JP2008094831A - New dideoxynucleoside derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new dideoxynucleoside derivative suitable for the raw material for synthesis of antisense nucleic acid, and to provide a thermally stable oligonucleotide analog into which the above derivative is introduced. <P>SOLUTION: The invention relates to a 5'-amino-2'-fluoro-2', 5'-dideoxynucleoside derivative represented by formula [1] (wherein R<SB>1</SB>is a nucleic acid base which may have a protecting group; R<SB>2</SB>is H or a protecting group of an amino group; and R<SB>3</SB>is H or a protecting group of a hydroxy group). There are also provided a dideoxynucleoside-insoluble carrier bound substance obtained by binding the above derivative to an insoluble carrier, and an oligonucleotide analog into which the derivative is introduced. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel dideoxynucleoside derivative, an amidite reagent using the same, and an oligonucleotide analog into which the dideoxynucleoside derivative is introduced.

  In the following description of the present specification, when A represents nucleobase, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil. The term “oligonucleotide” may also include “polynucleotide”.

  When double-stranded RNA is introduced into a cell, mRNA with a complementary base sequence (messenger RNA) is degraded and mRNA is inactivated (abbreviated as “RNA interference”, RNA interference, or “RNAi”). Is known to occur. This phenomenon is considered to occur by the following mechanism.

  That is, when a relatively long double-stranded RNA (dsRNA) is first introduced into a cell, it is degraded to a size of 21 to 23 bases by an RNaseIII-like nuclease called Dicer to produce siRNA. The small siRNA then binds to a plurality of proteins to form a complex called RISC (RNA induced silencing complex). This complex recognizes and binds to mRNA having the same sequence as siRNA, and mRNA is cleaved at the center of siRNA. As a result, it is believed that the gene is inactivated.

  The RNAi method (RNA interference method) is a method that suppresses the expression of specific genes by introducing artificially synthesized RNA into cells using this phenomenon, and is a simple and powerful method for inhibiting gene function. (Fire, A. et. Al., Nature, 1998, vol.391, pp.806-811, Svobada, P. et al., Development, 2000, vol.127, pp.4147- 4156, Elbashir, SM, Lendeckel, W. and Tuschl, T., Genes and Dev., 2001, vol.15, pp.188-200, Zamore, PD et al., Cell, 2000, vo.101, pp. 25-33, Bernstein, E. et al., Nature, 2001, vol.409, pp.363-366).

  In addition, compared to the antisense method described later, the RNAi method uses a mechanism inherent in living organisms, so it can efficiently suppress expression at low concentrations, has low toxicity, and has high specificity by nucleotide sequence. There are advantages such as easy. It has also been widely used for knockdown of endogenous genes in cells. Furthermore, it is expected to be used for gene therapy by specifically degrading mRNA of abnormal genes.

  However, on the other hand, conventionally used low molecular weight RNAs such as dsRNA and siRNA are easily decomposed by the action of nucleases such as nucleases, so that they are difficult to handle.

  On the other hand, a technique called an antisense method is also known as another method for controlling gene expression.

  The base sequence of mRNA that synthesizes protein (instructs synthesis) is called a sense sequence, and the base sequence complementary to this sequence is called antisense. A nucleic acid having an antisense base sequence is called an antisense nucleic acid.

  Antisense is a method in which an antisense nucleic acid complementary to mRNA transcribed from a target gene is administered to a cell, a double strand is formed between the mRNA and the administered antisense nucleic acid, and only the target gene is expressed. Is one of the gene expression control methods which intend to suppress the nucleotide sequence specifically. By examining the inhibitory effect of the target gene by the antisense nucleic acid, it is possible to know the function of the target gene.

  Antisense methods can also be applied in the field of pharmaceuticals. For example, by binding an antisense nucleic acid to mRNA involved in the synthesis process of a protein that is a factor causing a disease, it can be expected to prevent the gene from functioning at the gene level. Accordingly, it is expected that treatment using an antisense nucleic acid to suppress the function of a gene causing a disease can be performed. For example, a drug such as formivirsen currently targeted to cytomegalovirus retinitis, for which ISIS Pharmaceuticals Inc. has been approved by the US FDA, is one example.

