JP6429264B2 - Boranophosphate compounds and nucleic acid oligomers - Google Patents
Boranophosphate compounds and nucleic acid oligomers Download PDFInfo
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- JP6429264B2 JP6429264B2 JP2013234384A JP2013234384A JP6429264B2 JP 6429264 B2 JP6429264 B2 JP 6429264B2 JP 2013234384 A JP2013234384 A JP 2013234384A JP 2013234384 A JP2013234384 A JP 2013234384A JP 6429264 B2 JP6429264 B2 JP 6429264B2
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- nucleic acid
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- boranophosphate
- acid oligomer
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- 230000003287 optical effect Effects 0.000 description 1
- 125000003452 oxalyl group Chemical group *C(=O)C(*)=O 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- RLOWWWKZYUNIDI-UHFFFAOYSA-N phosphinic chloride Chemical compound ClP=O RLOWWWKZYUNIDI-UHFFFAOYSA-N 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000003746 solid phase reaction Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- ZMANZCXQSJIPKH-UHFFFAOYSA-O triethylammonium ion Chemical compound CC[NH+](CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-O 0.000 description 1
- KYWVDGFGRYJLPE-UHFFFAOYSA-N trimethylazanium;acetate Chemical compound CN(C)C.CC(O)=O KYWVDGFGRYJLPE-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Saccharide Compounds (AREA)
Description
本発明は、ボラノホスフェート化合物、及び核酸オリゴマーに関する。 The present invention relates to a boranophosphate compound and a nucleic acid oligomer.
標的核酸と相補的な塩基配列を有するアンチセンス分子は、標的核酸と相補的な二重鎖を形成し、標的核酸からのタンパク質生成を阻害することができる。標的核酸として疾病関連遺伝子を選択した場合には、アンチセンス分子は、疾病関連遺伝子に直接働きかけるため、遺伝子治療に有効な医薬として注目されている。 An antisense molecule having a base sequence complementary to the target nucleic acid forms a duplex complementary to the target nucleic acid, and can inhibit protein production from the target nucleic acid. When a disease-related gene is selected as a target nucleic acid, an antisense molecule directly acts on a disease-related gene, and thus has attracted attention as a drug effective for gene therapy.
アンチセンス分子（核酸オリゴマー）は、標的となるタンパク質の生成を効率よく阻害する観点から主として、細胞膜透過性、ヌクレアーゼ耐性、体内（例えば、ｐＨ７．４の環境下）での化学的安定性、及び特定の塩基配列とのみ安定な二重鎖を形成する性質を有することが求められる。アンチセンス分子としては、例えば、ホスホロチオエート化合物を用いて得られる核酸オリゴマー（以下、「ホスホロチオエート型核酸オリゴマー」と称する）が知られている（非特許文献１）。 Antisense molecules (nucleic acid oligomers) are mainly composed of cell membrane permeability, nuclease resistance, chemical stability in the body (for example, in an environment of pH 7.4), and from the viewpoint of efficiently inhibiting the production of a target protein. It is required to have a property of forming a stable duplex only with a specific base sequence. As an antisense molecule, for example, a nucleic acid oligomer obtained using a phosphorothioate compound (hereinafter referred to as “phosphorothioate-type nucleic acid oligomer”) is known (Non-patent Document 1).
アンチセンス分子のなかでも、ボラノホスフェート化合物を用いて得られる核酸オリゴマー（以下、「ボラノホスフェート型核酸オリゴマー」と称する）は、優れた水溶性、脂溶性、及び生体内のヌクレアーゼ耐性を有し、かつホスホロチオエート型核酸オリゴマーに比べ、細胞毒性が低いことなどの利点を有することが知られている（非特許文献２）。この観点から、ボラノホスフェート化合物は、アンチセンス分子（核酸オリゴマー）を構成するモノマーとしての使用が提案され、多くの側面から種々検討されている。 Among antisense molecules, nucleic acid oligomers obtained using boranophosphate compounds (hereinafter referred to as “boranophosphate nucleic acid oligomers”) have excellent water solubility, fat solubility, and in vivo nuclease resistance. However, it is known to have advantages such as low cytotoxicity compared to phosphorothioate-type nucleic acid oligomers (Non-patent Document 2). From this point of view, boranophosphate compounds have been proposed for use as monomers constituting antisense molecules (nucleic acid oligomers), and various studies have been made on various aspects.
例えば、特許文献１には、ボラノホスフェート及びその他の含リン酸−ホウ素結合修飾型ＲＮＡの効率的な合成に有用なリボヌクレオチドＨ−ボラノホスホネート化合物が開示されている。また、特許文献２〜３及び非特許文献１〜５には、ボラノホスフェート型核酸オリゴマーの効率的な製造に有用なボラノホスフェート化合物及びそれを用いたオリゴヌクレオチド誘導体の製造方法が開示されている。 For example, Patent Document 1 discloses a ribonucleotide H-boranophosphonate compound useful for efficient synthesis of boranophosphate and other phosphate-boron bond-modified RNAs. Patent Documents 2-3 and Non-Patent Documents 1-5 disclose boranophosphate compounds useful for efficient production of boranophosphate-type nucleic acid oligomers and methods for producing oligonucleotide derivatives using the boranophosphate compounds. Yes.
しかしながら、ボラノホスフェート型核酸オリゴマーは、疎水的、かつ立体障害の大きな官能基であるボラノ基（ＢＨ３）を構造上有しているため、ボラノ基を構造上有さない核酸オリゴマーに比べ、相補鎖との二重鎖形成能が低い。 However, since the boranophosphate type nucleic acid oligomer has a borano group (BH 3 ) which is a hydrophobic and highly sterically hindered functional group in structure, compared with a nucleic acid oligomer which does not have a borano group in structure, Low ability to form double strand with complementary strand.
本発明は、従来のボラノホスフェート型核酸オリゴマーと比較して、相補鎖との二重鎖形成能に優れた核酸オリゴマーを提供することを課題とする。
また、本発明は、上記核酸オリゴマーを合成するのに適した新規ボラノホスフェート化合物を提供することを他の課題とする。
An object of the present invention is to provide a nucleic acid oligomer that is superior in ability to form a double strand with a complementary strand as compared with a conventional boranophosphate type nucleic acid oligomer.
Another object of the present invention is to provide a novel boranophosphate compound suitable for synthesizing the nucleic acid oligomer.
前記課題を解決するための具体的手段は以下の通りである。
＜１＞ 下記一般式（Ｉ）で示されるボラノホスフェート化合物。
Specific means for solving the above problems are as follows.
<1> A boranophosphate compound represented by the following general formula (I).
（一般式（Ｉ）中、Ｒ１は水素原子又は水酸基の保護基を表し、Ｂｓは保護基を有してもよい核酸塩基を表し、Ｘ＋はカウンターカチオンを表す。）
＜２＞ 一般式（Ｉ）におけるＢｓが、アデニン、ウラシル、グアニン、チミン又はシトシンである＜１＞に記載のボラノホスフェート化合物。
＜３＞ 一般式（Ｉ）におけるＲ１が、水酸基の保護基である＜１＞又は＜２＞に記載のボラノホスフェート化合物。
＜４＞ 少なくとも２つの塩基からなる塩基配列を有し、かつ、該塩基配列に含まれる少なくとも１つの塩基が、＜１＞〜＜３＞のいずれか１つに記載のボラノホスフェート化合物に由来するヌクレオシド構造中の塩基である核酸オリゴマー。
＜５＞ 一般式（II）で示される構造及び一般式（III）で示される構造のうち少なくとも１つを有する＜４＞に記載の核酸オリゴマー。
(In general formula (I), R 1 represents a hydrogen atom or a protecting group for a hydroxyl group, Bs represents a nucleobase which may have a protecting group, and X + represents a counter cation.)
<2> The boranophosphate compound according to <1>, wherein Bs in the general formula (I) is adenine, uracil, guanine, thymine, or cytosine.
<3> The boranophosphate compound according to <1> or <2>, wherein R 1 in the general formula (I) is a hydroxyl-protecting group.
<4> Having a base sequence composed of at least two bases, and at least one base contained in the base sequence is derived from the boranophosphate compound according to any one of <1> to <3> A nucleic acid oligomer that is a base in a nucleoside structure.
<5> The nucleic acid oligomer according to <4>, which has at least one of a structure represented by the general formula (II) and a structure represented by the general formula (III).
（一般式（II）中、Ｒ１は水素原子又は水酸基の保護基を表す。一般式（II）及び一般式（III）中、Ｂｓは保護基を有してもよい核酸塩基を表し、Ｘ＋はカウンターカチオンを表す。） (In the general formula (II), R 1 represents a hydrogen atom or hydroxyl protecting group. In the general formula (II) and general formula (III), Bs represents a nucleobase which may have a protecting group, and X + Represents a counter cation.)
本発明によれば、従来のボラノホスフェート型核酸オリゴマーと比較して、相補鎖との二重鎖形成能に優れた核酸オリゴマーを提供することができる。
また、本発明によれば、上記核酸オリゴマーを合成するのに適した新規ボラノホスフェート化合物を提供することができる。
According to the present invention, it is possible to provide a nucleic acid oligomer excellent in duplex forming ability with a complementary strand as compared with a conventional boranophosphate type nucleic acid oligomer.
Moreover, according to this invention, the novel boranophosphate compound suitable for synthesize | combining the said nucleic acid oligomer can be provided.
本発明のボラノホスフェート化合物は、上記一般式（Ｉ）で示される化合物である。この化合物は、核酸オリゴマーを製造するモノマーとして用いることができる。
また、本発明の核酸オリゴマーは、少なくとも２つの塩基からなる塩基配列を有し、かつ、塩基配列に含まれる少なくとも１つの塩基が、一般式（Ｉ）で示されるボラノホスフェート化合物に由来するヌクレオシド構造中の塩基である核酸オリゴマーである。
The boranophosphate compound of the present invention is a compound represented by the above general formula (I). This compound can be used as a monomer for producing a nucleic acid oligomer.
Moreover, the nucleic acid oligomer of the present invention has a base sequence composed of at least two bases, and at least one base contained in the base sequence is derived from a boranophosphate compound represented by the general formula (I) It is a nucleic acid oligomer that is a base in the structure.
本発明のボラノホスフェート化合物は、リボース構造の２位の炭素原子に結合する酸素原子と４位の炭素原子とがメチレン基を介して環化した所謂固定化核酸（locked nucleic acid：ＬＮＡ）構造を有するボラノホスフェート化合物である。そのため、このボラノホスフェート化合物をモノマーとして用いて得られた核酸オリゴマーは、従来のボラノホスフェート型核酸オリゴマーに比べ、核酸オリゴマーと標的核酸との二重鎖形成能を高めることができる。 The boranophosphate compound of the present invention has a so-called locked nucleic acid (LNA) structure in which an oxygen atom bonded to the second carbon atom of the ribose structure and a fourth carbon atom are cyclized via a methylene group. Boranophosphate compound having Therefore, the nucleic acid oligomer obtained using this boranophosphate compound as a monomer can enhance the ability to form a duplex between the nucleic acid oligomer and the target nucleic acid, as compared with the conventional boranophosphate type nucleic acid oligomer.
一般式（Ｉ）で示される固定化核酸（ＬＮＡ）構造を有するボラノホスフェート化合物を、本明細書では、適宜「ＬＮＡ修飾ボラノホスフェート化合物」と称する。
本発明において「モノマー」とは、核酸オリゴマーを製造する際に、核酸オリゴマーのヌクレオシド単位を導入するため使用される核酸を意味する。
The boranophosphate compound having an immobilized nucleic acid (LNA) structure represented by the general formula (I) is appropriately referred to as “LNA-modified boranophosphate compound” in the present specification.
In the present invention, the “monomer” means a nucleic acid used for introducing a nucleoside unit of a nucleic acid oligomer when producing a nucleic acid oligomer.
