layer was collected and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was diluted with a mixed solvent of diethyl ether: diisopropyl ether = 1: 1 and washed 5 times with 0.1 M aqueous sodium hydroxide solution. The powder precipitated at this time was dissolved in anhydrous tetrahydrofuran (5.6 mL), trifluoroacetic acid ethyl ester (267 μL, 2.24 mmol) and triethylamine (850 mL, 4.48 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and the residue was purified by recycle preparative HPLC (acetonitrile), and 5′-O- (4,4′-dimethoxytrityl) -5- [3- (N-trifluoroacetyl)- Aminopropyn-1-yl] -2′-O-methyluridine 3-O- (2-cyanoethyl) -N, N-diisopropyl phosphoramidite (compound represented by formula (2)) was obtained (200 mg). Yield: 40%).
1 H NMR (270 MHz, CDCl 3 ) δ0.96-0.98 (4H, m), 1.07-1.11 (10H, m), 2.29-2.31 (1H, m), 2.51-2.55 (1H, m), 3.31- 3.52 (6H, m), 3.52−3.83 (7H, m), 3.98−4.05 (1H, m), 4.13−4.22 (1H, m), 4.40−4.59 (1H, m), 5.57−5.86 (1H, m ), 6.75-6.78 (4H, m), 7.04 (1H, bs), 7.07-7.41 (9H, m), 8.15, 8.19 (1H, 2s,); 13 C NMR (67.8 MHz, CDCl 3 ) δ 7.47 , 20.27, 20.31, 20.37, 24.36, 24.43, 24.47, 24.54, 24.64, 30.12, 43.04, 43.23, 55.13, 58.18, 58.37, 58.40, 58.46, 58.83, 58.87, 75.34, 75.51, 76.53, 82.74, 86.64, 86.80, 86.84 , 86.86, 88.14, 88.60, 98.61, 98.91, 113.22, 113.33, 117.56, 117.59, 117.75, 126.81, 126.85, 127.90, 127.93, 128.10, 130.03, 135.32, 135.39, 135.44, 135.46, 143.40, 144.49, 144.63, 149.3 , 156.10, 156.65, 158.51, 158.55, 162.13, 162.27; 31 P NMR (109.4 MHz, CDCL 3 ) δ 150.58, 150.97; ESI-mass m / z Calcd for C 45 H 51 F 3 N 5 NaO 10 P 932.3223; Observed [M + Na] 932.3892
The synthesis reaction of Production Example 2 is represented by the following formula.
実施例１
製造例１及び２、比較製造例１で得られたヌクレオシドホスホロアミダイト化合物を用いて、配列５’−ＣＧＵＵＵ＊ＵＵＧＣ−３’（配列番号：１）の２’−Ｏ−メチル化された９量体中Ｕ＊の部位に修飾核酸を組み込んだオリゴヌクレオチドを合成した。オリゴヌクレオチドの合成は、通常のホスホロアミダイト法によりＡＢＩ３９２自動合成機（Applied Biosystems）により行い、固相担体からの切り出し、及び塩基部の脱保護操作を行った後、Ｓｅｐ−ｐａｋ Ｃ１８カートリッジカラムによりトリチルＯＮ簡易精製を行い、必要に応じて陰イオンＨＰＣＬによって更に精製を行った。合成した各オリゴヌクレオチドと、別途合成を行った相補鎖（配列５’−ＧＣＡＡＡＡＡＣＧ−３’（配列番号：２）又は５’−ＧＣＡＡＧＡＡＣＧ−３’（配列番号：３）とを混和し、溶液中二重鎖形成時に、二重鎖の濃度が２μＭの濃度となるように、５００μＬのリン酸バッファー（１０ｍＭリン酸ナトリウム、ｐＨ７．０、１５０ｍＭ ＮａＣｌ、０．１ｍＭ ＥＤＴＡ）に溶解させた測定試料を調整した。測定には、Ｐｈａｒｍａ Ｓｐｅｃ ＵＶ−１７００（島津製作所（株）製）を用いた。測定は、まず試料を８０℃の温度に３０分間保持し、オリゴヌクレオチドをランダムコイル状態とした後、１．０℃／分で温度を５℃まで変化させてアニーリングを行い、次いで、１．０℃／分の速度で昇温させて、１．０℃毎にＵＶ吸光度を測定することにより行った。
