JP5424236B2 - Oligonucleotide derivative, oligonucleotide construct using oligonucleotide derivative, compound for synthesizing oligonucleotide derivative, and method for producing oligonucleotide derivative - Google Patents

Description

  The present invention relates to an oligonucleotide derivative, an oligonucleotide construct using the oligonucleotide derivative, a compound for synthesizing the oligonucleotide derivative, and a method for producing the oligonucleotide derivative.

  In recent years, various oligonucleotides such as DNA and RNA have been used for therapeutic and diagnostic purposes. For example, in the diagnostic use, a DNA chip and a DNA microarray can be mentioned, and in the therapeutic use, in addition to the introduction of a treatment-related gene, expression suppression by knockdown of a disease-related gene can be mentioned. Attempts have also been made to use aptamers that are nucleic acid molecules or peptides that specifically bind to specific molecules as therapeutic agents.

  Nucleic acid technology that has received particular attention includes a knockdown method for specific genes using RNA interference (RNAi). RNAi refers to a phenomenon in which the action of a gene whose sequence is homologous to it is suppressed by the action of double-stranded RNA (dsRNA). In the suppression of gene expression by RNAi, dsRNA is recognized by Dicer, which is a member of the RNase III family, and cleaved into 21-23 mer siRNAs (short interfering RNAs), and this siRNA is converted into RISC (RNA-induced silencing complex). This is because mRNA that is taken in and subsequently homologous to the taken-in siRNA is cleaved at the center and degraded.

  However, exogenous DNA and RNA in the living body are exposed to various nucleases, and in particular, RNA is easily degraded by nucleases, so that the intended knockdown effect cannot be obtained sufficiently or the knockdown effect is stable. There was a problem that it was difficult to maintain.

  In order to solve these problems, it has been studied to improve nuclease resistance by chemically modifying RNA (Non-Patent Documents 1 to 3).

  For example, various chemical modifications have been attempted for siRNA as shown in FIG. 3 (Non-patent Document 4). In addition, the present inventor converted the phosphodiester binding portion of the 3 ′ end dangling end of siRNA to carbamate binding or urea binding, thereby eliminating the negative charge of the binding portion, and siRNA nuclease resistance and silencing activity. (Non-Patent Document 5). The present inventor has also succeeded in enhancing nuclease resistance by introducing a unit having a benzene skeleton into a nucleic acid oligomer such as RNA instead of a nucleoside, and further, an amidite for introducing a benzene skeleton. The reagent was modified to CPG resin, and the modification of nucleotides using this was also successful (Patent Document 1).

WO2007 / 094135

(L. Beigelman., J.A McSwiggen., K.G.Draper et al., J Biolchem 270, 25702-25708 (1995) 27) S.P.Zinnen K. Domenico., M. Wilson et al., RNA 8,214-228 (2002) S.AgrawalandE.R.KandimaIla., Curr.CanGer Drug Targets., 1,197-209 (2001)) H. Hoshi FEBS Letters 521, 197-199 (2002)) Y. Ueno, T. Naito, K. Kawada, A. Shibata, Hye-SookKim Y. Wataya, Y. Kidade, Biochem Biophys Res Commun330, 1168-1175 (2005) J.J.Song., LiuN.H.Tolia., J.Schneiderman., S.K.Smith., R.A.Martienssen., G.J.Hannon and L.Joshuator., Nat.Struct.Biol ... 10,1026-1032 (2003) K.S.Yan., S.Yan., A.Farooq., A.Han., L.Zengand M.M.Zhou., Nature., 426,468-474 (2003) Zhang., F.A Kolb., L.Jaskiewicz., E.Westhofand W.Filipowicz Cell 118,57-68 (2003) (A. Linge B. Simon E. 1 zaurralde and M. Sattler Nature 426,465-469 (2003))

  The technology for introducing a unit having a benzene skeleton into a nucleic acid oligomer such as RNA described in Patent Document 1 described above has nuclease resistance and silencing activity by chemically modifying the 3 ′ end dangling end of siRNA. Although it has been confirmed that it can be increased, there has been a demand for further enhancing the effect.

In addition, if positron emission tomography (PET), which enables molecular elucidation of local sites and metabolism of drugs in vivo, can be applied to oligonucleotide derivatives, it will be a powerful weapon for the development of RNA drug discovery. It is thought that it becomes. However, the labeling elements used in PET have a short half-life (20 minutes at ¹¹ C, 110 minutes at ¹⁸ F), and therefore, an oligonucleotide derivative that can be synthesized in a short time, which can be applied to the PET method. There was a need for a production method.

  The present invention has been made in view of the above-described conventional circumstances, and has an oligonucleotide derivative having good nuclease resistance, an oligonucleotide construct using the oligonucleotide derivative, a compound for synthesizing the oligonucleotide derivative, and an oligonucleotide One object is to provide a method for producing a derivative. The present invention also provides an oligonucleotide derivative having improved gene expression suppression effect by RNAi, an oligonucleotide construct using the oligonucleotide derivative, a compound for synthesizing the oligonucleotide derivative, and a method for producing the oligonucleotide derivative. Is another purpose. Furthermore, the present invention provides an oligonucleotide derivative capable of introducing a labeling element used in the PET method in a short time, an oligonucleotide construct using the oligonucleotide derivative, a compound for synthesizing the oligonucleotide derivative, and a method for producing the oligonucleotide derivative One purpose is to provide

In the oligonucleotide derivative of the present invention, at least one unit represented by the following formula (1) (however, instead of hydrogen directly bonded to the benzene ring of the following formula (1), a substituent other than hydrogen is bonded. Including those that are present).

  A fluoromethyl group is bonded to the benzene ring of the above formula (1), and it has been elucidated by the present inventors that an oligonucleotide derivative in which this fluoromethyl group is changed to hydrogen has nuclease resistance ( Patent Document 1). The oligonucleotide derivative of the present invention has further excellent nuclease resistance due to the fluoromethyl group bonded to the benzene ring. In addition, the unit represented by the above formula (1) binds to a target site of various nucleases (specific sites such as 3 ′ end, 5 ′ end, non-3 ′ end and non-5 ′ end depending on the type of nuclease). Can be made.

  Further, by binding the unit of the above formula (1) to the 3 ′ dangling end of siRNA, the gene expression suppressing action by RNAi is increased. The reason for this is not clear, but can be inferred as follows. RISC, which plays an important role in RNAi, is known as a multidomain protein involved in the process of degradation of RNAi target mRNA. Recently, X-ray crystal structure analysis of co-crystal of PAZ domain and siRNA in RISC (JBMa K. Ye and DJPatel Nature 429, 318-322 (2004)). As a result, it has been clarified that the PAZ domain recognizes the 3 ′ terminal dangling end of siRNA, and 2 nucleotides of the 3 ′ terminal dangling end enter the hydrophobic pocket of the PAZ domain. . The oligonucleotide derivative of the present invention has a fluoromethyl group having strong hydrophobicity, and therefore, the affinity with PAZ and the recognition by PAZ are improved, thereby improving the silencing effect. It is considered a thing. For this reason, siRNA provided with the above unit at the 3 'end dangling end can have excellent nuclease resistance and excellent silencing effect.