  Properties required for an antisense nucleic acid include formation of a stable double strand with a target RNA, ability to recognize a base sequence that does not bind to a sequence containing mismatched bases, resistance to nucleases, and permeability to a cell membrane. In addition, in order to use an antisense nucleic acid as a pharmaceutical product, specific base sequence recognition ability, nuclease resistance, metabolism, and intracellular migration are important.

  However, when a natural oligonucleotide is used as an antisense nucleic acid, there are problems that the above-mentioned required properties such as nuclease resistance are not satisfied.

  Thus, many nucleic acid modifications have been attempted to date to obtain oligonucleotides that overcome the disadvantages of natural oligonucleotides and that also satisfy other properties. For example, modification of the nucleobase site (N. Haginoya et al., Bioconjugate Chem., 1997, vol.8, pp.271-280.), Modification of ribose (M. Aoyagi et al., Bioorg. Med. Chem. Lett., 1996, vol.6, pp.1573-1576., Modification of the ribose ring itself (A. Kakefuda et al., Tetrahedron, 1996, vol.52, pp.2863-2876.), To phosphodiester Modification (M. Shimizu et al., 2006, vol. 71, pp. 4262-4269.), Modification of phosphodiester bond (A. Waldner et al., Bioorg. Med. Chem. Lett., 1994, vol. .4, pp.405-408). By combining these, diversification of antisense nucleic acids satisfying the above properties can be expected. For example, SM Gryaznov et al. Have synthesized 5′-phosphoramidate type DNA (Non-patent Document 1), which is resistant to nucleases such as nucleases. However, there are problems such as insufficient binding affinity for the target mRNA. S. Obika et al. Introduced an amino group at the 5'-position of the sugar moiety and cyclized the 2 ', 4'-position via an oxygen atom to solve the problem of binding affinity for the target mRNA. 5'-amino-2 ', 4'-bridged nucleic acid (BNA) containing nucleotides has been synthesized (Non-patent Document 2). Similarly, S. Obika et al. Synthesized 5′-amino-3 ′, 5′-BNA in which the 5′-amino group and the 3′-position are bridged with a methylene group (Non-patent Document 3). These BNAs have a high binding affinity for the target mRNA and are resistant to nucleolytic enzymes such as nucleases. However, protein inhibitory activity when these BNAs are introduced into siRNA has not been reported.

WO03 / 093472 Publication S. M. Gryaznov et al., Nucleic Acids Res., 1992, vol.20, pp.3403-3409 S. Obika, et.al., Chem. Commun., 2003, pp.2202-2203 S. Obika, et al., Angew. Chem. Int. Ed., 2005, vol.44, pp.1945-1947 A. M. Kawasaki et al., J. Med. Chem., 1993, vol.36, pp.831-841 S. Helmling et al., Nucleosides, Nucleotides & Nucleic Acids, 2003, vol.22, Nos.5-8, pp.1035-1038 J. Shi et al., Bioorg. Med. Chem., 2005, vol.13, pp.1641-1652 S. M. Gryaznov et al., Nucleic Acids Res., 1992, vol.20, pp.3403-3409

  The present invention has been made in view of the circumstances as described above, and provides a novel dideoxynucleoside derivative suitable as a synthetic raw material for antisense nucleic acid, and a thermally stable oligonucleotide analog incorporating the dideoxynucleoside derivative. The task is to do.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基、Ｒ_３は水素原子又は水酸基の保護基を表す。）
で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシド誘導体。 The present invention has been made for the purpose of solving the above-described problems, and has the following configuration.
(1) The following formula [1]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a protecting group for a hydrogen atom or an amino group, and R ₃ represents a protecting group for a hydrogen atom or a hydroxyl group.)
A 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by:

（式中、Ｒ₁は保護基を有していていもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。）
で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシド誘導体を不溶性担体に結合させてなる、ジデオキシヌクレオシド−不溶性担体結合物。 (2) The following formula [6]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group.)
A dideoxynucleoside-insoluble carrier conjugate obtained by binding a 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula:

（式中、Ｒ₁は保護基を有していていもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基、Ｒ_３は水素原子又は水酸基の保護基を表す。）
で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシド誘導体を導入したオリゴヌクレオチドアナログ。 (3) The following formula [1]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group, and R ₃ represents a hydrogen atom or a hydroxyl protecting group.)
An oligonucleotide analog into which a 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula:

  That is, the present inventors have conducted intensive research aimed at synthesizing new oligonucleotides that are stable and can be used in antisense methods and RNAi methods.