測定により得られたＵＶ吸光度を温度変化に対してプロットし、二重鎖融解曲線とした。Ｓｔａｖｉｌｚｋｙ−Ｇｏｌａｙ法（２５point）を用いて融解曲線をスムージングした後、曲線を一次微分することにより変曲点を求め変曲点を二重鎖融解温度（Ｔｍ値）とした。なお、二重鎖のアニーリング時の曲線から得られる値と二重鎖融解時の曲線から得られる値において、１．０℃以上値のずれが観測された場合には、各測定温度における系内の平衡が不完全であるとみなし、変温レートを０．５℃／分に変更する等、適宜条件を変更して再度測定を行った。
Example 1
Using the nucleoside phosphoramidite compounds obtained in Production Examples 1 and 2 and Comparative Production Example 1, 2′-O-methylated 9 of sequence 5′-CGUUU * UUGC-3 ′ (SEQ ID NO: 1) An oligonucleotide incorporating a modified nucleic acid at the U * site in the monomer was synthesized. Oligonucleotides were synthesized by an ordinary ABI 392 synthesizer (Applied Biosystems) by the usual phosphoramidite method, cut out from the solid phase carrier, and deprotected at the base, and then separated by a Sep-pak C18 cartridge column. Trityl ON simple purification was performed, and further purification was performed with anion HPCL as necessary. Each synthesized oligonucleotide and a separately synthesized complementary strand (sequence 5′-GCAAAACG-3 ′ (SEQ ID NO: 2) or 5′-GCAAGACG-3 ′ (SEQ ID NO: 3)) are mixed and mixed in a solution. At the time of duplex formation, a measurement sample dissolved in 500 μL of phosphate buffer (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 0.1 mM EDTA) so that the concentration of the duplex becomes 2 μM. For the measurement, Pharma Spec UV-1700 (manufactured by Shimadzu Corporation) was used, and the measurement was performed by first holding the sample at a temperature of 80 ° C. for 30 minutes, and then setting the oligonucleotide in a random coil state. Annealing is performed by changing the temperature to 5 ° C. at 1.0 ° C./min, and then the temperature is increased at a rate of 1.0 ° C./min, and UV absorbance is measured every 1.0 ° C. It was carried out by.
The UV absorbance obtained by the measurement was plotted against the temperature change to obtain a double chain melting curve. After smoothing the melting curve using the Stabilzky-Golay method (25 points), the curve was linearly differentiated to obtain the inflection point, and the inflection point was defined as the double chain melting temperature (Tm value). In addition, in the value obtained from the curve at the time of annealing of the double chain and the value obtained from the curve at the time of melting the double chain, when a deviation of 1.0 ° C. or more is observed, The equilibrium was considered to be incomplete, and the measurement was performed again while changing the conditions as appropriate, such as changing the temperature change rate to 0.5 ° C./min.