The oligonucleotide derivative of the present invention may have at least one unit represented by the following formula (2).

式中、R¹及びR²は、それぞれ独立して水素又はヒドロキシル保護基を表し、ａ、ｂ及びｃは独立して０以上の整数であって少なくとも１つが1以上であり、Ａ及びＢは独立して改変されてもよいオリゴヌクレオチドであってＡとＢとを合わせた鎖長が３以上のオリゴヌクレオチドを表す。ただし、Ａ及びＢにおいてオリゴヌクレオチドの３’末端と５’末端の水酸基を含まない。 The oligonucleotide derivative of the present invention may have a structure represented by the following formula (3).

Wherein R ¹ and R ² each independently represent hydrogen or a hydroxyl protecting group, a, b and c are each independently an integer of 0 or more, at least one is 1 or more, and A and B are An oligonucleotide which may be independently modified and has a chain length of A and B combined is 3 or more. However, A and B do not include the hydroxyl groups at the 3 ′ end and 5 ′ end of the oligonucleotide.

Furthermore, in the oligonucleotide derivative of the present invention, R ¹ and R ² can be H in the above formula (3). Here, b may be 0. Furthermore, both a and b may be 0. Further, c may be 1 or more and 5 or less.

  Further, A and B may be oligonucleotide derivatives having a partial sequence of a predetermined gene mRNA or a complementary sequence thereof. Further, the chain length of A and B together may be 10 or more and 35 or less. Furthermore, A and B may be oligoribonucleotides.

The oligonucleotide construct of the present invention is an oligonucleotide construct for regulating gene expression, characterized by having any of the above oligonucleotide derivatives. This oligonucleotide construct can be an oligonucleotide construct selected from single and double stranded DNA, single and double stranded RNA, DNA / RNA chimeras and DNA / RNA hybrids, and its function. From the aspect, it can be selected from antigene, antisense, aptamer, siRNA, miRNA, shRNA and lipozyme. Furthermore, the dangling end portion can have a unit represented by the following formula (4).

  Moreover, it is siRNA, Comprising: In the said oligonucleotide derivative, a and b are 0, c is 1 or 2, 3 'terminal dangling end part contains the unit represented by said Formula (4). Can do.

  Furthermore, according to the present invention, an oligonucleotide construct for gene diagnosis having any of the above-described oligonucleotide derivatives can be obtained. Furthermore, the construct can be a probe or primer.

（式中Ｗ^１はヒドロキシル保護基を表し、Ｗ^２はＨ、ホスホロアミダイト基又は固相担体に結合される若しくは結合された連結基を表す。
この化合物は、ｓｉＲＮＡのダングリングエンドの化学修飾用のユニットとして用いることができる。 The oligonucleotide derivative of the present invention can be synthesized by utilizing the compound of the present invention. That is, the compound of the present invention is represented by the following formula (5) (however, in place of hydrogen directly bonded to the benzene ring of formula (5), a substituent other than hydrogen is bonded). Including that which is).

Wherein W ¹ represents a hydroxyl protecting group and W ² represents H, a phosphoramidite group or a linking group bound to or bound to a solid support.
This compound can be used as a unit for chemical modification of the dangling end of siRNA.

Oligonucleotides can be modified using one or more selected from the above compounds of the present invention. That is, at least one unit represented by the following formula (1) (however, instead of hydrogen directly bonded to the benzene ring of the following formula (1), a substituent other than hydrogen is bonded to the oligonucleotide. Any of these may be added, substituted and inserted, or a combination thereof.

(Oligonucleotide derivatives)
The oligonucleotide derivative of the present invention has a unit represented by the above formula (1) in at least one location of the oligonucleotide derivative. Due to the presence of this unit, excellent nuclease resistance can be obtained. Such a unit can be introduced into the oligonucleotide of known sequence or unknown sequence, either by addition, substitution or insertion, or a combination thereof, and thus can be used as the oligonucleotide derivative of the present invention. it can. In addition, the oligonucleotide here is an oligonucleotide that may be modified.

In the unit represented by the above formula (1), the two “—CH ₂ O—” groups and the fluoromethyl group bonded to the benzene ring may be bonded to any position. Moreover, it replaces with the hydrogen directly couple | bonded with a benzene ring and substituents other than hydrogen may couple | bond together. Such a substituent is preferably a hydrophobic group having a small polarity, for example, an alkyl group having 1 to 4 carbon atoms. That is, methyl group, ethyl group, n-propyl group, n-butyl group, isobutyl group and tert-butyl group can be mentioned.

  In the oligonucleotide derivative of the present invention, the unit represented by the above formula (1) is bound to a part of the oligonucleotide via an appropriate linking group. As the linking group, for example, a known linking group used for linking oligonucleotide nucleotide units such as a phosphodiester bond can be used. When the unit is at the end of the oligonucleotide, hydrogen or a known hydroxyl protecting group may be bonded to one oxygen atom of the unit.

式中、R¹及びR²は、それぞれ独立して水素又はヒドロキシル保護基を表す。ヒドロキシル保護基としては、該保護基により置換されるヒドロキシル基中の酸素を意図しない反応から保護する基であればよい。好ましくは、保護基は、オリゴヌクレオチド誘導体の活性を維持し、脱離容易なものである。こうしたヒドロキシル保護基としては、特に限定されなくて、従来公知の各種のヒドロキシル保護基を用いることができる。本発明の好ましい保護基として、例えばフルオレニルメトキシカルボニル（ＦＭＯＣ）、ジメトキシトリチル（ＤＭＴ）、モノメトキシトリチル、トリフルオロアセチル、レブリニル、シリル基等である。例えば、ヌクレオチド又はオリゴヌクレオチドの５’末端にあるヒドロキシル基についての好ましい保護基は、トリチル基、ジメトキシトリチル基から選択される。 Moreover, the compound represented by following formula (3) is mentioned as one aspect | mode of the oligonucleotide derivative of this invention.

In the formula, R ¹ and R ² each independently represent hydrogen or a hydroxyl protecting group. The hydroxyl protecting group may be any group that protects oxygen in the hydroxyl group substituted by the protecting group from an unintended reaction. Preferably, the protecting group maintains the activity of the oligonucleotide derivative and is easily removed. Such hydroxyl protecting groups are not particularly limited, and various conventionally known hydroxyl protecting groups can be used. Preferred protective groups of the present invention are, for example, fluorenylmethoxycarbonyl (FMOC), dimethoxytrityl (DMT), monomethoxytrityl, trifluoroacetyl, levulinyl, silyl group and the like. For example, preferred protecting groups for the hydroxyl group at the 5 ′ end of nucleotides or oligonucleotides are selected from trityl groups, dimethoxytrityl groups.