  The nucleic acid double-stranded helical structure mainly has A type and B type, and in the single-stranded state, there are fluctuations in the conformation, but by hybridizing with the complementary strand, the A type and B type A conformation such as a mold is fixed. In order to adopt an A-type helical structure, a nucleic acid in which the conformation of the sugar moiety is fixed to the N-type (3'-endo) is required. Conversely, in order to adopt a B-type helical structure, a nucleic acid immobilized on S-type (2'-endo) is required. DNA-DNA duplexes tend to be B-type, and RNA-RNA duplexes tend to be A-type. In the case of RNA-DNA double strand, it takes A type.

  Since the target is RNA in the case of antisense nucleic acid, the present inventors fixed the conformation of the antisense nucleic acid in the A type, that is, the conformation of the sugar moiety of the nucleoside constituting the antisense nucleic acid. We thought that fixing to N-type (3'-endo) would improve the stability (binding affinity) of double strands of antisense nucleic acid and complementary RNA.

  Then, the present inventors introduced an electron-withdrawing substituent at the 2′-position of the sugar moiety of the nucleoside constituting DNA, and fixed the conformation of the sugar moiety to the N type. From the knowledge that it has the effect of stabilizing the double strand (Non-patent Document 4), if an electron-withdrawing fluorine atom is introduced at the 2′-position of the sugar moiety of the 5′-amino nucleoside, the sugar moiety of the nucleoside It was speculated that the conformation may be fixed to the N type, and that the stability of the double strand might be improved by fixing.

  As nucleoside derivatives having a fluorine atom introduced into the sugar moiety, 2'-fluoro-L-uridine, 2'-fluoro-L-cytidine phosphoramidite (Non-patent Document 5, Patent Document 1), D- and L- 2′-deoxy-2′-fluororibonucleoside (Non-patent Document 6) and the like are known.

  In addition, there is a report that the nuclease resistance of a double-stranded nucleic acid is improved by changing the phosphodiester bond of an oligonucleotide to a phosphate amidate bond (Non-patent Document 7). However, the oligonucleotide having a 5′-phosphate amidate bond has a problem that the stability (binding affinity) of a double strand with a complementary strand is lower than that of an oligonucleotide having only a phosphodiester bond.

  Therefore, the present inventors introduced both of the above-described modifications to the oligonucleotide so that the oligonucleotide has nuclease resistance and further satisfies the duplex stability (binding affinity) with the complementary strand. As a result of further intensive studies, 5′-amino-2′-fluoro-2 in which a —NH— group was introduced at the 5′-position and a fluorine atom was introduced into the 2′-position of the sugar moiety was obtained. A ', 5'-dideoxynucleoside derivative was synthesized, and an oligonucleotide analog into which this dideoxynucleoside derivative was introduced was synthesized by a conventional method. And the stability of the double-stranded nucleic acid of this oligonucleotide analog and complementary strand DNA and complementary strand RNA was measured. Then, the double-stranded nucleic acid of the oligonucleotide analog into which this 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative is introduced and the complementary strand nucleic acid is thermally stabilized, It has been found that the binding affinity of this chain is also satisfactory, and the present invention has been completed. Furthermore, since this oligonucleotide analog has a phosphate amidate bond, it is expected that the oligonucleotide analog and the double-stranded nucleic acid obtained using the oligonucleotide analog are also excellent in nuclease resistance.

  The oligonucleotide analog introduced with the 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative of the present invention is excellent in thermal stability and also has a binding affinity in the case of a double strand. high. It is also expected to have excellent nuclease resistance. Furthermore, the 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative of the present invention can be used as an amidite reagent used in nucleic acid synthesis, or by binding this amidite reagent to a solid phase. It can be used as a starting material used in the solid phase synthesis method of nucleic acids.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基、Ｒ_３は水素原子又は水酸基の保護基を表す。）
で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシド誘導体である（以下、「本発明のジデオキシヌクレオシド誘導体」と略記する。）。 The dideoxynucleoside derivative of the present invention has the following formula [1].