比較製造例１で得られた化合物を用いてＡ型ＲＮＡ二重鎖に組み込んだ場合、２’−Ｏ−メチルウリジンが組み込まれた場合と比較し、融解温度の差（ΔＴｍ値）は−１．２℃であり、比較製造例１で得られた化合物をＡ型ＲＮＡ二重鎖に組み込んだ場合でも二重鎖の安定化は認められなかった。製造例１で得られた化合物をＡ型ＲＮＡ二重鎖に組み込んだ場合、２’−Ｏ−メチルウリジンが組み込まれた場合と比較し、融解温度の差（ΔＴｍ値）は＋３．２℃であり、Ａ型ＲＮＡ二重鎖が安定化されることがわかった。これは、二重鎖を歪ませることなく、アミノ基とリン酸基とが近づいていることを示し、Ａ型ＲＮＡ二重鎖を安定化させるためには、炭素数３のリンカーを導入することがよいことが示される。また、製造例２で得られた化合物をＡ型ＲＮＡ二重鎖に組み込んだ場合、２’−Ｏ−メチルウリジンが組み込まれた場合と比較し、融解温度の差（ΔＴｍ値）は＋６．９℃であり、大幅にＡ型ＲＮＡ二重鎖を安定化することが認められた。 When the compound obtained in Comparative Production Example 1 was incorporated into an A-type RNA duplex, the difference in melting temperature (ΔTm value) was −1 compared to the case where 2′-O-methyluridine was incorporated. The temperature was 2 ° C., and even when the compound obtained in Comparative Production Example 1 was incorporated into the A-type RNA duplex, duplex stabilization was not observed. When the compound obtained in Production Example 1 was incorporated into the A-type RNA duplex, the difference in melting temperature (ΔTm value) was + 3.2 ° C. compared to the case where 2′-O-methyluridine was incorporated. It was found that the A-type RNA duplex was stabilized. This indicates that the amino group and the phosphate group are close to each other without distorting the duplex, and in order to stabilize the A-type RNA duplex, a linker having 3 carbon atoms must be introduced. Is shown to be good. Further, when the compound obtained in Production Example 2 was incorporated into an A-type RNA duplex, the difference in melting temperature (ΔTm value) was +6.9 compared to the case where 2′-O-methyluridine was incorporated. It was observed that the temperature of the A-type RNA duplex was greatly stabilized.
実施例２
製造例１及び２、比較製造例１の相補的位置の塩基がＡの時のＴｍ値から、相補的位置の塩基がＧの時のＴｍ値を差し引き、その値を塩基識別能力とした。
Example 2
The Tm value when the base at the complementary position was G was subtracted from the Tm value when the base at the complementary position in Production Examples 1 and 2 and Comparative Production Example 1 was A, and the value was defined as the base discrimination ability.
結果を図１に示す。図１は、塩基識別能力の測定結果を示すグラフであり、比較製造例１で得られたヌクレオシドホスホロアミダイト化合物においては、ΔＴｍＡ−Ｇは７．４℃であり、製造例１及び製造例２で得られたヌクレオシドホスホロアミダイト化合物においては、ΔＴｍＡ−Ｇは、それぞれ、８．１℃及び１０．３℃であった。Ａ型ＲＮＡ二重鎖を安定化する修飾体においては、しばしば塩基識別能力が低下することが知られているが、本発明のヌクレオシドホスホロアミダイト化合物を用いた場合、塩基識別能力が低下せず、むしろ塩基識別能力が向上していることがわかった。 The results are shown in FIG. FIG. 1 is a graph showing measurement results of base discrimination ability. In the nucleoside phosphoramidite compound obtained in Comparative Production Example 1, ΔTm AG is 7.4 ° C., and Production Example 1 and Production Example In the nucleoside phosphoramidite compound obtained in 2, ΔTm AG was 8.1 ° C. and 10.3 ° C., respectively. It is known that in the modified form that stabilizes the A-type RNA duplex, the base discrimination ability is often lowered. However, when the nucleoside phosphoramidite compound of the present invention is used, the base discrimination ability is not lowered. Rather, it was found that the base discrimination ability was improved.