  The oligonucleotide derivative of this embodiment has any one or more of units 1 to 3 represented by the three types of parentheses shown in the above formula (3). Hereinafter, in the formula (3), the unit arranged at the 5 ′ end is referred to as unit 1, the unit arranged at the 3 ′ end is referred to as unit 3, and is arranged at the non-5 ′ end and the non-3 ′ end. The unit is referred to as unit 2. Although it differs depending on the function to be added to the oligonucleotide derivative, Unit 1 is arranged at the 5 ′ end of the oligonucleotide, so it is effective for imparting exonuclease resistance, for example, at the 5 ′ end. is there. Unit 3 is also effective for exonucleases acting on the 3 'end. Unit 2 is also effective in conferring endonuclease resistance at the non-3'end non-5'end of the oligonucleotide. Furthermore, when the unit 3 is formed at the 3 'end dangling end portion of the double-stranded RNA, it is effective for improving the silencing effect by RNAi. Thus, for example, a and b may be 0 when it is desired to improve 3'-end acting nuclease resistance. Conversely, b and c may be 0 for the purpose of increasing 5'-end acting nuclease resistance. Moreover, in order to obtain nuclease resistance, it is sufficient that at least one of a, b, and c is present, but it is also possible to have two or more in combination with each other.

  The number and site of unit 1, unit 2 and unit 3 are determined in consideration of nuclease resistance and the influence on oligonucleotide derivatives by introducing non-nucleoside units 1 to unit 3. For example, when siRNA or shRNA is constructed using the oligonucleotide derivative of the present invention, a and b are 0, and nucleotides corresponding to the dangling end site at the 3 ′ end of these constructs (for example, dT (deoxythymidine 1) to 3 (c = 1 to 3), preferably 1 or 2 (c = 1, 2) units 3 may be added to the 3 ′ end of One or two of dT may be replaced with unit 3 (c = 1, 2). Further, the unit 2 may be inserted into the 3 'terminal dangling end. The form in which the existing nucleotide is replaced with the unit 3 has an advantage that the chain length of siRNA is not extended.

  Moreover, when constructing an antigene, an antisense, an aptamer, a miRNA and a ribozyme using the oligonucleotide derivative of the present invention, a unit represented by the formula (1) or units 1 to 3 are provided as necessary. What should I do? For example, in the antisense RNA, the units represented by the formula (1), unit 1 and unit 3 can be formed on the 3 ′ end side and 5 ′ end side. In aptamers and ribozymes, the unit represented by the formula (1) or the unit 2 at the non-5 ′ end non-3 ′ end may be effective. In the probe, the unit represented by the formula (1), the unit 3 and / or the unit may be provided on the 3 ′ end side and / or the 5 ′ end side, or the probe may be provided on the solid phase carrier. When it is fixed, the unit represented by the formula (1), the unit 1 or the unit 3 can be provided on the free end side. Furthermore, the primer may be appropriately provided with a unit represented by the formula (1) or units 1 to 3 as necessary.

  In the oligonucleotide derivative of the present invention, in the above formula (3), A and B each independently represent an oligonucleotide which may be the same or different but may be modified. Here, the term “oligonucleotide” as used in the present specification means a polymer having a plurality of monomer units in which nucleotides, which are monomers constituting oligonucleotides and polynucleotides, are used as monomer units. The oligonucleotide means deoxyribonucleotide and / or ribonucleotide as a monomer unit. In general, a polymer having deoxyribonucleotides as monomer units as nucleotides is referred to as DNA, and a polymer having ribonucleotides as monomer units is referred to as RNA, but the oligonucleotide derivative of the present invention is generally used for DNA and RNA, These oligomer unit oligomers are included. Oligonucleotides also include RNA / DNA chimeras. The oligonucleotides that may be modified include not only oligonucleotides comprising only natural bases such as guanine, cytosine, thymine, adenine, uracil or methylcytosine, which are purines and pyrimidines, but also various portions of oligonucleotide That is, oligonucleotides having one or more nucleotides with some chemical modification in the base, sugar moiety and phosphate ester moiety.

  In the oligonucleotide derivative of the present invention, the base sequence of the oligonucleotide A and the base sequence of the oligonucleotide B in the above formula (3) are the sense strand of the DNA of a predetermined gene, or the antisense strand thereof, respectively or in combination thereof Alternatively, it may have a partial sequence of mRNA or a complementary sequence thereof. By having such complementarity, it is possible to hybridize with various target nucleic acids, thereby expressing the intended function of the oligonucleotide derivative. In the oligonucleotide derivative of the present invention, the lengths of A and B are not particularly limited and can be set according to the intended use. However, considering the ease of oligonucleotide synthesis and the expected effect, 10 It is preferable to set it as 35 or more. In the case of antisense, it can be about 10 or more and 30 or less. In the case of siRNA, the total chain length of A and B is preferably 15 or more and 35 or less, more preferably 30 or less. . In the case of a primer, it is preferably 10 or more and 30 or less, and in the case of a probe, it is preferably 10 or more and 30 or less.

  When the oligonucleotide derivative of the present invention is used for, for example, siRNA, shRNA, antisense, ribozyme and aptamer, the monomer units of A and B can be oligonucleotide ribonucleotides which may be modified.

(Oligonucleotide construct)
The oligonucleotide construct of the present invention has one or more of the oligonucleotide derivatives of the present invention. Depending on the type and combination of the oligonucleotide derivative in the oligonucleotide construct, the construct can be a single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, DNA / RNA chimera, DNA / RNA hybrid, etc. Each form or a combination form can be adopted. As already described, since the oligonucleotide part constituting the oligonucleotide derivative contains a modified oligonucleotide, the oligonucleotide construct may also contain a modified form in the oligonucleotide construct.

  It is preferable that the oligonucleotide construct which takes such various forms is provided with the unit represented by Formula (1) or the unit 1-the unit 3 in the site | part which may become a target of a nuclease. The unit represented by the formula (1) or the units 1 to 3 can be provided for terminal mismatch or dangling end. In consideration of exonucleotide resistance, it is preferable to provide the unit represented by the formula (1), the unit 1 and the unit 2 at the dangling end. Moreover, the unit represented by Formula (1) or the unit 2 can be provided in a bulge, a mismatch internal loop, a hairpin loop, or the like.

  Since the oligonucleotide construct of the present invention has improved nuclease resistance, it can be used for gene expression regulation or for various uses for research and diagnosis. Examples of gene expression regulation applications include antigene, antisense, aptamer, siRNA, miRNA, shRNA and ribozyme. In particular, in siRNA and shRNA, both the nuclease resistance and the silencing activity are improved by introducing the unit represented by the formula (1) or the unit 2 into the 3 ′ terminal overhang site dT by substitution or addition. be able to.