本明細書において「工程」との語は、独立した工程だけではなく、他の工程と明確に区別できない場合であってもその工程の所期の目的が達成されれば、本用語に含まれる。
また本明細書において「〜」は、その前後に記載される数値をそれぞれ最小値および最大値として含む範囲を示すものとする。
さらに本明細書において組成物中の各成分の量は、組成物中に各成分に該当する物質が複数存在する場合、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。
以下、本発明について説明する。
In this specification, the term “process” is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
In the present specification, “to” indicates a range including the numerical values described before and after the values as a minimum value and a maximum value, respectively.
Furthermore, in this specification, the amount of each component in the composition is the total amount of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. means.
The present invention will be described below.
＜ＬＮＡ修飾ボラノホスフェート化合物＞
本発明のＬＮＡ修飾ボラノホスフェート化合物は、下記一般式（Ｉ）で示される。一般式（Ｉ）中、Ｒ１は水素原子又は水酸基の保護基を表し、Ｂｓは保護基を有してもよい核酸塩基を表し、Ｘ＋はカウンターカチオンを表す。
<LNA modified boranophosphate compound>
The LNA-modified boranophosphate compound of the present invention is represented by the following general formula (I). In general formula (I), R 1 represents a hydrogen atom or a protecting group for a hydroxyl group, Bs represents a nucleobase which may have a protecting group, and X + represents a counter cation.
Ｒ１は、水素原子又は水酸基の保護基を表す。水酸基の保護基としては、例えば、アセチル基、フェノキシアセチル基、ピバロイル基、ベンジル基、４−メトキシベンジル基、ベンゾイル基、トリフェニルメチル基、４，４’−ジメトキシトリチル（ＤＭＴｒ）基、４−メトキシトリチル（ＭＭＴｒ）基、９−フェニルキサンテニル基、トリメチルシリル基、シアノメトキシメチル基、２−（シアノエトキシ）エチル基、シアノエトキシメチル基等を挙げることができる。これらの水酸基の保護基のうち、４，４’−ジメトキシトリチル基が、酸性条件下で容易に除去可能であり、固相合成における鎖長伸長に適しているため、より好ましい。 R 1 represents a hydrogen atom or a hydroxyl-protecting group. Examples of the hydroxyl-protecting group include acetyl group, phenoxyacetyl group, pivaloyl group, benzyl group, 4-methoxybenzyl group, benzoyl group, triphenylmethyl group, 4,4′-dimethoxytrityl (DMTr) group, 4- Examples include methoxytrityl (MMTr) group, 9-phenylxanthenyl group, trimethylsilyl group, cyanomethoxymethyl group, 2- (cyanoethoxy) ethyl group, cyanoethoxymethyl group and the like. Of these hydroxyl protecting groups, the 4,4′-dimethoxytrityl group is more preferable because it can be easily removed under acidic conditions and is suitable for chain length extension in solid phase synthesis.
Ｂｓは保護基を有してもよい核酸塩基を表す。核酸塩基としては、アデニン、ウラシル、チミン、グアニン、及びシトシンからなる群から選ばれる核酸塩基の他、例えば、リボチミジン、５−メチルウリジン等の修飾された核酸塩基を挙げることができる。これらのうち、アデニン、ウラシル、グアニン、チミン及びシトシンが好ましい。 Bs represents a nucleobase which may have a protecting group. Examples of the nucleobase include nucleobases selected from the group consisting of adenine, uracil, thymine, guanine, and cytosine, and modified nucleobases such as ribothymidine and 5-methyluridine. Of these, adenine, uracil, guanine, thymine and cytosine are preferred.
核酸塩基が保護基を有する場合、保護基の種類は特に限定されない。核酸塩基として、アミノ基を有するアデニン、グアニン、及びシトシンが選択される場合、核酸塩基のアミノ基を保護する観点から、保護基として、例えば、ベンジル基、４−メトキシベンゾイル基、アセチル基、プロピオニル基、ブチリル基、イソブチリル基、フェニルアセチル基、４−ｔｅｒｔ−ブチルフェノキシアセチル基、４−イソプロピルフェノキシアセチル基、（ジメチルアミノ）メチレン基等を挙げることができる。 When the nucleobase has a protecting group, the type of protecting group is not particularly limited. When adenine, guanine, and cytosine having an amino group are selected as the nucleobase, from the viewpoint of protecting the amino group of the nucleobase, examples of the protecting group include benzyl group, 4-methoxybenzoyl group, acetyl group, propionyl. Group, butyryl group, isobutyryl group, phenylacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylphenoxyacetyl group, (dimethylamino) methylene group and the like.
Ｘ＋はカウンターカチオンを表す。カウンターカチオンとしては、例えば、有機アミン化合物由来のカチオン、又は金属カチオン等を挙げることができる。有機アミン化合物由来のカチオンとしては、有機溶媒への溶解度、及びカウンターカチオンの揮発性の観点から、例えば、三級アルキルアミン化合物由来のカチオンを挙げることができる。三級アルキルアミン化合物由来のカチオンとしては、例えば、トリエチルアミン由来のカチオン（ＨＮＥｔ３ ＋）を挙げることができる。金属カチオンとしては、Ｎａ＋、Ｌｉ＋、Ｋ＋等が挙げられる。 X + represents a counter cation. Examples of the counter cation include a cation derived from an organic amine compound or a metal cation. Examples of the cation derived from the organic amine compound include a cation derived from a tertiary alkylamine compound from the viewpoint of solubility in an organic solvent and volatility of the counter cation. Examples of the cation derived from the tertiary alkylamine compound include a cation derived from triethylamine (HNEt 3 + ). Examples of the metal cation include Na + , Li + , K + and the like.
一般式（Ｉ）で表されるＬＮＡ修飾ボラノホスフェート化合物は、光学的に純粋な化合物、ラセミ体、ジアステレオ混合物等、任意の形態の物質であってもよい。 The LNA-modified boranophosphate compound represented by the general formula (I) may be any form of a substance such as an optically pure compound, a racemate, a diastereo mixture, and the like.
ＬＮＡ修飾ボラノホスフェート化合物は、公知の方法で製造することができる。具体的には、ＬＮＡ修飾ボラノホスフェート化合物の製造方法は、ＬＮＡ修飾デオキシリボ核酸をＨ−ボラノホスフィニル化剤を用いてＨ−ボラノホスフェート化する工程（ボラノホスフェート化工程）を含むことができる。 The LNA-modified boranophosphate compound can be produced by a known method. Specifically, the method for producing an LNA-modified boranophosphate compound includes a step of converting LNA-modified deoxyribonucleic acid into H-boranophosphate using an H-boranophosphinylating agent (boranophosphate formation step). be able to.
ＬＮＡ修飾デオキシリボ核酸とは、ＬＮＡ構造を有するデオキシリボ核酸を意味し、ＬＮＡ修飾デオキシリボ核酸の有する官能基又は核酸塩基が保護基を有するＬＮＡ修飾デオキシリボ核酸の誘導体も含まれる。 An LNA-modified deoxyribonucleic acid means a deoxyribonucleic acid having an LNA structure, and includes a derivative of an LNA-modified deoxyribonucleic acid having a functional group or a nucleobase having a protecting group.
ボラノホスフェート化工程で使用されるＨ−ボラノホスフィニル化剤としては、例えば、Ｈ−ボラノホスホネートの有機アミン塩等を挙げることができ、Ｈ−ボラノホスホネートの有機アミン塩としては、例えば、ピリジニウム Ｈ−ボラノホスホネート、トリエチルアンモニウム Ｈ−ボラノホスホネート等を挙げることができる。Ｈ−ボラノホスフェート化反応におけるＬＮＡ修飾デオキシリボ核酸とＨ−ボラノホフィニル化剤との量比は、Ｈ−ボラノホスフェート化反応が進行すれば特に限定されないが、ＬＮＡ修飾デオキシリボ核酸に対して、Ｈ−ボラノホスフィニル化剤が１．２当量以上２．０当量以下、好ましくは２．０当量で使用することができる。 Examples of the H-boranophosphinylating agent used in the boranophosphate conversion step include organic amine salts of H-boranophosphonate, and the organic amine salts of H-boranophosphonate. Examples thereof include pyridinium H-boranophosphonate and triethylammonium H-boranophosphonate. The amount ratio between the LNA-modified deoxyribonucleic acid and the H-boranophofinylating agent in the H-boranophosphate reaction is not particularly limited as long as the H-boranophosphate reaction proceeds. The boranophosphinylating agent can be used in an amount of 1.2 equivalents to 2.0 equivalents, preferably 2.0 equivalents.
Ｈ−ボラノホスフェート化反応は、副反応の進行を抑制する観点から、縮合剤の存在下で行うことができる。縮合剤としては、例えば、Ｎ，Ｎ−ビス（２−オキソ−３−オキサゾリジニル）ホスフィン酸クロライド（ＢｏｐＣｌ）、３−ニトロ−１，２，４−トリアゾール−１−イル−トリス（ピロリジン−１−イル）ホスホニウム・ヘキサフルオロホスフェート（ＰｙＮＴＰ）等を挙げることができ、核酸オリゴマーの伸長効率の点で、Ｎ，Ｎ−ビス（２−オキソ−３−オキサゾリジニル）ホスフィン酸クロライド（ＢｏｐＣｌ）であることが好ましい。 The H-boranophosphate reaction can be carried out in the presence of a condensing agent from the viewpoint of suppressing the progress of side reactions. Examples of the condensing agent include N, N-bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl), 3-nitro-1,2,4-triazol-1-yl-tris (pyrrolidine-1- Yl) phosphonium hexafluorophosphate (PyNTP) and the like, and N, N-bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BopCl) in terms of the elongation efficiency of the nucleic acid oligomer. preferable.
Ｈ−ボラノホスフェート化反応は、必要に応じて、さらに求核触媒の存在下で行ってもよい。求核触媒としては、例えば、３−ニトロトリアゾール等を挙げることができる。 The H-boranophosphate reaction may be carried out in the presence of a nucleophilic catalyst as necessary. Examples of the nucleophilic catalyst include 3-nitrotriazole.
Ｈ−ボラノホスフェート化反応は、氷冷下（０℃）以上室温（２５℃）以下、数分以上数時間以下、例えば３０分以上１時間以下の範囲で行うことができる。 The H-boranophosphate reaction can be carried out under ice cooling (0 ° C.) or more and room temperature (25 ° C.) or less and several minutes or more and several hours or less, for example, 30 minutes or more and 1 hour or less.
反応溶媒としては、例えば、含窒素有機溶媒を挙げることができる。含窒素有機溶媒としては、例えば、ピリジン等を挙げることができる。 Examples of the reaction solvent include nitrogen-containing organic solvents. Examples of the nitrogen-containing organic solvent include pyridine.
Ｈ−ボラノホスフェート化工程は、Ｈ−ボラノホスフェート化反応後、反応溶液を不活性有機溶媒で希釈し、その反応溶液を添加して、反応系中に生じた副生成物（例えば、塩化ピリジン、ＢｏｐＣｌの残渣等）を中和して反応を停止することができる。不活性有機溶媒としては、例えば、クロロホルム、ジクロロメタン等を挙げることができる。また、保護基の脱離を抑制する観点から、反応を停止する前に、緩衝溶液を添加することが好ましい。緩衝溶液としては、例えば、炭酸水素トリエチルアンモニウム（ＴＥＡＢ）溶液、炭酸水素ナトリウム溶液（ＮａＨＣＯ３）、炭酸アンモニウム水溶液（（ＮＨ４）２ＣＯ３）等を挙げることができる。不活性溶媒としては、例えば、クロロホルム、ジクロロメタン等を挙げることができる。
緩衝溶液を添加するのは、反応を停止する前に反応溶媒を留去すると副生成物の酸性によって保護基が脱離する場合があるからである。
In the H-boranophosphate conversion step, after the H-boranophosphate conversion reaction, the reaction solution is diluted with an inert organic solvent, and the reaction solution is added to produce a by-product (for example, chloride) in the reaction system. The reaction can be stopped by neutralizing pyridine, BopCl residue, etc.). Examples of the inert organic solvent include chloroform and dichloromethane. Further, from the viewpoint of suppressing the elimination of the protecting group, it is preferable to add a buffer solution before stopping the reaction. Examples of the buffer solution include triethylammonium hydrogen carbonate (TEAB) solution, sodium hydrogen carbonate solution (NaHCO 3 ), aqueous ammonium carbonate solution ((NH 4 ) 2 CO 3 ), and the like. Examples of the inert solvent include chloroform and dichloromethane.