Claims (9)
A primer for starting amplification of a specific gene using the oligonucleic acid according to claim 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005067121A JP2006248975A (en) | 2005-03-10 | 2005-03-10 | Nucleoside phosphoroamidite compound |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005067121A JP2006248975A (en) | 2005-03-10 | 2005-03-10 | Nucleoside phosphoroamidite compound |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2006248975A true JP2006248975A (en) | 2006-09-21 |
Family
ID=37089841
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2005067121A Pending JP2006248975A (en) | 2005-03-10 | 2005-03-10 | Nucleoside phosphoroamidite compound |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2006248975A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8871737B2 (en) | 2010-09-22 | 2014-10-28 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| US8916538B2 (en) | 2012-03-21 | 2014-12-23 | Vertex Pharmaceuticals Incorporated | Solid forms of a thiophosphoramidate nucleotide prodrug |
| US8980865B2 (en) | 2011-12-22 | 2015-03-17 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| US9012427B2 (en) | 2012-03-22 | 2015-04-21 | Alios Biopharma, Inc. | Pharmaceutical combinations comprising a thionucleotide analog |
-
2005
- 2005-03-10 JP JP2005067121A patent/JP2006248975A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8871737B2 (en) | 2010-09-22 | 2014-10-28 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| US9278990B2 (en) | 2010-09-22 | 2016-03-08 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| US8980865B2 (en) | 2011-12-22 | 2015-03-17 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| US9605018B2 (en) | 2011-12-22 | 2017-03-28 | Alios Biopharma, Inc. | Substituted nucleotide analogs |
| US8916538B2 (en) | 2012-03-21 | 2014-12-23 | Vertex Pharmaceuticals Incorporated | Solid forms of a thiophosphoramidate nucleotide prodrug |
| US9394330B2 (en) | 2012-03-21 | 2016-07-19 | Alios Biopharma, Inc. | Solid forms of a thiophosphoramidate nucleotide prodrug |
| US9856284B2 (en) | 2012-03-21 | 2018-01-02 | Alios Biopharma, Inc. | Solid forms of a thiophosphoramidate nucleotide prodrug |
| US9012427B2 (en) | 2012-03-22 | 2015-04-21 | Alios Biopharma, Inc. | Pharmaceutical combinations comprising a thionucleotide analog |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109937042B (en) | Synthesis of backbone modified morpholino oligonucleotides and chimeras using phosphoramidite chemistry | |
| RU2708237C2 (en) | Modified oligonucleotides and method for production thereof | |
| EP2370451B1 (en) | Method for the synthesis of phosphorus atom modified nucleic acids | |
| CN110831951B (en) | Multiple coupling and oxidation process | |
| JP5520818B2 (en) | Thiocarbon protecting group for RNA synthesis | |
| US9751909B2 (en) | Synthesis and uses of nucleic acid compounds with conformationally restricted monomers | |
| EP4219516A2 (en) | Chiral control | |
| JP5030998B2 (en) | Nucleoside analogues and oligonucleotide derivatives containing the nucleotide analogues thereof | |
| WO2002018388A1 (en) | Novel nucleoside analogs and oligonucleotide derivatives containing these analogs | |
| Hari et al. | Synthesis and properties of 2′-O, 4′-C-methyleneoxymethylene bridged nucleic acid | |
| WO2006078278A2 (en) | Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety | |
| JP2013520438A (en) | Phosphoramidites for reverse synthetic RNA | |
| AU2017203685B2 (en) | 3'end caps for RNAi agents for use in RNA interference | |
| BR112020010090A2 (en) | gapmer oligonucleotide, pharmaceutically acceptable salt thereof, conjugate, pharmaceutical composition and use thereof | |
| CN111868244A (en) | Antisense oligonucleotides with reduced toxicity | |
| JP2006248975A (en) | Nucleoside phosphoroamidite compound | |
| JP2000512630A (en) | 2'-substituted nucleoside and oligonucleotide derivatives | |
| EP2006293B1 (en) | 2'-hydroxyl-modified ribonucleoside derivative | |
| GB2366290A (en) | Hexitol Nucleosides | |
| WO2023131170A1 (en) | Double-stranded rna having nucleotide analog | |
| Seio et al. | Short-RNA selective binding of oligonucleotides modified using adenosine and guanosine derivatives that possess cyclohexyl phosphates as substituents | |
| EP2854813B1 (en) | Pyrazolotriazolyl nucleoside analogues and oligonucleotides comprising them | |
| BR112020012427A2 (en) | oligonucleotide, pharmaceutically acceptable salt, conjugate, pharmaceutical composition, process for making an oligonucleotide, manufactured oligonucleotide and invention | |
| JP7173467B2 (en) | Nucleoside derivative and its use | |
| JP5424236B2 (en) | Oligonucleotide derivative, oligonucleotide construct using oligonucleotide derivative, compound for synthesizing oligonucleotide derivative, and method for producing oligonucleotide derivative |