  FIG. 1 shows an example of siRNA having unit 3 at the 3 'end. Moreover, a probe and a primer are mentioned as a diagnostic use or research use. A probe is an oligonucleotide that, by design or selection, has a sequence that is specifically defined for a target nucleic acid and that has been obtained to allow them to hybridize under a given stringency. Since the nuclease resistance is improved by using this oligonucleotide derivative in the probe, the effect of nuclease mixed in the sample containing the target nucleic acid is suppressed or avoided, and even if the nuclease removal level is low or nuclease removal treatment Sample preparation without omission becomes possible. As a result, genetic diagnosis and testing can be performed easily. Such hybridization between the probe and the target can be performed by immobilizing the probe on an appropriate glass substrate, a plastic substrate, or a solid phase carrier such as beads. The present invention also includes a solid phase carrier on which a probe containing the present oligonucleotide derivative is immobilized.

(Method for producing oligonucleotide derivative)
The compound represented by the formula (5) is a preferable compound for using the oligonucleotide derivative of the present invention for production.

These compounds are preferable compounds for introducing any one of substitution, addition and insertion of the units 1 to 3 in the oligonucleotide or a combination thereof in the oligonucleotide derivative of the present invention. These compounds have a fluorine element applicable to the PET method ( ¹⁸ F has a half-life of 110 minutes, and can take about 1 hour for synthesis and purification). ^Since it can be introduced into an oligonucleotide derivative in a short time by binding it to a phosphoramidite group or a solid phase carrier, it can be used as a raw material for an oligonucleotide derivative that can be applied to the PET method. .

In formula (5), W ¹ represents a hydroxyl protecting group. The hydroxyl protecting group may be any group that protects oxygen in the hydroxyl group substituted by the protecting group from an unintended reaction. Preferably, the protecting group is removed while maintaining the activity of the oligonucleotide derivative. Such hydroxyl protecting groups are not particularly limited, and various conventionally known hydroxyl protecting groups can be used. Preferred protecting groups of the present invention are fluorenylmethoxycarbonyl group (FMOC), dimethoxytrityl group (DMT), monomethoxytrityl group, trifluoroacetyl group, levulinyl group, silyl group and the like. Preferred protecting groups are a trityl group and a dimethoxytrityl group.

W ² represents H, a phosphoramidite group, or a linking group bound to or bound to a solid phase carrier. A compound in which W ² is H (hereinafter also referred to as compound I) can be used as a precursor compound for nucleic acid synthesis. In addition, a compound in which W ² is a phosphoramidite group (hereinafter, also referred to as compound II) is used as a phosphoramidite reagent by the phosphoramidite method, and unit 1 and unit 2 in the formula (1) with respect to the oligonucleotide An oligonucleotide derivative having a unit introduced therein can be obtained. In the present invention, the phosphoramidite group can be represented by the following formula (6).

（式（６）中、各Ｙ^ｌは独立して、同−であっても異なっていてもよく、分枝していてもよい炭素数１〜５個のアルキル基を表し、Ｙ^２は、分枚状又は直鎖状の炭素数１〜５偶のアルキル基又は置換されていてもよいアルコキシル基を表す。）上記式（６）において、Ｙ^ｌは、特に限定しないがイソプロピル基が好ましいものとして挙げられ、またＹ^２としては、一ＯＣＨ_３、−ＯＥｔｃＮ、−ＯＣＨ_２ＣＨＣＨ_２等が挙げられる。また、式（５）においてＷ^２が固相担体に結合される連結基である化合物（以下、化合物IIIともいう）は、当該連結基とアミノ基など固相担体上の所定の官能基とを結合させることにより、固相担体に保持される。そして、式（５）において、Ｗ^２が固相担体に結合された連結基である化合物（以下、化合物ｌＶともいう）は、連結基を介して式（１）のユニットが固相担体に結合されているため、各種の核酸固相合成法の出発材料として用いることができる。この出発材料を用いることで、ユニット３を有するオリゴヌクレオチド誘導体を製造することができる。ここで固相担体とは、一般に高分子担体が用いられ、例えば、ＣＰＧ（controlled pored glass）ややＨＣＰ（highly cross-1inked polystyren）や、ある種のゲルなどが挙げられる。また同相担体には適切なスペーサーを有していてもよい。連結基は固相担体と本化合物とを連結するリンカーである。こうした連結基としては、以下に示すコハク酸エステルリンカー、シュウ酸エステルリンカー、シランジイルリンカー、シリルリンカーなどを用いることができる。

(In the formula (6), each Y ¹ is independently the same or different, and may represent a branched alkyl group having 1 to 5 carbon atoms, and Y ² represents Represents a split or straight-chain alkyl group having 1 to 5 carbon atoms or an optionally substituted alkoxyl group.) In the above formula (6), Y ^l is not particularly limited but is preferably an isopropyl group Y ² includes mono-OCH ₃ , —OEtcN, —OCH ₂ CHCH _{2 and the} like. In addition, the compound in which W ² is a linking group bonded to the solid phase carrier in the formula (5) (hereinafter, also referred to as compound III) has the linking group and a predetermined functional group on the solid phase carrier such as an amino group By binding, it is held on a solid phase carrier. In the formula (5), a compound in which W ² is a linking group bonded to a solid phase carrier (hereinafter, also referred to as compound 1V) has a unit of formula (1) bound to the solid phase carrier via the linking group. Therefore, it can be used as a starting material for various nucleic acid solid phase synthesis methods. By using this starting material, an oligonucleotide derivative having unit 3 can be produced. Here, as the solid phase carrier, a polymer carrier is generally used, and examples thereof include CPG (controlled pored glass), HCP (highly cross-1 inked polystyren), and a certain kind of gel. The in-phase carrier may have an appropriate spacer. The linking group is a linker that links the solid phase carrier and the present compound. As such a linking group, the following succinate ester linker, oxalate ester linker, silanediyl linker, silyl linker, and the like can be used.

(Method for producing oligonucleotide derivative)
The method for producing an oligonucleotide derivative of the present invention is characterized by using the above-mentioned compounds I to IV of the present invention. Since the oligonucleotide can be obtained by a conventionally known nucleic acid synthesis method, the compound of the present invention is suitably used at the site where the unit of formula (1) or unit 1 to unit 3 is to be introduced during the oligonucleotide synthesis step of such a nucleic acid synthesis method. By utilizing I to IV, the oligonucleotide derivative of the present invention can be produced.