The buffer solution is added because if the reaction solvent is distilled off before the reaction is stopped, the protecting group may be eliminated due to the acidity of the by-product.
また、本発明のＬＮＡ修飾ボラノホスフェート化合物の製造方法は、必要に応じて、ボラノホスフェート化工程で得られた一般式（Ｉ）で示されるＬＮＡ修飾ボラノホスフェート化合物から水酸基の保護基を脱離する工程（脱離工程）を含むことができる。保護基の脱離工程に用いられる脱離化剤の種類、脱離反応に適用されるｐＨ及びその他の反応条件等については、ＬＮＡ修飾ボラノホスフェート化合物中の保護基の種類により適宜選択することができる。 In addition, the method for producing an LNA-modified boranophosphate compound of the present invention can be obtained by adding a protecting group for a hydroxyl group from the LNA-modified boranophosphate compound represented by the general formula (I) obtained in the boranophosphate conversion step, if necessary. A desorption step (desorption step) can be included. The type of desorbing agent used in the deprotecting step of the protecting group, the pH applied to the desorbing reaction, and other reaction conditions should be appropriately selected according to the type of protecting group in the LNA-modified boranophosphate compound. Can do.
本発明のＬＮＡ修飾ボラノホスフェート化合物の製造方法の一例について説明する。ただし、本発明はこれに限定されるものではない。
本発明のＬＮＡ修飾ボラノホスフェート化合物（３ａ−ｄ）は、例えば、以下のスキーム１に従って製造される。なお、スキーム１では、縮合剤としてＮ，Ｎ−ビス（２−オキソ−３−オキサゾリジニル）ホスフィン酸クロライド（Ｂｏｐ−Ｃｌ）を用いている。また、出発物質として、Ｈ−ボラノホスフィニル化剤（２）及びリボース構造の３位の炭素原子に水酸基が結合したＬＮＡ修飾デオキシリボ核酸の誘導体（１ａ−ｄ）を用いている。
An example of the method for producing the LNA-modified boranophosphate compound of the present invention will be described. However, the present invention is not limited to this.
The LNA-modified boranophosphate compound (3a-d) of the present invention is produced, for example, according to the following scheme 1. In Scheme 1, N, N-bis (2-oxo-3-oxazolidinyl) phosphinic chloride (Bop-Cl) is used as a condensing agent. As starting materials, an H-boranophosphinylating agent (2) and an LNA-modified deoxyribonucleic acid derivative (1a-d) in which a hydroxyl group is bonded to the 3-position carbon atom of the ribose structure are used.
スキーム１におけるＢｐｒｏは保護基を有する核酸塩基を表し、Ｒ１１及びＸ＋は各々一般式（Ｉ）中のＲ１及びＸ＋と同義である。
ＬＮＡ修飾ボラノホスフェート化合物（３ａ−ｄ）は、ＬＮＡ修飾デオキシリボ核酸の誘導体（１ａ−ｄ）をＨ−ボラノホスフィニル化剤（２）によりＨ−ボラノホスフェート化することによって製造することができる。
B pro in Scheme 1 represents a nucleobase having a protecting group, and R 11 and X + have the same meanings as R 1 and X + in formula (I), respectively.
The LNA-modified boranophosphate compound (3a-d) is produced by converting a derivative (1a-d) of an LNA-modified deoxyribonucleic acid into H-boranophosphate with an H-boranophosphinylating agent (2). Can do.
＜核酸オリゴマー＞
本発明の核酸オリゴマーは、少なくとも２つの塩基からなる塩基配列を有し、かつ、塩基配列に含まれる少なくとも一つの塩基が、一般式（Ｉ）で示されるＬＮＡ修飾ボラノホスフェート化合物に由来するヌクレオシド構造中の塩基である核酸オリゴマーである。核酸オリゴマーは、ＬＮＡ修飾ボラノホスフェート化合物をモノマーの１つとして用いて、このモノマーを縮合することにより得られる構造を有する。
<Nucleic acid oligomer>
The nucleic acid oligomer of the present invention has a base sequence composed of at least two bases, and at least one base contained in the base sequence is derived from an LNA-modified boranophosphate compound represented by the general formula (I) It is a nucleic acid oligomer that is a base in the structure. The nucleic acid oligomer has a structure obtained by condensing this monomer using an LNA-modified boranophosphate compound as one of the monomers.
核酸オリゴマーの長さ（塩基長）については、２つ以上の塩基を有する核酸オリゴマーであれば特に制限はなく、標的核酸の種類、長さ等に応じて所望の長さを適宜選択できる。本発明の核酸オリゴマーの長さは、特に限定されないが、アンチセンス医薬への応用の観点から、１０量体以上３０量体以下であることが好ましく、１０量体以上２１量体以下が好ましい。
核酸オリゴマーの塩基配列は、標的核酸の塩基配列に基づいて設定される。二重鎖形成能の観点から、核酸オリゴマーの塩基配列は、標的核酸の塩基配列に対して相補的な塩基配列となるように適宜選択されるが、場合によって、異なる塩基を１つ以上含む塩基配列であってもよい。
The length (base length) of the nucleic acid oligomer is not particularly limited as long as it is a nucleic acid oligomer having two or more bases, and a desired length can be appropriately selected according to the type and length of the target nucleic acid. The length of the nucleic acid oligomer of the present invention is not particularly limited, but is preferably 10-mer or more and 30-mer or less, more preferably 10-mer or 21-mer, from the viewpoint of application to antisense medicine.
The base sequence of the nucleic acid oligomer is set based on the base sequence of the target nucleic acid. From the viewpoint of the ability to form a double strand, the base sequence of the nucleic acid oligomer is appropriately selected so as to be a base sequence complementary to the base sequence of the target nucleic acid, but in some cases, the base includes one or more different bases. It may be an array.
核酸オリゴマーは、ＬＮＡ修飾ボラノホスフェート化合物のみに由来する核酸オリゴマーであってもよく、ＬＮＡ修飾ボラノホスフェート化合物と、ＬＮＡ修飾ボラノホスフェート化合物中のヌクレオシド構造以外のヌクレオシド構造を有する核酸を含む核酸オリゴマー（キメラオリゴマー）であってもよい。本明細書では、ＬＮＡ修飾ボラノホスフェート化合物中のヌクレオシド構造以外のヌクレオシド構造を有し、核酸オリゴマーを製造する際にモノマーとして使用可能な核酸を、単に「核酸誘導体」と称する場合がある。 The nucleic acid oligomer may be a nucleic acid oligomer derived only from an LNA-modified boranophosphate compound, and a nucleic acid comprising a LNA-modified boranophosphate compound and a nucleic acid having a nucleoside structure other than the nucleoside structure in the LNA-modified boranophosphate compound It may be an oligomer (chimeric oligomer). In the present specification, a nucleic acid having a nucleoside structure other than the nucleoside structure in the LNA-modified boranophosphate compound and usable as a monomer when producing a nucleic acid oligomer is sometimes simply referred to as “nucleic acid derivative”.
キメラオリゴマーの場合、ＬＮＡ修飾ボラノホスフェート化合物の位置については、特に制限はなく、核酸オリゴマー中に連続して存在してもよく、前記核酸誘導体を介在させて不連続に存在してもよい。 In the case of a chimeric oligomer, the position of the LNA-modified boranophosphate compound is not particularly limited, and may be continuously present in the nucleic acid oligomer, or may be discontinuously present with the nucleic acid derivative interposed.
核酸オリゴマーは、５’末端及び３’末端の少なくとも一方にＬＮＡ構造を有することが好ましく、５’及び３’の両末端にＬＮＡ構造を有することがより好ましい。５’末端及び３’末端の両末端に存在しうるＬＮＡ構造を有するヌクレオシドは、それぞれ独立に、リボース構造の２’位の炭素原子にフッ素原子、水酸基、及びメトキシ基から選択されるいずれか１つが結合したヌクレオシドであってもよい。 The nucleic acid oligomer preferably has an LNA structure at at least one of the 5 'end and the 3' end, and more preferably has an LNA structure at both the 5 'and 3' ends. The nucleoside having an LNA structure that can exist at both ends of the 5 ′ end and the 3 ′ end is independently any one selected from a fluorine atom, a hydroxyl group, and a methoxy group at the 2′-position carbon atom of the ribose structure. It may be a nucleoside with one attached.
核酸オリゴマーが他の核酸誘導体を含む場合、ＬＮＡ修飾ボラノホスフェート化合物の核酸オリゴマー全体における含有数については、特に制限はなく、核酸オリゴマーの長さによって異なるが、１０量体以上３０量体以下の核酸オリゴマーの場合、ＬＮＡ修飾ボラノホスフェート化合物由来のヌクレオシドの数は３以上１０以下が好ましい。 When the nucleic acid oligomer contains other nucleic acid derivatives, the content of the LNA-modified boranophosphate compound in the entire nucleic acid oligomer is not particularly limited, and varies depending on the length of the nucleic acid oligomer, but it may be 10-mer or more and 30-mer or less. In the case of the nucleic acid oligomer, the number of nucleosides derived from the LNA-modified boranophosphate compound is preferably 3 or more and 10 or less.
核酸オリゴマーは、核酸オリゴマーにおけるＨ−ボラノホスフェート基の塩基性溶液中での安定性の観点から、一般式（Ｉ）で示される構造のＨ−ボラノホスフェート基（−Ｏ−（ＢＨ３）ＰＨ（Ｏ-Ｘ＋）−）を酸化した酸化型のボラノホスフェート基（−Ｏ−（ＢＨ３）Ｐ（Ｏ-Ｘ＋）−Ｏ−：以下、酸化型ボラノホスフェート基と称する場合がある）を有するヌクレオシド構造中の塩基を含むことが好ましい。 From the viewpoint of stability of the H-boranophosphate group in the nucleic acid oligomer in a basic solution, the nucleic acid oligomer is an H-boranophosphate group (—O— (BH 3 ) having a structure represented by the general formula (I). Oxidized boranophosphate group (—O— (BH 3 ) P (O − X + ) —O— in which PH (O − X + ) —) is oxidized may be referred to as an oxidized boranophosphate group hereinafter. It is preferred to include a base in the nucleoside structure having
前記酸化型のボラノホスフェート基を含む核酸オリゴマーは、以下の構造、すなわち一般式（II）で示される構造及び一般式（III）で示される構造のうち少なくとも１つを有することができる。なお、一般式（II）で示される構造は、ＬＮＡ修飾ボラノホスフェート化合物に由来するヌクレオシド構造中の塩基が、核酸オリゴマーの５’末端の塩基である場合を意味する。一般式（II)で示される構造が核酸オリゴマー中に含まれる場合、核酸オリゴマーは通常、一般式（II)で示される構造を１つのみ含む。一般式（III）で示される構造は、ＬＮＡ修飾ボラノホスフェート化合物に由来するヌクレオシド構造中の塩基が、核酸オリゴマーの５’末端及び３’末端以外の塩基である場合を意味する。一般式（III)で示される構造が核酸オリゴマー中に含まれる場合、核酸オリゴマーは、一般式（III)で示される構造を１つ以上含むことができる。 The nucleic acid oligomer containing the oxidized boranophosphate group can have at least one of the following structures: a structure represented by the general formula (II) and a structure represented by the general formula (III). The structure represented by the general formula (II) means that the base in the nucleoside structure derived from the LNA-modified boranophosphate compound is the 5 ′ terminal base of the nucleic acid oligomer. When the structure represented by the general formula (II) is contained in the nucleic acid oligomer, the nucleic acid oligomer usually contains only one structure represented by the general formula (II). The structure represented by the general formula (III) means that the base in the nucleoside structure derived from the LNA-modified boranophosphate compound is a base other than the 5 'end and 3' end of the nucleic acid oligomer. When the structure represented by the general formula (III) is included in the nucleic acid oligomer, the nucleic acid oligomer can include one or more structures represented by the general formula (III).