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a protecting group for a hydrogen atom or an amino group, and R ₃ represents a protecting group for a hydrogen atom or a hydroxyl group.)
5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the following (hereinafter abbreviated as “dideoxynucleoside derivative of the present invention”)

In the formula [1], the nucleobase of the nucleobase which may have a protecting group represented by R ₁ is a natural nucleobase selected from adenine, guanine, cytosine, thymine, uracil, 1- Examples include nucleobase minor components such as methyladenine, 1-methylguanosine, xanthine, 5-methylcytosine, and dihydrouracil, and artificial nucleobases such as 6-methoxypurine, 2-aminopurine, and pseudoisocytosine.

  Further, the nucleobase may be appropriately protected with a protecting group known per se that is usually used in nucleic acid synthesis, if necessary. Specific examples include amino-protecting groups such as benzoyl group and acetyl group, but are not particularly limited.

Examples of the amino-protecting group for R ₂ include amino-protecting groups for nucleoside synthesis that are usually used in this field, such as 4-Methoxytriphenylmethyl (MMTr) group, 4,4′-Dimethoxytriphenylmethyl. (DMTr) group, Triphenylmethyl (Tr) group, etc. are mentioned.

Examples of the hydroxyl-protecting group for R ₃ include those usually used in this field for the synthesis of nucleosides, such as 4,4′-Dimethoxytriphenylmethyl group, 2-cyanoethoxyldiisopropylaminophosphinyl group, tert-Butyldimethylsilyl Groups and the like.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。）で表されるものが挙げられる。Ｒ₁で表される保護基を有していてもよい核酸塩基の、保護基及び核酸塩基の具体例は前記したとおりである。 As the dideoxynucleoside derivative of the present invention, for example, the following formula [2]

(Wherein R ₁ represents a nucleobase optionally having a protecting group). Specific examples of the protecting group and the nucleobase of the nucleobase optionally having the protecting group represented by R ₁ are as described above.

  Specific examples of the compound represented by the formula [2] include 5′-amino-2′-fluoro-2 ′, 5′-dideoxyuridine, 5′-amino-2′-fluoro-2 ′, 5′-. Dideoxyadenosine, 5'-amino-2'-fluoro-2 ', 5'-dideoxyguanosine, 5'-amino-2'-fluoro-2', 5'-dideoxycytidine, 5'-amino-2'-fluoro -2 ', 5'-dideoxythymidine and the like.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。）
で表されるものが挙げられる。Ｒ₁で表される保護基を有していてもよい核酸塩基の保護基及び核酸塩基の具体例、及びＲ_２におけるアミノ基の保護基の具体例は前記したとおりである。 As another example of the dideoxynucleoside derivative of the present invention, for example, the following formula [3]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group.)
The thing represented by is mentioned. Specific examples of the nucleobase protecting group and nucleobase optionally having a protecting group represented by R ₁ , and specific examples of the amino group protecting group in R ₂ are as described above.

  Specific examples of the compound represented by the formula [3] include 5'-amino-2'-fluoro-2 ', 5'-dideoxy-5'-N-monomethoxytrityluridine, 5'-amino-2'-fluoro- 2 ', 5'-dideoxy-5'-N-monomethoxytrityladenosine, 5'-amino -2'-fluoro-2', 5'-dideoxy-5'-N-monomethoxytritylguanosine, 5'-amino -2'-fluoro- 2 ', 5'-dideoxy-5'-N-monomethoxytritylcytidine, 5'-amino-2'-fluoro-2', 5'-dideoxy-5'-N-monomethoxytritylthymidine, and the like.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。２個のＲ_４はそれぞれ独立して炭素数１〜５の低級アルキル基を表す。Ｒ_５は炭素数１〜３の低級アルキレン基を表す。
で表されるものが挙げられる。 As another example of the dideoxynucleoside derivative of the present invention, for example, the following formula [4]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a protecting group for a hydrogen atom or an amino group. Two R _{4 s} each independently have 1 to 5 carbon atoms. R ₅ represents a lower alkylene group having 1 to 3 carbon atoms.
The thing represented by is mentioned.

Specific examples of the nucleobase substituent and nucleobase which may have a substituent represented by R ₁ , and specific examples of the amino group protecting group in R ₂ are as described above.