  For example, to obtain an oligonucleotide derivative having a unit of the formula (1) at the 5 ′ end, a compound II which is a phosphoramidite reagent derived from the above compound I at the 5 ′ end of an oligonucleotide obtained by a conventional nucleic acid synthesis method. And other introduction reagents can be used to introduce the unit of the formula (1). Furthermore, a plurality of units of the formula (1) can be continuously introduced by continuously connecting the compound II and the like to the unit of the formula (1) introduced as necessary. In this way, an oligonucleotide derivative having 1 or 2 or more units of the formula (1) on the 5 'terminal side can be obtained.

In addition, in order to obtain an oligonucleotide derivative having the unit of the formula (1) on the 3 ′ end side, the compound IV derived from the compound III is used as a starting material, and the oligonucleotide is obtained by various nucleic acid synthesis methods including the amidite method. The product is synthesized and cut out from the solid phase carrier in a form containing the unit of the formula (1). In order to obtain an oligonucleotide derivative having a plurality of units of the formula (1) continuously on the 3 ′ terminal side, compound IV was used as a starting material, and compound II was introduced into this by the amidite method or derived from compound I By introducing another introduction reagent, an oligonucleotide derivative having a plurality of consecutive units of the formula (1) at the 3 ′ end can be obtained. Furthermore, in order to obtain an oligonucleotide derivative having a unit of the formula (1) at the non-3 ′ terminal non-5 ′ terminal, other introduction reagents derived from compound II or compound I during the conventional oligonucleotide synthesis may be used. .
Furthermore, the oligonucleotide derivative which has 2 or more types among the unit of Formula (1), and unit 1-unit 3 can be obtained by combining this compound I-IV.

(Modification method of oligonucleotide)
As a method for modifying an oligonucleotide, at least one unit of the formula (1) can be added to a known or unknown sequence of oligonucleotide, introduced, substituted or inserted, or a combination thereof. By such modification, an RNA construct having a high silencing effect in addition to nuclease resistance can be obtained. The introduction of the unit of formula (1) may be carried out according to the method for producing an oligonucleotide derivative.

(Use of oligonucleotide derivatives)
The oligonucleotide derivative of the present invention can be used as a gene expression inhibitor by being constructed so as to function as siRNA or antisense. Further, the oligonucleotide derivative of the present invention can be used as an active ingredient of a pharmaceutical composition for prevention / treatment of diseases in humans and non-human animals. For example, for a disease associated with gene expression, the oligonucleotide derivative of the present invention constructed as a gene expression inhibitor is effective for the prevention and treatment of such diseases.

  Furthermore, the oligonucleotide derivative of the present invention can be used as a test reagent or a diagnostic reagent such as a probe or a primer by constructing it so as to exhibit its hybridization function. Furthermore, what hold | maintained these oligonucleotide constructs to solid supports, such as a chip | tip and a bead, can be utilized as a test | inspection apparatus, a diagnostic apparatus, or these one part. Furthermore, such test reagents and diagnostic agents can be used as test or diagnostic kits in combination with other reagents, diagnostic agents or devices.

The oligonucleotide derivative of the present invention can also be used for a gene expression suppression method using the gene expression suppression action of an oligonucleotide construct containing the oligonucleotide derivative of the present invention. Furthermore, it can also be used in a gene detection method using the hybridization function of the oligonucleotide construct of the present invention.
In the present specification, the following abbreviations and abbreviations are used.
Ar: Argon
BzCl ＝ benzoyl chloride
CPG: Controlled pore glass
DEAD: Diethyl azodicarboxylate
DIPEA: diisopropylethylamine
DMAP: 4-dimethylaminopyrideine (4-dimethylaminopyrideine)
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
DMTrCl: dimethoxytritylchloride
dsRNA: double strand RNA
EDTA: Ethylenediaminetetraacetic acid
HPLC: high performance liquid chromatography
HRHS: high-resolution mass spectrometry
MS: Mass Spectroscopy
NMR: nuclear magnetic resonance
PHO: phosphoramidite morpholino oligonucleotide
oligonuoleotide)
RISC: RNA-induced Silencing Complex
WSC: 1-ethyl-3- (3-dimethylaminobrovir) -carpositimide hydrochloride (1-methy-3- (3-dimethylaminopropyl) -carbodiimide, hydrochloride)

  Hereinafter, the present invention will be specifically described by comparing examples with comparative examples, but these examples do not limit the present invention.

(Example)
<Synthesis of fluoromethylbenzene unit>

In the Examples, Compound 13 that can be used as an amidite body and Compound 14 bound to a CPG carrier were synthesized according to the above synthesis route. That is, trimethyl 1,3,5-tricarboxylate (Compound 7) was used as a starting material, and a reduction reaction was performed with LiAlH ₄ to obtain Compound 8 as a triol form. Next, two of the three hydroxyl groups were protected with a benzoyl group to obtain compound 9, and then the remaining one hydroxyl group was converted to F using DAST to obtain compound 10. Thereafter, the benzoyl group was deprotected to obtain a diol compound 11 and then one hydroxyl group was converted to DMTr to obtain a trityl compound 12. Furthermore, the amidite of compound 13 was obtained by phosphorylating the remaining hydroxyl group of compound 12. Further, Compound 14 which is a CPG carrier was obtained from Compound 12 through succinylation. Details of each step are as follows.

1,3,5-
Synthesis of Trihydroxymethylbenzene (8)
Trimethyl-1,3,5-Benzene-Tricarboxylate (4.00g, 15.9 mmol) in dryTHF25
Dissolved in mL, LiAlH ₄ (1.80 g, 3 eq) was suspended in 75 mL of dryTHF and ice-cooled. Under ice cooling, the starting material / THF was added dropwise to LiAlH ₄ / THF and stirred. After 8 hours, the reaction was quenched with ethanol in an ice bath. The reaction solution was filtered through Celite to remove the precipitate, and the filtrate was concentrated. The target substance was isolated from the concentrate by silica gel column chromatography (CHCl ₃ : MeOH = 7: 1 → 5: 1) to obtain white crystalline compound 8 (2.34 g, 13.9 mmol, 88%).
¹ H NMR (400 MHz, DMSO-d ₆ ) δ [ppm]: 7.11 (3H, s, Ph-2, 4, 6), 5.13 (3H, s, OH-1 ′, 3 ′, 5 ′), 4.46 (6H, s, -CH ₂ -1 ', 3', 5 ')

Synthesis of 1-hydroxymethyl- 3,5-dibenzoyloxymethylbenzene (9) Compound 8 (1.52 g, 9.01 mmol), which was vacuum-dried overnight, was dissolved in dry CH ₂ Cl ₂ (45 mL, 0.2 M solution) and dry pyridine (15 mL) I let you. Next, BzCl (2.20 mL, 2.1 eq.) Was added and stirred for about 8 hours under Ar atmosphere. Progress of the reaction was confirmed by TLC (CHCl ₃ : MeOH = 30: 1). After evaporating the solvent under reduced pressure, it was isolated and purified by silica gel chromatography (CHCl ₃ : MeOH = 70: 1 → 30: 1 → 10: 1) to obtain the target compound 9 (2.06 g, 5.46 mmol, 61%). It was.
¹ H NMR (400 MHz, CDCl ₃ ) δ [ppm]: 8.08 (4H, d, -COPh-2,6), 7.57 (2H, t, -COPh-4), 7.46-7.42 (7H, m, Ph- 2,4,6 and -COPh-3,5), 5.39 (4H, s, -CH ₂ -3 ', 5'), 4.75 (2H, s, -CH ₂ -1 ')