一般式（II）及び（III）中、Ｒ１、Ｂｓ及びＸ＋は、一般式（Ｉ）におけるＲ１、Ｂｓ及びＸ＋と同義である。 In the general formula (II) and (III), R 1, Bs and X + is, R 1, is Bs and X + synonymous in the general formula (I).
核酸オリゴマーは、ＬＮＡ修飾ボラノホスフェート化合物以外のモノマー（以下、単に「他のモノマー」と称する場合がある）に由来する他のヌクレオシド構造中の塩基を含むことができる。
核酸オリゴマーを得る際に用いる他のモノマーについては特に制限はなく、例えば、以下の一般式（IV）で表されるボラノホスフェート化合物（以下、ＬＮＡ未修飾ボラノホスフェート化合物」と称する）、一般式（Ｖ）で表される核酸、一般式（VI）で表されるＬＮＡ修飾核酸などを挙げることができる。
Nucleic acid oligomers can contain bases in other nucleoside structures derived from monomers other than LNA modified boranophosphate compounds (hereinafter sometimes referred to simply as “other monomers”).
Other monomers used for obtaining the nucleic acid oligomer are not particularly limited. For example, boranophosphate compounds represented by the following general formula (IV) (hereinafter referred to as LNA unmodified boranophosphate compounds), Examples thereof include a nucleic acid represented by the formula (V) and an LNA modified nucleic acid represented by the general formula (VI).
一般式（IV）〜(VI）中、Ｒ２、Ｒ３及びＲ５は一般式（Ｉ）中のＲ１と同義であり、Ｒ６は、水素原子、保護基を有する水酸基、ハロゲン原子（例えば、フッ素原子）、アルコキシ基（例えば、メトキシ基）を表し、Ｂｓ２は一般式（Ｉ）中のＢｓと同義であり、Ｘ＋は一般式（Ｉ）中のＸ＋と同義である。 In general formulas (IV) to (VI), R 2 , R 3 and R 5 have the same meaning as R 1 in general formula (I), and R 6 represents a hydrogen atom, a hydroxyl group having a protecting group, a halogen atom ( for example, a fluorine atom), an alkoxy group (e.g., represents a methoxy group), Bs 2 has the same meaning as Bs in the general formula (I), X + has the same meaning as X + in the general formula (I).
前記他のモノマーの由来するヌクレオシド構造は、核酸オリゴマーのいずれの位置に存在してもよい。例えば、一般式（VI）で表されるモノマーは、Ｒ５が水素原子を表す場合、核酸オリゴマーの３’末端のヌクレオシドとして使用されてもよい。 The nucleoside structure derived from the other monomer may be present at any position of the nucleic acid oligomer. For example, the monomer represented by the general formula (VI) may be used as a nucleoside at the 3 ′ end of the nucleic acid oligomer when R 5 represents a hydrogen atom.
本発明の核酸オリゴマーは、標的核酸の塩基配列と相補的な塩基配列となるように、ＬＮＡ修飾ボラノホスフェート化合物に由来するヌクレオシド構造中の塩基を少なくとも１つ以上を用いて、公知の製造方法を用いて得ることができる。具体的には、本発明の核酸オリゴマーの製造方法は、３’末端のＬＮＡ修飾ボラノホスフェート化合物を少なくとも１つ含むモノマーから、標的核酸の塩基配列に相補的となる塩基配列となるようにモノマーを選択して、３’末端の塩基となる塩基を含むモノマーに他のモノマーを順次縮合させる工程（縮合工程）を含む。 The nucleic acid oligomer of the present invention is a known production method using at least one base in a nucleoside structure derived from an LNA-modified boranophosphate compound so that the base sequence is complementary to the base sequence of the target nucleic acid. Can be used. Specifically, in the method for producing a nucleic acid oligomer of the present invention, a monomer having a base sequence complementary to the base sequence of a target nucleic acid is obtained from a monomer containing at least one LNA-modified boranophosphate compound at the 3 ′ end. And a step (condensation step) of sequentially condensing another monomer with a monomer containing a base that becomes a base at the 3 ′ end.
前記縮合工程では、核酸オリゴマーのヌクレオシド構造中のリボース構造の５’位の炭素原子に結合する水酸基とモノマーのリン酸部とが縮合する。
前記縮合工程では、縮合剤を用いることができる。
縮合剤としては、例えば、Ｎ，Ｎ−ビス（２−オキソ−３−オキサゾニル）ホスフィン酸クロライド、３−ニトロ−１，２，４−トリアゾール−１−イル−トリス（ピロリジン−１−イル）ホスホニウム・ヘキサフルオロホスフェート（ＰｙＮＴＰ）、又は１，３、−ジメチル−２−（３−ニトロ−１，２，４−トリアゾール−１−イル）−２−ピロリジン−１−イル−１，２，３−ジアザホスホリジウム・ヘキサフルオロホスフェート（ＭＮＴＰ）等を挙げることができる。このうち、副反応を抑制し、かつ反応が速やかに完結する観点から、ＭＮＴＰが好ましい。
In the condensation step, the hydroxyl group bonded to the 5′-position carbon atom of the ribose structure in the nucleoside structure of the nucleic acid oligomer is condensed with the phosphate portion of the monomer.
In the condensation step, a condensing agent can be used.
Examples of the condensing agent include N, N-bis (2-oxo-3-oxazonyl) phosphinic chloride, 3-nitro-1,2,4-triazol-1-yl-tris (pyrrolidin-1-yl) phosphonium. Hexafluorophosphate (PyNTP) or 1,3, -dimethyl-2- (3-nitro-1,2,4-triazol-1-yl) -2-pyrrolidin-1-yl-1,2,3- Examples thereof include diazaphosphoridium hexafluorophosphate (MNTP). Among these, MNTP is preferable from the viewpoint of suppressing side reactions and completing the reaction quickly.
前記縮合工程、さらに塩基性化合物の存在下で行うことができる。
塩基性化合物としては、例えば、１，８−ビス（ジメチルアミノ）ナフタレン（ＤＭＡＮ）、２，２，６，６−テトラメチルピペリジン（ＴＭＰ）、ジイソプロピルエチルアミン（ＤＩＰＥＡ）、２，６−ルチジン、２，４,６−トリメチルピリジン等を挙げることができる。このうち、副反応を抑制する観点から、２，６−ルチジンが好ましい。
The condensation step can be performed in the presence of a basic compound.
Examples of the basic compound include 1,8-bis (dimethylamino) naphthalene (DMAN), 2,2,6,6-tetramethylpiperidine (TMP), diisopropylethylamine (DIPEA), 2,6-lutidine, , 4,6-trimethylpyridine and the like. Among these, 2,6-lutidine is preferable from the viewpoint of suppressing side reactions.
前記縮合工程における縮合反応は、氷冷下（０℃）以上室温（２５℃）以下、好ましくは２０℃以上２５℃以下の範囲で、数分以上数時間以下、例えば１５分以上１時間以下の範囲で行うことができる。 The condensation reaction in the condensation step is performed under ice-cooling (0 ° C.) or more and room temperature (25 ° C.) or less, preferably 20 ° C. or more and 25 ° C. or less, several minutes to several hours, for example, 15 minutes to 1 hour. Can be done in a range.
反応溶媒としては、例えば、不活性溶媒等を挙げることができる。不活性溶媒のうち、アセトニトリル等を挙げることができる。 As a reaction solvent, an inert solvent etc. can be mentioned, for example. Among the inert solvents, mention may be made of acetonitrile and the like.
本発明の核酸オリゴマーの製造方法は、固相担体を用いた反応により核酸オリゴマーを製造することができる（固相法）。
固相反応により核酸オリゴマーを製造する場合、核酸オリゴマーの３’末端の塩基を含むモノマーは、リボース構造の３’位の炭素原子に結合する酸素原子を介して、固相担体と結合することができる。この場合、モノマーと固相担体との間には、必要に応じてリンカーが存在してもよい。
In the method for producing a nucleic acid oligomer of the present invention, a nucleic acid oligomer can be produced by a reaction using a solid phase carrier (solid phase method).
In the case of producing a nucleic acid oligomer by a solid phase reaction, a monomer containing a 3 ′ terminal base of the nucleic acid oligomer can be bound to a solid phase carrier through an oxygen atom that binds to the 3 ′ carbon atom of the ribose structure. it can. In this case, a linker may be present between the monomer and the solid phase carrier as necessary.
前記固相担体の種類は特に限定されず、例えば、定孔ガラス、オキサリル化−定孔ガラス、ＴｅｎｔａＧｅｌ支持体−アミノポリエチレングリコール誘導体化支持体、高架橋アミノメチルポリスチレン、Ｐｒｏｓ−ポリスチレン／ジビニルベンゼンの共重合体等を挙げることができる。前記固相担体とモノマーとの連結には、固相担体上のアミノ基を利用することができる。前記固相担体上のアミノ基としては、３−アミノプロピル基、長鎖アルキルアミノ基（ＬＣＡＡ）等を挙げることができる。前記固相担体とモノマーとの間には、リンカーが存在してもよい。前記リンカーとしては、スクニシル基、オキサリル基等を挙げることができる。 The kind of the solid phase carrier is not particularly limited, and examples thereof include constant pore glass, oxalylated-constant pore glass, TentaGel support-aminopolyethylene glycol derivatized support, highly crosslinked aminomethylpolystyrene, and Pros-polystyrene / divinylbenzene. A polymer etc. can be mentioned. An amino group on the solid phase carrier can be used for linking the solid phase carrier and the monomer. Examples of the amino group on the solid phase carrier include 3-aminopropyl group, long-chain alkylamino group (LCAA) and the like. A linker may be present between the solid phase carrier and the monomer. Examples of the linker include a succinyl group and an oxalyl group.
本発明の核酸オリゴマーの製造方法は、縮合工程で得られた核酸オリゴマーを酸化剤により酸化する酸化工程を含んでもよい。
酸化剤としては、例えば、（＋）−カンフォリルスルホニルオキサジリジン（ＣＳＯ）（＋）−（８，８−ジクロロカンフォリルスルホニル）−オキサジリジン（ＤＣＳＯ）、メチルエチルケトン過酸化物、及びポジティブハロゲン試薬（四塩化炭素、四臭化炭素、ヨウ素、Ｎ−クロロスクシンイミド、Ｎ−ブロモスクシンイミド、Ｎ−ヨードスクシンイミド等）などを挙げることができる。
酸化反応は塩基性化合物存在下で行ってもよい。塩基性化合物としては、例えば、ジイソプロピルエチルアミン等を挙げることができる。
The method for producing a nucleic acid oligomer of the present invention may include an oxidation step of oxidizing the nucleic acid oligomer obtained in the condensation step with an oxidizing agent.
Examples of the oxidizing agent include (+)-camphorylsulfonyloxaziridine (CSO) (+)-(8,8-dichlorocamphorylsulfonyl) -oxaziridine (DCSO), methyl ethyl ketone peroxide, and positive halogen. Examples include reagents (carbon tetrachloride, carbon tetrabromide, iodine, N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, and the like).
The oxidation reaction may be performed in the presence of a basic compound. Examples of basic compounds include diisopropylethylamine.
本発明の核酸オリゴマーの製造における縮合工程は、核酸オリゴマーのヌクレオチド構造中のリボース構造の５’位の炭素原子に結合する水酸基とモノマーのリン酸部とが縮合する反応を含む。このため、５’位の炭素原子に結合する水酸基が保護基を有する場合、脱保護試薬と反応させて保護基を脱離させる工程（第１の保護基脱離工程）を含む。第１の保護基脱離工程は、核酸オリゴマーの製造における縮合反応の前に行う。
前記第１の保護基脱離工程には、脱保護剤を用いることができる。第１の保護基脱離工程で使用し得る脱保護剤としては、例えば、ハロゲン化アルキルカルボン酸等を挙げることができる。ハロゲン化アルキルカルボン酸としては、例えば、トリフルオロ酢酸、トリクロロ酢酸、ジクロロ酢酸等を挙げることができる。保護基を有する核酸オリゴマーが２〜１００当量の脱保護剤と反応することが好ましい。
The condensation step in the production of the nucleic acid oligomer of the present invention includes a reaction in which the hydroxyl group bonded to the 5′-position carbon atom of the ribose structure in the nucleotide structure of the nucleic acid oligomer and the phosphate portion of the monomer are condensed. For this reason, when the hydroxyl group couple | bonded with the carbon atom of 5'-position has a protecting group, the process (1st protecting group elimination process) which makes it react with a deprotection reagent and remove | eliminates a protecting group is included. The first protecting group elimination step is performed before the condensation reaction in the production of the nucleic acid oligomer.
A deprotecting agent can be used in the first protecting group elimination step. Examples of the deprotecting agent that can be used in the first protecting group elimination step include a halogenated alkylcarboxylic acid. Examples of the halogenated alkylcarboxylic acid include trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid and the like. It is preferred that the nucleic acid oligomer having a protecting group reacts with 2 to 100 equivalents of a deprotecting agent.
また、核酸オリゴマーの製造方法に用いられるモノマー中の塩基に保護基を有する場合、前記製造方法は、塩基の保護基を脱離させる工程（第２の保護基脱離工程）を更に含む。第２の保護基脱離工程は、最終的に得られる核酸オリゴマー中の塩基が保護基を有さないようにすれば、どの段階で行うかは特に限定されない。
第２の保護基脱離工程では、例えば、モノマー又は核酸オリゴマーをアンモニア水で処理することができる。第２の保護基脱離工程で用いられるアンモニア水としては、例えば、２５質量％アンモニア水又は２５質量％アンモニア水−エタノール混合溶液（３：１、ｖ／ｖ）が挙げられる。
In addition, when the base used in the method for producing the nucleic acid oligomer has a protecting group, the production method further includes a step of removing the protecting group of the base (second protecting group elimination step). The stage in which the second protecting group elimination step is performed is not particularly limited as long as the base in the finally obtained nucleic acid oligomer does not have a protecting group.
In the second protecting group elimination step, for example, the monomer or nucleic acid oligomer can be treated with aqueous ammonia. Examples of the ammonia water used in the second protecting group elimination step include 25 mass% ammonia water or a 25 mass% ammonia water-ethanol mixed solution (3: 1, v / v).
得られた核酸オリゴマーは、例えば、逆相高速液体クロマトグラフィー（逆相ＨＰＬＣ）、イオン交換ＨＰＬＣ、カラムクロマトグラフィー、再結晶等の公知の精製方法により精製することができる。 The obtained nucleic acid oligomer can be purified by a known purification method such as reverse phase high performance liquid chromatography (reverse phase HPLC), ion exchange HPLC, column chromatography, recrystallization and the like.
本発明の核酸オリゴマーの製造方法の一例について説明する。ただし、本発明はこれに限定されるものではない。以下のスキーム２は、固相担体に連結された核酸オリゴマー（スキーム２では二量体）を製造する工程の一例を示す。 An example of the method for producing the nucleic acid oligomer of the present invention will be described. However, the present invention is not limited to this. Scheme 2 below shows an example of a process for producing a nucleic acid oligomer (dimer in Scheme 2) linked to a solid support.
ＬＮＡ修飾ボラノホスフェート化合物（３ａ−ｄ）と固相担体に連結した他のモノマー（４）とを塩基の存在下で縮合剤を用いて、反応させて核酸オリゴマー（ＬＮＡＰＢＨ−ＤＮＡ（二量体））（５ａ−ｄ）を得る。 The LNA-modified boranophosphate compound (3a-d) and another monomer (4) linked to a solid support were reacted in the presence of a base using a condensing agent to react with a nucleic acid oligomer ( LNA PBH-DNA (dimeric) Body)) (5a-d).
他のモノマー（４）のＢｐｒｏはＬＮＡ修飾ボラノホスフェート化合物（３ａ−ｄ）の有するＢｐｒｏと同義である。
他のモノマー（４）の有するＲ６は、水酸基、保護基を有する水酸基、ハロゲン原子（例えば、フッ素原子）、又はアルコキシ基（例えば、メトキシ基）を表す。Ｒ６が保護基を有する水酸基を表す場合、その保護基は、一般式（Ｉ）中のＲ１の表す水酸基の保護基と同義である。
核酸オリゴマー（ＬＮＡＰＢＨ−ＤＮＡ（二量体））（５ａ−ｄ）中のＲ１１及びＢｐｒｏは、各々一般式（Ｉ）中のＲ１及びＬＮＡ修飾ボラノホスフェート化合物（３ａ−ｄ）中のＢｐｒｏと同義である。
核酸オリゴマー（５ａ−ｄ）は、その後、標的核酸と相補鎖な塩基配列となるように、モノマーを適宜選択し、この反応を繰り返して行うことにより、所望の配列を有する核酸オリゴマーを得ることができる。
B pro of other monomers (4) has the same meaning as B pro with LNA modified Boranophosphate compound of (3a-d).
R 6 of the other monomer (4) represents a hydroxyl group, a hydroxyl group having a protecting group, a halogen atom (for example, a fluorine atom), or an alkoxy group (for example, a methoxy group). When R 6 represents a hydroxyl group having a protecting group, the protecting group has the same meaning as the hydroxyl protecting group represented by R 1 in formula (I).
R 11 and B pro in the nucleic acid oligomer ( LNA PBH-DNA (dimer)) (5a-d) are R 1 and LNA modified boranophosphate compounds (3a-d) in the general formula (I), respectively. It is synonymous with B pro .
For the nucleic acid oligomer (5a-d), a monomer is appropriately selected so that the base sequence is complementary to the target nucleic acid, and this reaction is repeated to obtain a nucleic acid oligomer having a desired sequence. it can.
また、縮合工程で得られた核酸オリゴマーは、更に酸化して酸化型の核酸オリゴマーとしてもよい。一例として、以下のスキーム３に、上記スキーム２で得られた二量体の核酸オリゴマー（ＬＮＡＰＢＨ−ＤＮＡ）から、酸化型の核酸オリゴマー（ＬＮＡＰＢＯ−ＯＤＮ）（６ａ−ｄ）を得る工程を示す。
核酸オリゴマー（ＬＮＡＰＢＯ−ＯＤＮ（二量体））（６ａ−ｄ）中のＲ１１、Ｒ６及びＢｐｒｏは、各々核酸オリゴマー（ＬＮＡＰＢＨ−ＤＮＡ（二量体））（５ａ−ｄ）中のＲ１１、Ｒ６及びＢｐｒｏと同義である。
The nucleic acid oligomer obtained in the condensation step may be further oxidized to form an oxidized nucleic acid oligomer. As an example, a step of obtaining an oxidized nucleic acid oligomer ( LNA PBO-ODN) (6a-d) from the dimer nucleic acid oligomer ( LNA PBH-DNA) obtained in the above Scheme 2 in Scheme 3 below. Show.
R 11 , R 6 and B pro in the nucleic acid oligomer ( LNA PBO-ODN (dimer)) (6a-d) are each in the nucleic acid oligomer ( LNA PBH-DNA (dimer)) (5a-d). R 11 , R 6 and B pro are the same.
本発明の核酸オリゴマーは、標的核酸の塩基配列に相補的となるように設計することにより、標的核酸に対する二本鎖形成能に優れたアンチセンス分子として使用することができる。例えば、標的核酸が疾患関連遺伝子の部分配列に相当する場合には、本発明の核酸オリゴマーは、翻訳阻害能の高いアンチセンス医薬等の医薬用途に好ましく用いられる。 The nucleic acid oligomer of the present invention can be used as an antisense molecule excellent in the ability to form a double strand for a target nucleic acid by designing it to be complementary to the base sequence of the target nucleic acid. For example, when the target nucleic acid corresponds to a partial sequence of a disease-related gene, the nucleic acid oligomer of the present invention is preferably used for pharmaceutical use such as an antisense drug having a high translation inhibitory ability.
以下、本発明を実施例にて具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
各種分析器は以下に示した機種を用いた。
１Ｈ−核磁気共鳴スペクトル（１Ｈ−ＮＭＲ）：バリアン Ｍｅｒｃｕｒｙ ３００（３００ＭＨｚ）
１３Ｃ−核磁気共鳴スペクトル（１３Ｃ−ＮＭＲ）：バリアン Ｍｅｒｃｕｒｙ ３００（７５ＭＨｚ）
３１Ｐ−核磁気共鳴スペクトル（３１Ｐ−ＮＭＲ）：バリアン Ｍｅｒｃｕｒｙ ３００（１２１．５ＭＨｚ）
なお、１Ｈ−ＮＭＲにはテトラメチルシラン（ＴＭＳ）を、１３Ｃ−ＮＭＲにはＣＤＣｌ３（δ７７．１６ｐｐｍ）を内部標準として用い、３１Ｐ−ＮＭＲは８５％Ｈ３ＰＯ４を外部標準として用いた。
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.
The various analyzers shown below were used.
1 H-nuclear magnetic resonance spectrum ( 1 H-NMR): Varian Mercury 300 (300 MHz)
13 C-nuclear magnetic resonance spectrum ( 13 C-NMR): Varian Mercury 300 (75 MHz)
31 P-nuclear magnetic resonance spectrum ( 31 P-NMR): Varian Mercury 300 (121.5 MHz)
Tetramethylsilane (TMS) is used for 1 H-NMR, CDCl 3 (δ77.16 ppm) is used for 13 C-NMR as an internal standard, and 31 P-NMR uses 85% H 3 PO 4 as an external standard. Using.
ＭＡＬＤＩ−ＴＯＦ ＭＳ： ＡＢ ＳＣＩＥＸ ＴＯＦ／ＴＯＦＴＭ ５８００ Ｓｙｓｔｅｍ
紫外可視分光光度計：ＪＡＳＣＯ Ｖ−５５０ ＵＶ／ＶＩＳ ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｅｒ
イオン交換ＨＰＬＣ：ＧＥ Ｈｅａｌｔｈｃａｒｅ ＡＫＴＡ ｐｕｒｉｆｉｅｒ １０Ｓ
カラムクロマトグラフィーに充填するシリカゲルには、ＫＡＮＴＯ ＣＨＥＭＩＣＡＬのＳｉｌｉｃａ Ｇｅｌ ６０Ｎを用いた。
逆相ＨＰＬＣに使用したカラム（分析）：ｗａｔｅｒ μ−ＢＯＵＮＤＡＳＰＨＥＲＥ，Ｃ１８ ５μｍ，１００Å，３．９ｍｍ×１５９ｍｍ
イオン交換ＨＰＬＣに使用したカラム（分析・分取）：ＧＥ Ｈｅａｌｔｈｃａｒｅ ＭｉｎｉＱＴＭ ４．６／５０ＰＥ， ３μｍ，４．６ｍｍ×５０ｍｍ）
MALDI-TOF MS: AB SCIEX TOF / TOFTM 5800 System
UV-visible spectrophotometer: JASCO V-550 UV / VIS spectrophotometer
Ion exchange HPLC: GE Healthcare AKTA purifier 10S
Silica Gel 60N from KANTO CHEMICAL was used as the silica gel packed in the column chromatography.
Column (analysis) used for reverse phase HPLC: water μ-BOUNDASPHERE, C18 5 μm, 100 mm, 3.9 mm × 159 mm
Column used for ion exchange HPLC (analysis and preparative): GE Healthcare MiniQ ™ 4.6 / 50PE, 3 μm, 4.6 mm × 50 mm)
なお、特に断りのない限り、「％」は質量基準である。実施例中、ＨＮＥｔ３はトリエチルアミンを示し、Ｂｐｒｏは保護基を有する核酸塩基を示し、ＴｂｚはＮ３−ベンゾイルチミン−１−イルを示し、ＡｂｚはＮ６−ベンゾイルアデニン−９−イルを示し、ＣｉｂｕはＮ４−イソブチリルシトシン−１−イルを示し、Ｇｃｅ，ｉｂｕはＯ６−シアノエチル−Ｎ２−イソブチリルグアニン−９−イルを示し、ＤＭＴｒは４，４’−ジメトキシトリチルを示し、Ｂｏｐ−Ｃｌはビス（２−オキソ−３−オキサゾリジニル）ホスフィン酸クロライドを示し、その他の略号は上記説明中のものと同義である。 Unless otherwise specified, “%” is based on mass. In the examples, HNEt 3 represents triethylamine, Bpro represents a nucleobase having a protecting group, T bz represents N 3 -benzoylthymin-1-yl, and A bz represents N 6 -benzoyladenine-9-yl. C ibu represents N 4 -isobutyrylcytosin -1-yl, G ce, ibu represents O 6 -cyanoethyl- N 2 -isobutyrylguanin -9-yl, DMTr represents 4,4′- Dimethoxytrityl, Bop-Cl represents bis (2-oxo-3-oxazolidinyl) phosphinic acid chloride, and other abbreviations are as defined above.