1-
Synthesis of fluoromethyl-3,5-dibenzoyloxymethylbenzene (10) Compound 9 (2.06 g, 5.46 mmol), which had been vacuum-dried overnight, was dissolved in dry CH ₂ Cl ₂ (54.6 mL, 0.1 M solution). Subsequently, DAST (1.43 mL, 2 eq) was gradually added in an ice bath and stirred for 2 hours. Progress of the reaction was confirmed by TLC (Hex: EtOAc = 2: 1). After quenching with MeOH, the solution was concentrated once, extracted with EtOAc and sat. NaHCO ₃ aq., The organic layer was washed with sat. NaCl aq., Dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (Hex: EtOAc = 5: 1) to obtain the target compound 10 (1.24 g, 3.28 mmol, 62%).
¹ H NMR (400 MHz, DMSO-d ₆ ) δ [ppm]: 8.07 (4H, d, -COPh-2,6), 7.58 (2H, t, -COPh-4), 7.52 (3H, s, Ph- 2,4,6), 7.48 (4H, t, COPh-3,5), 5.43 (2H, br, -CH ₂ -1 '), 4.75 (4H, s, -CH ₂ -3', 5 ')

Synthesis of 1-fluoromethyl-3,5-dihydroxymethylbenzene (11) Compound 10 (1.24 g, 3.28 mmol), which was vacuum-dried overnight, was dissolved in dry CHCl ₃ (10 mL) and dryMeOH (50 mL). Subsequently, CH ₃ ONa in MeOH (2.5 mL, 0.052 eq) was gradually added in an ice bath, and the mixture was stirred for 24 hours under an Ar atmosphere. Progress of the reaction was confirmed by TLC (Hex: EtOAc = 4: 1). Thereafter, 80% acetic acid was added to confirm pH = 7. After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (EtOAc only) to obtain the target compound 11 (0.40 g, 2.36 mmol, 72%).
¹ H NMR (400 MHz, DMSO-d ₆ ) δ [ppm]: 7.21 (3H, s, Ph-2, 4, 6), 5.38 (2H, d, -CH ₂ -1 '), 4.21 (2H, t , OH-3 ', 5'), 4.49 (4H, d, -CH ₂ -3 ', 5')

1-
Synthesis of fluoromethyl-3- (4,4'-dimethoxytrityloxy) methyl-5-hydroxymethylbenzene (12) Compound 11 (400mg, 2.4mmol), which was vacuum-dried overnight, was dissolved in dry pyridine (24mL, 0.1M solution) 4,4′-dimethoxytrityl chloride (880 mg, 1.1 eq) was added in an ice bath, and the mixture was stirred for 3 and a half hours under an Ar atmosphere. Progress of the reaction was confirmed by TLC (Hex: EtOAc = 1: 1). Thereafter, extraction was performed with EtOAc, water, and sat. NaHCO ₃ aq., And the organic layer was washed with sat. NaCl aq. And then dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by silica gel chromatography (Hex: EtOAc = 1: 1) to obtain Compound 12 (0.31 g, 0.66 mmol, 28%).
¹ H NMR (400 MHz, CDCl ₃ ) δ [ppm]: 7.51 to 6.82 (16H, m, Tr and Ph-2, 4, 6), 5.39 (2H, d, -CH ₂ -1 '), 4.72 (2H , d, -CH ₂ -5 '), 4.20 (2H, s, -CH ₂ -3'), 3.80 (6H, s, -Tr-OCH ₃ )
¹⁹ F NMR (372 MHz, CDCl ₃ ) δ [ppm]: -143.37

Synthesis of 1-fluoromethyl-3- (4,4'-dimethoxytrityloxy) methyl-5-{(2-cyanoethoxy)-(N, N-diisopropylamino)}-phosphinyloxymethylbenzene (13) Reaction under completely anhydrous conditions in a gloveback The operation was performed. Compound 12 (150 mg, 0.31 mmol), which was vacuum-dried overnight, was dissolved in dry THF (3.2 mL, 0.1 M solution), and Hunig's Base (0.16 mL, 3 eq) and phosphorylating reagent (0.11 mL, 1.5 eq) were added. added. Then, it was taken out from the glove bag and stirred for 0.5 hour under Ar atmosphere. The disappearance of the starting material was confirmed by TLC (Hex: EtOAc = 1: 1). Thereafter, extraction was performed with CHCl ₃ and satNaHCO ₃ aq, the organic layer was washed with sat NaClaq, and dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated and purified by silica gel chromatography (Hex: EtOAc = 1: 1) to obtain the target compound 13 (0.19 g, 0.27 mmol, 87%).
³¹ P NMR (160MHz, CDCl ₃ )
δ [ppm]: 149.04

Synthesis of 1-fluoromethyl-3- (4,4'-dimethoxytrityloxy) methyl-5-succinyloxymethylbenzene (12 → 14 intermediate) Compound 12 (160 mg, 0.33 mmol), which was vacuum-dried overnight, was dry DMF (3.3 mL, 0.1 MMAP), DMAP (4 mg, 0.1 eq) and succinic anhydride (100 mg, 3 eq) were added thereto, and the mixture was stirred at room temperature for 32 hours in an Ar atmosphere. The disappearance of the starting material was confirmed by TLC (Hex: EtOAc = 1: 1). Then, extraction was performed with EtOAc and sat.NaHCO ₃ aq., And the organic layer was washed with sat. NaCl aq. And dried over anhydrous Na ₂ SO ₄ . The solvent was distilled off under reduced pressure to obtain the target intermediate compound by a mixture.

Synthesis of 1-fluoromethyl-3- (4,4'-dimethoxytrityloxy) methyl-5- (CPG-longchain-alkylamino-succinyloxymethyl) benzene (14) Overnight vacuum-dried intermediate (mixture; succinylation progressed 100% Assuming that 0.33 mmol, CPG: 4 eq) was dissolved in dryDMF (8.3 mL, CPG: 0.01 Msolution) under Ar atmosphere, and CPG resin (77 μmol / g, 1.07 g, 0.083 mmol) was added to blend with the solution. . After that, WSC (250 mg, CPG: 4 eq) was added and shaken overnight at room temperature. The reaction solution was washed with pyridine, and the resin was recovered. Then, 30 mL of 0.1M DMAP in (pyridine: acetic anhydride = 9: 1) was added thereto, and the mixture was shaken overnight at room temperature. On the next day, the reaction solution was further washed with pyridine, EtOH and acetonitrile, and the recovered resin was vacuum-dried overnight. The activity of the obtained CPG resin 14 was measured. The activity value was 45.2 μmol / g.