＜ＬＮＡ修飾ボラノホスフェート化合物の合成＞
以下、スキーム４に従って、ＬＮＡ修飾ボラノホスフェート化合物（９）を合成した。
<Synthesis of LNA-modified boranophosphate compound>
Hereinafter, an LNA-modified boranophosphate compound (9) was synthesized according to Scheme 4.
（１Ｒ，３Ｒ，４Ｒ，７Ｓ）−３−（Ｎ３−ベンゾイルチミン−１−イル）−１−ジメトキシトリチルオキシメチル−７−ヒドロキシ−２，５−ジオキサビシクロ[２．２．１]へプタン（７）０．６９ｇ（１．０ｍｍｏｌ）とピリジニウム Ｈ−ボラノホスホネート（８）０．３０ｇ（２．０ｍｍｏｌ）をアルゴン雰囲気下で、ピリジン（２ｍｌ×３）で共沸乾燥してピリジン（１０ｍｌ）に溶解した。得られたピリジン溶液を氷浴下（０℃）にて撹拌しつつＢｏｐ−Ｃｌ：０．５１ｇ（２．０ｍｍｏｌ）を加え、その後反応温度を室温（２５℃）に上げ、２０分間撹拌した。この溶液をジクロロメタン５０ｍｌで希釈した後、溶液に１Ｍ 炭酸アンモニウム水溶液 （５０ｍＬ）を添加した。水層をジクロロメタン（２００ｍｌ×２）で逆抽出した。有機層に再度、１Ｍ ＴＥＡＢ緩衝溶液（１００ｍｌ×３）を添加した。水層をジクロロメタン（５００ｍｌ×１）で抽出し、有機層中の水分を無水硫酸ナトリウムを用いて除去した。次いで、有機層の溶媒を留去し、得られた残渣をシリカゲルクロマトグラフィー（酢酸エチル−ヘキサン（２：１＝ｖ／ｖ）中トリエチルアミン０．５％〜ジクロロメタン−メタノール０．５％、トリエチルアミン０．５％）で分離精製することにより、目的化合物を得た。収率７５％。 (1R, 3R, 4R, 7S) -3- (N3-Benzoylthymin-1-yl) -1-dimethoxytrityloxymethyl-7-hydroxy-2,5-dioxabicyclo [2.2.1] heptane (7) 0.69 g (1.0 mmol) and pyridinium H-boranophosphonate (8) 0.30 g (2.0 mmol) were azeotropically dried with pyridine (2 ml × 3) under an argon atmosphere, and pyridine (10 ml) ). While stirring the obtained pyridine solution in an ice bath (0 ° C.), Bop-Cl: 0.51 g (2.0 mmol) was added, and then the reaction temperature was raised to room temperature (25 ° C.) and stirred for 20 minutes. After diluting this solution with 50 ml of dichloromethane, 1M aqueous ammonium carbonate solution (50 mL) was added to the solution. The aqueous layer was back extracted with dichloromethane (200 ml × 2). To the organic layer again, 1M TEAB buffer solution (100 ml × 3) was added. The aqueous layer was extracted with dichloromethane (500 ml × 1), and water in the organic layer was removed using anhydrous sodium sulfate. Subsequently, the solvent of the organic layer was distilled off, and the obtained residue was chromatographed on silica gel (0.5% triethylamine to 0.5% dichloromethane-methanol in ethyl acetate-hexane (2: 1 = v / v), 0% triethylamine). And 5%) to obtain the target compound. Yield 75%.
１Ｈ−ＮＭＲ（ＣＤＣｌ３）δ・８．００−７．９３（ｍ，２Ｈ），７．８２（ｄｄ，Ｊ＝７．２，１．１Ｈｚ，１Ｈ），７．６４（ｍ，１Ｈ），７．５３−７．４６（ｍ，４Ｈ），７．４１−７．３０（ｍ，６Ｈ），７．３６（ｂｒ ｄ，１ＪＰＨ＝３９９Ｈｚ，１Ｈ），７．２４−７．２１（ｍ，１Ｈ），６．８９−６．８５（ｍ，４Ｈ），６．２１（ｍ，１Ｈ），５．６３（ｓ，１Ｈ），４．７９（ｄｄ，Ｊ＝７８，６．６Ｈｚ，１Ｈ），４．６５（ｄ，Ｊ＝１１Ｈｚ，１Ｈ），３．９８（ｑ，Ｊ＝３．９Ｈｚ，１Ｈ），３．８０（ｓ，６Ｈ），３．７８−３．７２（ｍ，１Ｈ），３．５１（ｄｄ，Ｊ＝２７，１１Ｈｚ，２Ｈ），２．９０（ｑ，Ｊ＝７．２Ｈｚ，７．５Ｈ），１．６７（ｄｄ，，Ｊ＝６．０，０．９Ｈｚ，３Ｈ），１．２１（ｔ，Ｊ＝７．２Ｈｚ，１１Ｈ），０．９０−０．００（ｂｒ，３Ｈ）；１３Ｃ−ＮＭＲ（ＣＤＣｌ３）δ１６９．２，１６３．２，１５８．９，１４９．１，１４９．１，１４４．７，１４４．６，１３５．７，１３５．６，１３５．５，１３５．３，１３５．３，１３４．７，１３４．７，１３１．７，１３１．６，１３０．９，１３０．８，１３０．４，１３０．３，１３０．２，１２９．５，１２９．４，１２８．４，１２８．４，１２８．３，１２８．２，１２７．３，１１３．７，１１３．６，１１０．７，１１０．７，８８．４，８８．４，８８．３，８７．８，８６．９，７９．０，７８．９，７７．８，７７．６，７７．４，７７．０，７５．０，７４．８，７２．６，７２．４，７１．９，５８．２，５７．９，５５．５，４５．７，１３．０，１２．９，８．７；３１Ｐ−ＮＭＲ（ＣＤＣｌ３）δ１０７．５（ｍ）．
ＥＳＩ−ＨＲＭＳ：Ｃａｌｃｄ ｆｏｒＣ３９Ｈ３９ＢＮ２Ｏ１０Ｐ−［Ｍ−Ｈ］−；７３７．２４４１，ｆｏｕｎｄ；７３７．２４４３．
1 H-NMR (CDCl 3 ) δ 8.00-7.93 (m, 2H), 7.82 (dd, J = 7.2, 1.1 Hz, 1H), 7.64 (m, 1H) 7.53-7.46 (m, 4H), 7.41-7.30 (m, 6H), 7.36 (br d, 1 J PH = 399 Hz, 1H), 7.24-7.21 (M, 1H), 6.89-6.85 (m, 4H), 6.21 (m, 1H), 5.63 (s, 1H), 4.79 (dd, J = 78, 6.6 Hz , 1H), 4.65 (d, J = 11 Hz, 1H), 3.98 (q, J = 3.9 Hz, 1H), 3.80 (s, 6H), 3.78-3.72 (m , 1H), 3.51 (dd, J = 27, 11 Hz, 2H), 2.90 (q, J = 7.2 Hz, 7.5H), 1.67 (dd, J = 6.0, 0) .9Hz, 3H), 1.21 (t, J = 7.2 Hz, 11H), 0.90-0.00 (br, 3H); 13 C-NMR (CDCl 3 ) δ 169.2, 163.2, 158.9, 149. 1, 149.1, 144.7, 144.6, 135.7, 135.6, 135.5, 135.3, 135.3, 134.7, 134.7, 131.7, 131.6, 130.9, 130.8, 130.4, 130.3, 130.2, 129.5, 129.4, 128.4, 128.4, 128.3, 128.2, 127.3, 113. 7, 113.6, 110.7, 110.7, 88.4, 88.4, 88.3, 87.8, 86.9, 79.0, 78.9, 77.8, 77.6, 77.4, 77.0, 75.0, 74.8, 72.6, 72.4, 71.9, 58.2, 57. , 55.5,45.7,13.0,12.9,8.7; 31 P-NMR ( CDCl 3) δ107.5 (m).
ESI-HRMS: Calcd forC 39 H 39 BN 2 O 10 P - [M-H] -; 737.2441, found; 737.2443.
＜ＰＢＯ−ＤＮＡの合成方法＞
下記スキーム５に従って、上記化合物（７）に代えてデオキシリボ核酸（１０ａ−ｄ）を用いること以外はＬＮＡ修飾ボラノホスフェート化合物と同様にして、アデニン、グアニン、シトシンまたはチミンの各塩基を有するＬＮＡ未修飾ボラノホスフェート化合物（１１ａ−ｄ）をそれぞれ合成した。
<Method of synthesizing PBO-DNA>
According to the following scheme 5, LNA having an adenine, guanine, cytosine or thymine base is used in the same manner as the LNA-modified boranophosphate compound except that deoxyribonucleic acid (10a-d) is used instead of the compound (7). Modified boranophosphate compounds (11a-d) were respectively synthesized.
＜ＬＮＡＰＢＯ−ＯＤＮ（１２量体）の合成方法−固相法−＞
アポＢ（ａｐｏＢ）蛋白質をコードするｍＲＮＡの一部、アポＢ蛋白質ｍＲＮＡ（３’−ＣＧＵ ＡＡＣ ＣＡＵ ＡＡＧ−５’：相補鎖ＲＮＡ、配列番号１）（アポ蛋白質Ｂ−１００：Ｎａｔｕｒｅ，２００６，Ｖｏｌ．４４１，ｐｐ１１１−１１４）と、対応するアポＢ蛋白質ＤＮＡ（３’−ＣＧＴ ＡＡＣ ＣＡＴ ＡＡＧ−５’：相補鎖ＤＮＡ、配列番号２）を標的核酸として選択した。相補鎖ＲＮＡ及び相補鎖ＤＮＡの塩基配列に相補的な塩基配列を有し、ＬＮＡ修飾ボラノホスフェート化合物由来の塩基を含むアンチセンス分子、ＬＮＡＰＢＯ−ＯＤＮを以下のようにして合成した。
<Synthesis Method of LNA PBO-ODN (12-mer) -Solid Phase Method->
A part of mRNA encoding Apo B (apoB) protein, Apo B protein mRNA (3′-CGU AAC CAU AAG-5 ′: complementary strand RNA, SEQ ID NO: 1) (Apoprotein B-100: Nature, 2006, Vol. .441, pp111-114) and the corresponding apo B protein DNA (3′-CGT AAC CAT AAG-5 ′: complementary DNA, SEQ ID NO: 2) were selected as target nucleic acids. An antisense molecule, LNA PBO-ODN, having a base sequence complementary to the base sequence of complementary strand RNA and complementary strand DNA and containing a base derived from an LNA-modified boranophosphate compound was synthesized as follows.
５’-Ｏ−ＤＭＴｒ−Ｎ３-ベンゾイルチミジンをスクシニルリンカーを介して担持させた固相担体（０．５μｍｏｌ）を、３％ジクロロ酢酸／ジクロロメタン溶液（１ｍＬ）で１５秒間反応させて保護基を脱離させた後、反応溶液を除去した。同様の操作を４回繰り返した後、ジクロロメタン（１ｍＬ）で４回、アセトニトリル（１ｍＬ）で４回洗浄を行い、固相担体の真空乾燥を行った。 A solid support (0.5 μmol) carrying 5′-O-DMTr-N 3 -benzoylthymidine via a succinyl linker was reacted with a 3% dichloroacetic acid / dichloromethane solution (1 mL) for 15 seconds to remove the protecting group. After desorption, the reaction solution was removed. The same operation was repeated 4 times, followed by washing 4 times with dichloromethane (1 mL) and 4 times with acetonitrile (1 mL), and vacuum drying of the solid support.
次に、以下の（ｉ）工程と（ii）の組み合わせを１１回繰り返すことでオリゴマーの鎖長伸長反応を行った。なお、モノマーは、標的核酸の塩基配列に対して相補的な塩基配列となる順序で、塩基配列におけるチミジンには上記で合成したＬＮＡ修飾ボラノホスフェート化合物（９）を選択し、塩基配列におけるチミジン以外の塩基には上記合成のＬＮＡ未修飾ボラノホスフェート化合物（１１ａ−ｄ）を選択し、５’末端となるモノマーは、５’位に遊離の水酸基と、塩基としてグアニンを有するヌクレオシドとした。 Next, the combination of the following steps (i) and (ii) was repeated 11 times to carry out an oligomer chain length extension reaction. The monomers are in the order of the base sequence complementary to the base sequence of the target nucleic acid, and the LNA-modified boranophosphate compound (9) synthesized above is selected as the thymidine in the base sequence, and the thymidine in the base sequence is selected. As the base other than the above, the LNA unmodified boranophosphate compound (11a-d) synthesized above was selected, and the 5 ′ terminal monomer was a nucleoside having a free hydroxyl group at the 5 ′ position and guanine as the base.
（ｉ）工程： 配列に応じて選択されたモノマー（２０μｍoｌ）、ＭＮＴＰ（５０μｍoｌ)、及び２，６−ルチジン（１００μｍoｌ）を、アセトニトリル（０．２ｍＬ）で混合し、１分間反応させた。１分後、反応溶液を除去し、反応後の固相担体を、アセトニトリルで４回、ジクロロメタンで４回洗浄を行った。 (I) Step: Monomers (20 μmol), MNTP (50 μmol), and 2,6-lutidine (100 μmol) selected according to the sequence were mixed with acetonitrile (0.2 mL) and allowed to react for 1 minute. After 1 minute, the reaction solution was removed, and the solid support after the reaction was washed 4 times with acetonitrile and 4 times with dichloromethane.
（ii）工程： 前記（ｉ）工程後の固相担体を、３％ジクロロ酢酸／ジクロロメタン―トリエチルシラン（１：１、ｖ／ｖ）溶液（１ｍＬ）で５秒間反応させた。同様の操作を６回繰り返した後、ジクロロメタン（１ｍＬ）で４回、アセトニトリル（１ｍＬ）で４回洗浄を行い、固相担体を真空乾燥させた。 Step (ii): The solid phase carrier after the step (i) was reacted with a 3% dichloroacetic acid / dichloromethane-triethylsilane (1: 1, v / v) solution (1 mL) for 5 seconds. The same operation was repeated 6 times, followed by washing 4 times with dichloromethane (1 mL) and 4 times with acetonitrile (1 mL), and the solid support was vacuum dried.
鎖長伸長後の固相担体に、トリエチルアミン（０．１Ｍ）／四塩化炭素−２，６−ルチジン−Ｈ２Ｏ（５：１２．５：１，ｖ／ｖ／ｖ）溶液を０．２ｍＬ加え、１時間反応させた。その後、反応後の固相担体を、アセトニトリル（１ｍＬ）で４回、ジクロロメタン（１ｍＬ）で洗浄した。
その後、洗浄後の固相担体に対して、２５％アンモニア水−エタノール（３：１、ｖ／ｖ）を５０℃下１２時間反応させることで、核酸塩基部位の保護基の除去と固相担体からの核酸オリゴマーの切り出しを行った。固相担体をろ別し、ろ液を回収した後、減圧留去した。残留物を４ｍＬのＨ２Ｏに溶解し、クロロホルム（５００μＬ）で６回洗浄した。
0.2 mL of a triethylamine (0.1 M) / carbon tetrachloride-2,6-lutidine-H 2 O (5: 12.5: 1, v / v / v) solution is applied to the solid phase carrier after chain length extension. The mixture was further reacted for 1 hour. Thereafter, the solid support after the reaction was washed with acetonitrile (1 mL) four times and with dichloromethane (1 mL).
Thereafter, the solid phase carrier after washing is reacted with 25% aqueous ammonia-ethanol (3: 1, v / v) at 50 ° C. for 12 hours to remove the protecting group at the nucleobase site and the solid phase carrier. The nucleic acid oligomer was excised from The solid phase carrier was filtered off and the filtrate was recovered and then distilled off under reduced pressure. The residue was dissolved in 4 mL of H 2 O and washed 6 times with chloroform (500 μL).
得られた核酸オリゴマー（１２量体、ＬＮＡＰＢＯ−ＯＤＮ）を、イオン交換ＨＰＬＣにて分離精製した。分離精製前後のイオン交換ＨＰＬＣの結果を図１に示す。イオン交換ＨＰＬＣ及び分離精製は、室温下、０〜１Ｍ ＮａＣｌ、５０〜２５％アセトニトリルの１０ｍＭ トリスＨＣｌ緩衝溶液（ｐＨ８．０）を流速０．８ｍｌ／分、２０分間の条件で行われた。図１（Ａ）に分精精製前のイオン交換ＨＰＬＣのグラフを示す。図１（Ａ）のメインピークに相当するフラクションを分取し精製した。図１（Ｂ）に分離精製後のイオン交換ＨＰＬＣのグラフを示す。
収率７％。ＭＡＬＤＩ−ＴＯＦ ＭＳ： Ｃａｌｃｄ ｆｏｒ ［Ｍ−Ｈ］−； ３７６７．０２， ｆｏｕｎｄ； ３７６７．０６．
The obtained nucleic acid oligomer (12-mer, LNAPBO-ODN) was separated and purified by ion exchange HPLC. The results of ion exchange HPLC before and after separation and purification are shown in FIG. Ion exchange HPLC and separation / purification were performed at room temperature under conditions of 0 to 1 M NaCl, 50 to 25% acetonitrile in 10 mM Tris-HCl (pH 8.0) at a flow rate of 0.8 ml / min for 20 minutes. FIG. 1 (A) shows a graph of ion exchange HPLC before purification. The fraction corresponding to the main peak in FIG. 1 (A) was collected and purified. FIG. 1B shows a graph of ion exchange HPLC after separation and purification.
Yield 7%. MALDI-TOF MS: Calcd for [M−H] − ; 3767.02, found; 3767.06.
得られた核酸オリゴマー（１２量体、ＬＮＡＰＢＯ−ＯＤＮ）は、ｄＧＣＡＴ'Ｔ'ＧＧＴ'ＡＴ'Ｔ'Ｃ（Ｔ'はＬＮＡ修飾したチミジンを表す。配列番号３）の塩基配列を有するアンチセンス分子である。 The obtained nucleic acid oligomer (12-mer, LNA PBO-ODN) has an antisense having the base sequence of dGCAT'T'GGT'AT'T'C (T 'represents LNA-modified thymidine; SEQ ID NO: 3). Is a molecule.
＜ＰＢＯ−ＤＮＡ（１２量体）の合成方法−固相法−＞
ＬＮＡＰＢＯ−ＯＤＮと同様にして標的核酸（アポＢ蛋白質）の塩基配列と相補的な配列となるようにＮ４−イソブチリルシチジンと、アデニン、グアニン、チミン又はシトシンを塩基として有するＬＮＡ未修飾ボラノホスフェート化合物（１１ａ−ｄ）を用いて、順次、保護基を有する水酸基の脱保護反応、縮合反応を１２量体が得られるまで繰り返し行った後、酸化反応及び核酸塩基の脱保護反応を行い、ＰＢＯ−ＤＮＡ（１２量体）を得た。
得られたＰＢＯ−ＤＮＡ（１２量体）を含む溶液を、２５％ＮＨ３ −エタノール水溶液（３：１ ｖ／ｖ）で処理し、エタノール（２ｍｌ×４）で洗浄した。水溶液は減圧下で溶媒を留去した。残渣を水（４ｍｌ）に溶解し、クロロホルム（５００μＬ×６）で洗浄した。逆相ＨＰＬＣにて分離精製した。逆相ＨＰＬＣ及び分離精製は、３０℃で、０〜６０％アセトニトリルの０．１Ｍ 酢酸トリメチルアンモニウム緩衝溶液（ｐＨ７．０）を流速０．５ｍｌ／分で６０分間行われた。
収率４４％。ＭＡＬＤＩ−ＴＯＦ ＭＳ： Ｃａｌｃｄ ｆｏｒ ［Ｍ−Ｈ］−； ３６２７．０５， ｆｏｕｎｄ； ３６２６．８２．
<Method of synthesizing PBO-DNA (12-mer) -solid phase method->
LNA unmodified having N 4 -isobutyryl cytidine and adenine, guanine, thymine, or cytosine as bases so as to be complementary to the base sequence of the target nucleic acid (apo B protein) in the same manner as LNA PBO-ODN Using the boranophosphate compound (11a-d), the deprotection reaction of the hydroxyl group having a protecting group and the condensation reaction were successively repeated until a 12-mer was obtained, and then the oxidation reaction and the deprotection reaction of the nucleobase were performed. And PBO-DNA (12-mer) was obtained.
The resulting solution containing PBO-DNA (12-mer) was treated with 25% NH 3 -ethanol aqueous solution (3: 1 v / v) and washed with ethanol (2 ml × 4). The solvent was distilled off from the aqueous solution under reduced pressure. The residue was dissolved in water (4 ml) and washed with chloroform (500 μL × 6). Separation and purification were performed by reverse phase HPLC. Reverse phase HPLC and separation / purification were performed at 30 ° C. in a 0.1 M trimethylammonium acetate buffer solution (pH 7.0) of 0 to 60% acetonitrile for 60 minutes at a flow rate of 0.5 ml / min.
Yield 44%. MALDI-TOF MS: Calcd for [M−H] −; 3627.05, found; 3626.82.
得られたＰＢＯ−ＤＮＡ（１２量体）は、ＬＮＡ修飾ボラノホスフェート化合物に由来するヌクレオシド構造を含まず、ＧＣＡＴＴＧＧＴＡＴＴＣの塩基配列を有するアンチセンス化合物である。 The obtained PBO-DNA (12-mer) is an antisense compound that does not contain a nucleoside structure derived from an LNA-modified boranophosphate compound and has a base sequence of GCATTGGTATTC.
＜実施例１＞
相補鎖ＤＮＡの最終モル数が０．６０ｎｍｏｌとなるように、及び上記合成方法で得たＬＮＡＰＢＯ−ＯＤＮ（１２量体）の最終濃度が４．０μＭとなるように、相補鎖ＤＮＡ及びＬＮＡＰＢＯ−ＯＤＮ（１２量体）を１０ｍＭ ＮａＨ２ＰＯ４−Ｎａ２ＨＰＯ４緩衝溶液（ｐＨ７．０）に溶解し、測定試料を調製した。この測定試料の塩濃度を１００ｍＭ ＮａＣｌに調整した。その後、測定試料を光路長１ｃｍのセルに入れ、可視紫外分光光度計により２６０ｎｍにおける吸光度を、０．５℃／ｍｉｎの速度で温度を０℃から９０℃まで変化させて測定した。そして、１００ｍＭ ＮａＣｌにおける相補鎖ＤＮＡとＬＮＡＰＢＯ−ＯＤＮ（１２量体）との融解曲線を得た。その結果を図２（Ａ）に示す。この融解曲線から、二重鎖融解温度（Ｔｍ）を得た。その結果を表１に示す。
なお、融解温度（Ｔｍ）は、中線法で求めた。具体的には、得られた融解曲線の前遷移領域及び後遷移領域にそれぞれにおいて２点を指定し、回帰計算によりベースラインを求め、２つのベースラインの中線と融解曲線との交点を融解温度とした。
<Example 1>
The complementary strand DNA and LNA PBO- were adjusted so that the final molar number of the complementary strand DNA was 0.60 nmol, and the final concentration of LNAPBO-ODN (12-mer) obtained by the synthesis method was 4.0 μM. ODN (12-mer) was dissolved in 10 mM NaH 2 PO 4 -Na 2 HPO 4 buffer solution (pH 7.0) to prepare a measurement sample. The salt concentration of this measurement sample was adjusted to 100 mM NaCl. Thereafter, the measurement sample was placed in a cell having an optical path length of 1 cm, and the absorbance at 260 nm was measured with a visible ultraviolet spectrophotometer at a rate of 0.5 ° C./min while changing the temperature from 0 ° C. to 90 ° C. And the melting curve of the complementary strand DNA and LNA PBO-ODN (12-mer) in 100 mM NaCl was obtained. The result is shown in FIG. From this melting curve, the double chain melting temperature (Tm) was obtained. The results are shown in Table 1.