<Synthesis of siRNA of Examples>
Using the amidite compound 13 and chemically modified CPG resin 14 obtained as described above, oligos having the sequences shown in Table 1 below (this sequence targets Renilla Luciferase) Nucleotides were synthesized by automated nucleic acid synthesis. The obtained oligonucleotide was used as an aqueous solution, and the absorbance (Abs ₂₆₀ ) at a wavelength of 260 was measured. In consideration of the optical path length (l), the concentration C (mol / L) was calculated using the following equation.

C = Abs ₂₆₀・ ε ₂₆₀ ⁻¹・ l
^-1
The antisense (as) strand and the sense (s) strand were combined in equimolar amounts and annealed to obtain the double-stranded siRNA of the example. Double strand Tm values (50% dissociation temperature) were measured. The results are shown in Table 1.

(Comparative example)
<Synthesis of methylbenzene unit>
In a comparative example, it replaced with the fluoromethylbenzene unit shown in the said Formula (1) used in the Example, and the same synthesis as the Example was performed using the methylbenzene unit. That is, as shown in the following synthesis route, 5-methylisophthalic acid (compound 1) was used as a starting material, and ion exchange resin DOWEX (registered trademark) (H ⁺ type) was used for esterification to obtain compound 2. . Next, this ester was reduced with LiBH ₄ to obtain Compound 3 as a diol. Further, one hydroxyl group of compound 3 was converted to DMTr to obtain compound 4 which is a trityl compound, and then phosphorylated to obtain compound 5 which is an amidite compound. Further, Compound 6 which is a CPG carrier was obtained from Compound 4 through succinylation. Details of each step are shown below.

3,5-
Synthesis of dimethylcarboxytoluene (2) 50 mL of DOWEX (registered trademark) activated under H ⁺ conditions by dissolving 5-methylisophthalic acid (560 mg, 3.35 mmol) in CH ₃ OH (100 mL). And stirred for 14 hours. The progress of the reaction was confirmed by TLC (EtOAc: MeOH = 3: 1) but no change was observed, so WSC (1.92 g, 3 eq) was added. After 90 minutes, the disappearance of the raw materials was confirmed. After removing DOWEX by cotton plug filtration, the product was isolated by silica gel chromatography (Hex: EtOAc = 1: 1), and the target compound 2 (520 mg,
2.68 mmol, 80%) was obtained.
¹ H NMR (400 MHz, CDCl ₃ ) δ [ppm]: 8.49 (1H, s, Ph-4), 8.05 (2H, s, Ph-2, 4, 6), 3.95 (6H, s, —OCH ₃ ) , 2.46 (3H, s, CH ₃ -Ph)

Synthesis of 1-methyl- 3,5-dihydroxymethylbenzene (3) Compound 2 (1.35 g, 6.95 mmol), which was vacuum-dried overnight, was dissolved in dry THF (34.8 mL, 0.2 M solution), and LiBH ₄ (0.76 g , 5eq) and stirred at 60 ° C for 16 hours. The progress of the reaction was confirmed by TLC (Hex: EtOAc = 1: 1), sat NaHCO ₃ aq was added to the reaction solution in an ice bath to deactivate LiBH ₄ , and then the solvent was distilled off under reduced pressure. Then, the cerite filtration was performed and the inorganic substance was removed. Finally, it was isolated by silica gel chromatography (Hex: EtOAc = 2: 1) to obtain the target compound 3 (0.75 g, 4.92 mmol, 71%).
¹ H NMR (400 MHz, DMSO-d) δ [ppm]: 7.11 (3H, s, Ph-2,4,6), 5.13 (3H, s, —CH ₂ -3 ′, 5 ′), 3.80 (6H , s, -OCH ₃ ), 2.11 (3H, s, CH ₃ -Ph)

Synthesis of 1-methyl-3- (dimethoxytrityloxy) methyl-5-hydroxymethylbenzene (4) Compound 3 (0.75 g, 4.9 mmol), which was vacuum-dried overnight, was dissolved in dry pyridine (35 mL, 0.15 M solution) and ice bath Below, 4,4'-dimethoxytrityl chloride (1.83g, 1.1eq) was added and stirred for 24 hours under Ar atmosphere. Progress of the reaction was confirmed by TLC (Hex: EtOAc = 1: 1). Thereafter, extraction was performed with EtOAc, water, and sat. NaHCO ₃ aq., And the organic layer was washed with sat. NaCl aq. And then dried over anhydrous Na ₂ SO ₄ . After evaporating the solvent under reduced pressure, the residue was isolated by neutral silica gel chromatography (Hex: EtOAc = 2: 1) to obtain Compound 4 (0.97 g, 2.14 mmol, 44%).
¹ H NMR (400 MHz, CDCl ₃ ) δ [ppm]: 7.52 to 6.82 (16H, m, Tr and Ph-2,4,6), 4.66 and 4.13 (4H, s, —CH ₂ -3 ′, 5 ′ ), 3.80 (6H, s, -OCH ₃ ), 2.11 (3H, s, CH ₃ -Ph)

Synthesis of 1-methyl-3- (4,4'-dimethoxytrityloxy) methyl-5-{(2-cyanoethoxy)-(N, N-diisopropylamino)}-phosphinyloxymethylbenzene (5) Operation in a gloveback under completely anhydrous conditions Went. Compound 4 (150 mg, 0.31 mmol), which was vacuum-dried overnight, was dissolved in dry THF (3.2 mL, 0.1 M solution), and Hunig's Base (0.16 mL, 3 eq) and phosphite reagent (0.11 mL, 1.5 eq) were added. added. Then, it was taken out from the glove bag and stirred for 0.5 hour under Ar atmosphere. The disappearance of the starting material was confirmed by TLC (Hex: EtOAc = 1: 1). Thereafter, the mixture was extracted with CHCl ₃ and sat.NaHCO ₃ aq., The organic layer was washed with sat. NaCl aq., And anhydrous Na ₂ SO ₄ was added and dried. After evaporating the solvent under reduced pressure, the residue was isolated and purified by neutral silica gel chromatography (Hex: EtOAc = 1: 1) to obtain the target compound 5 (0.19 g, 0.27 mmol, 87%).
³¹ P NMR (160MHz, CDCl ₃ )
δ [ppm]: 149.04

Synthesis of 1-methyl-3- (dimethoxytrityloxy) methyl-5-succinyl-hydroxymethylbenzene (4´) Compound 4 (0.97g, 2.14mmol), which was vacuum-dried overnight, was dissolved in dry DMF (21.4mL, 0.1Msolution) DMAP (26 mg, 0.1 eq) and succinic anhydride (0.64 g, 3 eq) were added thereto, and the mixture was stirred at room temperature for 43 hours in an Ar atmosphere. The disappearance of the starting material was confirmed by TLC (Hex: EtOAc = 1: 1). Then, extraction was performed with EtOAc and sat.NaHCO ₃ aq., And the organic layer was washed with sat. NaCl aq. And dried over anhydrous Na ₂ SO ₄ . The solvent was distilled off under reduced pressure to obtain the target intermediate compound 4 ′ (1.47 g, 2.71 mmol) by mixture.