The melting temperature (Tm) was determined by the midline method. Specifically, two points are specified in each of the front transition region and the rear transition region of the obtained melting curve, a baseline is obtained by regression calculation, and the intersection between the midline of the two baselines and the melting curve is melted. It was temperature.
相補鎖ＤＮＡを相補鎖ｍＲＮＡに変更した以外は上記と同様にして、１００ｍＭ ＮａＣｌにおける相補鎖ｍＲＮＡとＬＮＡＰＢＯ−ＯＤＮ（１２量体）との融解曲線を得て、二重鎖融解温度を決定した。それらの結果を図２（Ｂ）及び表１に示す。融解温度は、実施例１と同様に決定した。 In the same manner as described above except that the complementary strand DNA was changed to the complementary strand mRNA, a melting curve of the complementary strand mRNA in 100 mM NaCl and LNA PBO-ODN (12-mer) was obtained, and the duplex melting temperature was determined. . The results are shown in FIG. The melting temperature was determined in the same manner as in Example 1.
塩濃度を１Ｍに変更した以外は上記と同様にして、１Ｍ ＮａＣｌにおける相補鎖ＤＮＡ又は相補鎖ｍＲＮＡとＬＮＡＰＢＯ−ＯＤＮ（１２量体）との融解曲線を得て、二重鎖融解温度を決定した。それらの結果をそれぞれ図３（Ａ）及び図３（Ｂ）、並びに表１に示す。 Except that the salt concentration was changed to 1M, the melting curve of the complementary strand DNA or complementary strand mRNA in 1M NaCl and LNA PBO-ODN (12-mer) was obtained, and the double strand melting temperature was determined. did. The results are shown in FIGS. 3A and 3B and Table 1, respectively.
＜比較例１＞
ＬＮＡＰＢＯ−ＯＤＮ（１２量体）に代えて、標的核酸と相補的な塩基配列を有する天然型ＤＮＡを用いた以外は、実施例１と同様にして、１００ｍＭ又は１Ｍの塩濃度における相補鎖ＤＮＡ又は相補鎖ｍＲＮＡと天然型ＤＮＡとの融解曲線を得て、二重鎖融解温度を決定した。それらの結果を図２〜図３及び表１に示す。
<Comparative Example 1>
A complementary strand DNA at a salt concentration of 100 mM or 1 M in the same manner as in Example 1 except that natural DNA having a base sequence complementary to the target nucleic acid was used instead of LNA PBO-ODN (12-mer). Alternatively, a melting curve between complementary strand mRNA and natural DNA was obtained to determine the duplex melting temperature. The results are shown in FIGS.
＜比較例２＞
ＬＮＡＰＢＯ−ＯＤＮ（１２量体）に代えて、標的核酸と相補的な塩基配列を有するＰＢＯ−ＤＮＡ（１２量体）を用いた以外は、実施例１と同様にして、１００ｍＭ又は１Ｍの塩濃度における相補鎖ＤＮＡ又は相補鎖ｍＲＮＡとＰＢＯ−ＤＮＡ（１２量体）との融解曲線を得て、二重鎖融解温度を決定した。それらの結果を図２〜図３及び表１に示す。
<Comparative Example 2>
100 mM or 1M salt in the same manner as in Example 1 except that PBO-DNA (12-mer) having a base sequence complementary to the target nucleic acid was used instead of LNA PBO-ODN (12-mer). Melting curves of complementary strand DNA or complementary strand mRNA and PBO-DNA (12-mer) at concentrations were obtained to determine the duplex melting temperature. The results are shown in FIGS.
なお、表１中のΔＴｍ（℃）は天然型ＤＮＡの二重鎖融解温度との差を示す。図２〜図３の各図において、実線は天然型ＤＮＡを用いた場合の融解曲線を表し、点線はＰＢＯ−ＤＮＡ（１２量体）を用いた場合の融解曲線を表し、一点鎖線はＬＮＡＰＢＯ−ＯＤＮ（１２量体）を用いた場合の融解曲線を表す。 In Table 1, ΔTm (° C.) indicates a difference from the double-stranded melting temperature of natural DNA. 2 to 3, the solid line represents a melting curve when natural DNA is used, the dotted line represents a melting curve when PBO-DNA (12-mer) is used, and the one-dot chain line represents LNA PBO. -Represents a melting curve when ODN (12-mer) is used.
表１に示すように、１００ｍＭ ＮａＣｌ及び１Ｍ ＮａＣｌのいずれの条件下においても、ＰＢＯ−ＤＮＡ（比較例２）の相補鎖ＤＮＡ又は相補鎖ｍＲＮＡに対する二重鎖溶解温度は、天然型ＤＮＡ（比較例１）の相補鎖ＤＮＡ又は相補鎖ｍＲＮＡに対する二重鎖溶解温度に比べ低い。このことから、天然型ＤＮＡへのボラノ基（ＢＨ３）導入により二重鎖形成能が低減することがわかる。 As shown in Table 1, under both conditions of 100 mM NaCl and 1 M NaCl, the double strand melting temperature of PBO-DNA (Comparative Example 2) with respect to the complementary strand DNA or the complementary strand mRNA is the natural DNA (Comparative Example). Lower than double strand melting temperature for complementary strand DNA or complementary strand mRNA of 1). From this, it can be seen that double strand forming ability is reduced by introducing borano group (BH 3 ) into natural DNA.
これに対して、ＬＮＡＰＢＯ−ＯＤＮ（実施例１）は、１００ｍＭ ＮａＣｌ及び１Ｍ ＮａＣｌのいずれの条件下においても、天然型ＤＮＡ（比較例１）及びＰＢＯ−ＤＮＡ（比較例２）に比べ、高い二重鎖融解温度を示した。このことから、ＰＢＯ−ＤＮＡへのＬＮＡ修飾により、ボラノ基導入による二重鎖形成能の低減を抑制し、かつ二重鎖形成能を高めていることがわかる。 In contrast, LNA PBO-ODN (Example 1) is higher than natural DNA (Comparative Example 1) and PBO-DNA (Comparative Example 2) under both conditions of 100 mM NaCl and 1 M NaCl. The duplex melting temperature was indicated. From this, it can be seen that the LNA modification to PBO-DNA suppresses the reduction of the duplex formation ability due to the introduction of the borano group and enhances the duplex formation ability.
このように、ＬＮＡＰＢＯ−ＯＤＮは優れた二本鎖形成能を有する核酸オリゴマーであり、有用なアンチセンス分子として使用可能であることが示された。
従って、本発明によれば、従来のボラノホスフェート型核酸オリゴマーと比較して、相補鎖との二重鎖形成能に優れた核酸オリゴマーと、この核酸オリゴマーを合成するのに適した新規ボラノホスフェート化合物を提供することができる。
Thus, it was shown that LNA PBO-ODN is a nucleic acid oligomer having an excellent ability to form a double strand and can be used as a useful antisense molecule.
Therefore, according to the present invention, compared with a conventional boranophosphate type nucleic acid oligomer, a nucleic acid oligomer excellent in duplex forming ability with a complementary strand, and a novel borano suitable for synthesizing this nucleic acid oligomer. Phosphate compounds can be provided.
Claims (5)
（一般式（Ｉ）中、Ｒ１は水素原子又はアセチル基、フェノキシアセチル基、ピバロイル基、ベンジル基、４−メトキシベンジル基、ベンゾイル基、トリフェニルメチル基、４，４’−ジメトキシトリチル（ＤＭＴｒ）基、４−メトキシトリチル（ＭＭＴｒ）基、９−フェニルキサンテニル基、トリメチルシリル基、シアノメトキシメチル基、２−（シアノエトキシ）エチル基及びシアノエトキシメチル基からなる群より選ばれる水酸基の保護基を表し、Ｂｓは、ベンジル基、４−メトキシベンゾイル基、アセチル基、プロピオニル基、ブチリル基、イソブチリル基、フェニルアセチル基、４−ｔｅｒｔ−ブチルフェノキシアセチル基、４−イソプロピルフェノキシアセチル基及び（ジメチルアミノ）メチレン基からなる群より選ばれる保護基を有してもよいアデニン、グアニン及びシトシン並びにチミン及びウラシルからなる群より選ばれる核酸塩基を表し、Ｘ＋はカウンターカチオンを表す。） A boranophosphonate compound represented by the following general formula (I).
(In the general formula (I), R 1 is a hydrogen atom or an acetyl group, a phenoxyacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a triphenylmethyl group, 4,4′-dimethoxytrityl (DMTr ), 4-methoxytrityl (MMTr) group, 9-phenylxanthenyl group, trimethylsilyl group, cyanomethoxymethyl group, 2- (cyanoethoxy) ethyl group and cyanoethoxymethyl group. Bs represents benzyl group, 4-methoxybenzoyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, phenylacetyl group, 4-tert-butylphenoxyacetyl group, 4-isopropylphenoxyacetyl group and (dimethylamino). ) Protection selected from the group consisting of methylene groups May have an adenine, it represents a nucleobase selected from the group consisting of guanine and cytosine and thymine, and uracil, X + represents a counter cation.)
（一般式（II）中、Ｒ１は水素原子又はアセチル基、フェノキシアセチル基、ピバロイル基、ベンジル基、４−メトキシベンジル基、ベンゾイル基、トリフェニルメチル基、４，４’−ジメトキシトリチル（ＤＭＴｒ）基、４−メトキシトリチル（ＭＭＴｒ）基、９−フェニルキサンテニル基、トリメチルシリル基、シアノメトキシメチル基、２−（シアノエトキシ）エチル基及びシアノエトキシメチル基からなる群より選ばれる水酸基の保護基を表す。一般式（II）及び一般式（III）中、Ｂｓはベンジル基、４−メトキシベンゾイル基、アセチル基、プロピオニル基、ブチリル基、イソブチリル基、フェニルアセチル基、４−ｔｅｒｔ−ブチルフェノキシアセチル基、４−イソプロピルフェノキシアセチル基及び（ジメチルアミノ）メチレン基からなる群より選ばれる保護基を有してもよいアデニン、グアニン及びシトシン並びにチミン及びウラシルからなる群より選ばれる核酸塩基を表し、Ｘ＋はカウンターカチオンを表す。） The method further comprises an oxidation step of oxidizing the nucleic acid oligomer obtained in the condensation step with an oxidizing agent, and the nucleic acid oligomer obtained in the oxidation step is represented by the structure represented by the general formula (II) and the general formula (III) The method for producing a nucleic acid oligomer according to claim 4, which has at least one of structures.
(In the general formula (II), R 1 is a hydrogen atom or an acetyl group, a phenoxyacetyl group, a pivaloyl group, a benzyl group, a 4-methoxybenzyl group, a benzoyl group, a triphenylmethyl group, 4,4′-dimethoxytrityl (DMTr ), 4-methoxytrityl (MMTr) group, 9-phenylxanthenyl group, trimethylsilyl group, cyanomethoxymethyl group, 2- (cyanoethoxy) ethyl group and cyanoethoxymethyl group. In general formula (II) and general formula (III), Bs is a benzyl group, 4-methoxybenzoyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, phenylacetyl group, 4-tert-butylphenoxyacetyl. Group, 4-isopropylphenoxyacetyl group and (dimethylamino) methylene May have a protective group selected from the group consisting of adenine, it represents a nucleobase selected from the group consisting of guanine and cytosine and thymine, and uracil, X + represents a counter cation.)
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