Synthesis of 1-methyl-3- (dimethoxytrityloxy) methyl-5- (CPG-longchain-alkylamino-succinyl-hydroxymethyl) benzene (6) An intermediate (0.62 mmol, CPG: 4 eq) dried under vacuum overnight under Ar atmosphere It was dissolved in dryDMF (14.5 mL, CPG: 0.02 Msolution), and CPG resin (77 μmol / g, 3.73 g, 0.29 mmol) was added to blend with the solution. After that, WSC (220 mg, CPG: 4 eq) was added and shaken overnight at room temperature. This reaction solution was washed with pyridine and 0.1 M DMAP solution (pyridine: acetic anhydride = 9: 1)
30 mL was added and shaken overnight at room temperature. The next day, the reaction solution was further washed with pyridine, EtOH and acetonitrile and dried in vacuum overnight. The activity of the obtained CPG resin 6 was measured. The activity value was 33.2 μmol / g.

<Synthesis of siRNA of Comparative Example>
Using the compound 5 which is an amidite and the chemically modified CPG resin 6 obtained as described above, an oligonucleotide having the same sequence as that of Example was synthesized by automatic nucleic acid synthesis. The obtained oligonucleotide was used as an aqueous solution, and the absorbance (Abs ₂₆₀ ) at a wavelength of 260 was measured. The measurement method was the same as the measurement of siRNA in Examples. Furthermore, the antisense (as) strand and the sense (s) strand thus obtained were combined in equimolar amounts, respectively, and annealed to obtain a double-stranded siRNA of a comparative example. Double strand Tm values (50% dissociation temperature) were measured. The results are shown in Table 2.

-Evaluation-
Using the siRNA duplexes of Examples and Comparative Examples obtained as described above and the natural siRNA duplexes, a dual luciferase repoter assay using HeLa cells was performed to evaluate the knockdown effect. The synthesized siRNA targets Renilla Luciferase, and the knockdown effect was measured by simultaneously transfecting a HeRNA cell with a vector expressing this gene and a control gene (firefly Luciferase) and siRNA. The dangling end of the natural siRNA duplex consists of nucleotides with two thymine bases.

  As a result, as shown in FIG. 2, it was found that the siRNA of Examples into which fluoromethylbenzene was introduced had a knockdown effect almost equivalent to that of the natural type. On the other hand, the siRNA of the comparative example into which methylbenzene was introduced had a knockdown effect, but was found to be inferior to the natural knockdown effect.

Moreover, although siRNA of an Example is obtained by introduce | transducing fluoromethylbenzene into the terminal of a nucleic acid oligomer, this operation can be obtained only by letting it pass through the column of the compound 6 which is a chemically modified CPG carrier. For this reason, if compound 6 using ¹⁸ F as a labeling element is synthesized, siRNA labeled with ¹⁸ F can be obtained in a very short time. That is, a nucleic acid oligomer applicable to the PET method can be made into a PET probe, and by using this, it becomes possible to synthesize a nucleic acid oligomer using ¹⁸ F having various sequences as a labeling element in a short time. I understood.

  The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.

FIG. 4 shows an example of siRNA having unit 3 at the 3 ′ end. It is a graph which shows a knockdown effect about siRNA double strand of a natural type, an Example, and a comparative example. It is a figure which shows the example of the chemical modification of siRNA.

  INDUSTRIAL APPLICABILITY The present invention can provide a useful means in the medical field using nucleic acid oligomers such as RNA drug discovery that is expected to be developed into personalized medicine.

Claims (18)

An oligonucleotide derivative represented by the following formula (3).

Wherein R ¹ and R ² each independently represent hydrogen or a hydroxyl protecting group, a, b and c are each independently an integer of 0 or more, at least one is 1 or more, and A and B are An oligonucleotide which may be independently modified and has a chain length of A and B combined is 3 or more. However, A and B do not include the hydroxyl groups at the 3 ′ end and 5 ′ end of the oligonucleotide. The oligonucleotide derivative according to claim ^1, wherein R ¹ and R ² are H.   The oligonucleotide derivative according to claim 1 or 2, wherein b is 0.   The oligonucleotide derivative according to any one of claims 1 to 3, wherein a and b are both 0.   The oligonucleotide derivative according to claim 4, wherein c is 1 or more and 5 or less.   The oligonucleotide derivative according to any one of claims 1 to 5, wherein A and B have a partial sequence of a predetermined gene mRNA or a complementary sequence thereof.   The oligonucleotide derivative according to any one of claims 1 to 6, wherein the combined chain length of A and B is 10 or more and 35 or less.   The oligonucleotide derivative according to any one of claims 1 to 7, wherein A and B are oligoribonucleotides. An oligonucleotide construct for regulating gene expression comprising:
A construct comprising the oligonucleotide derivative according to any one of claims 1 to 8.   The construct according to claim 9, which is an oligonucleotide construct for regulating gene expression selected from single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, DNA / RNA chimera and DNA / RNA hybrid.   11. A construct according to claim 9 or 10, selected from antigenes, antisenses, aptamers, siRNAs, miRNAs, shRNAs and lipozymes. The structure according to any one of claims 9 to 11, which has a unit represented by the following formula (4) at a dangling end portion.

The siRNA, wherein in the oligonucleotide derivative, a and b are 0, c is 1 or 2, and a unit represented by the following formula (4) is included in the dangling end portion at the 3 ′ end: 12. The construct according to 12.

  An oligonucleotide construct for gene diagnosis, comprising the oligonucleotide derivative according to any one of claims 1 to 8.   15. The construct according to claim 14, which is a probe or a primer. Compounds represented by the following formula (5) (including those in which a substituent other than hydrogen is bonded instead of hydrogen bonded directly to the benzene ring in formula (5)).

In the formula, W ¹ represents a hydroxyl protecting group, and W ² is any one of H, phosphoramidite group, or succinate linker, oxalate linker, silanediyl linker, and silyl linker bound to or bonded to a solid support. representing the Kano linking group. The compound according to claim 16 , which is used for a dangling end unit of siRNA. A method for modifying an oligonucleotide, wherein one or more selected from the compound according to claim 16 is used.

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