Specific examples of the lower alkyl group having 1 to 5 carbon atoms represented by R ₄ include an ethyl group, a propyl group, and an isopropyl group.

Specific examples of the lower alkylene group having 1 to 3 carbon atoms represented by R ₅ include an ethylene group and a propylene group.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。i-Prはイソプロピル基を表す。）
で表されるものが挙げられる。Ｒ₁で表される置換基を有していてもよい核酸塩基の置換基及び核酸塩基の具体例、及びＲ_２におけるアミノ基の保護基の具体例は前記したとおりである。 As another example of the dideoxynucleoside derivative of the present invention, for example, the following formula [5]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group. I-Pr represents an isopropyl group.)
The thing represented by is mentioned. Specific examples of the nucleobase substituent and nucleobase which may have a substituent represented by R ₁ , and specific examples of the amino group protecting group in R ₂ are as described above.

  The compound represented by the formula [4] and the compound represented by the formula [5] can be used as a so-called amidite reagent in nucleic acid synthesis.

  Specific examples of the compound represented by the formula [5] include 5′-amino-N-monomethoxytrityl-2 ′, 5′-dideoxy-2′-fluorouridine-3′-O-[(2-cyanoethyl)-( N, N-diisopropyl)]-phosphoramidite, 5'-amino -N-monomethoxytrityl -2 ', 5'-dideoxy-2'-fluoroadenosine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl )]-phosphoramidite, 5'-amino -N-monomethoxytrityl -2 ', 5'-dideoxy-2'-fluoroguanosine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite, 5'-amino -N-monomethoxytrityl -2 ', 5'-dideoxy-2'-fluorocytidine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite, 5'-amino- N-monomethoxytrityl-2 ', 5'-dideoxy-2'-fluorothymidine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite, and the like.

  Examples of the method for synthesizing the dideoxynucleoside derivative of the present invention include the following methods.

First, the nucleobase used as a raw material is cyclized from the carbonyl group of the base part and the 2 ′ hydroxyl group of the sugar part using a condensing agent such as diphenyl carbonate ((PhO) ₂ CO), and 2,2′-anhydro- 1-β-D-arabinofuranosyl nucleoside is obtained.

  Next, in the presence of a catalyst such as 4-dimethylaminopyridine (DMAP), a reaction with a tritylating agent such as 4,4'-dimethoxytrityl chloride (DMTrCl) in an inert gas atmosphere is performed to produce 2,2'-anhydro- Tritylated 3 ', 5'-hydroxyl group of 1-β-D-arabinofuranosyl nucleoside, 2,2'-anhydro-3', 5'-di-O- (4,4'-dimethoxytrityl) -1-β-D-arabinofuranosyl nucleoside is obtained.

  This is then reacted with a base such as NaOH to open the ring to obtain 3 ′, 5′-di-O- (4,4′-dimethoxytrityl) -nucleoside.

  Furthermore, in an inert gas atmosphere, a fluorinating agent such as (dimethylamino) sulfur trifluoride (DAST) is reacted to form a 3 ', 5'-di-O- (4,4'-dimethoxytrityl) -nucleoside sugar moiety. Fluorination of the 2'-position yields 2'-deoxy-3 ', 5'-di-O- (4,4'-dimethoxytrityl) -2'-fluoro nucleoside.

  Next, this is reacted with an acid such as acetic acid to detritylate the 3′-position of the sugar moiety to obtain 2′-fluoro-2 ′, 5′-dideoxynucleoside.

Under inert gas atmosphere, 5'-hydroxyl of the sugar part was obtained using sodium azide, triphenylphosphine ((Ph) ₃ P) and CBr ₄ for the obtained 2'-fluoro-2 ', 5'-dideoxynucleoside. Via group bromination, azidation yields 5'-azido-2'-fluoro-2 ', 5'-dideoxynucleoside.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。）で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシドが得られる。 When the obtained 5′-azido-2′-fluoro-2 ′, 5′-dideoxynucleoside is reduced by reaction in the presence of a catalyst such as palladium carbon (Pd—C) in an H ₂ atmosphere, the present invention The following formula [2]

(Wherein R ₁ represents a nucleobase optionally having a protecting group), 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside represented by the following formula is obtained.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。）
で表される5'-アミノ-2'-フルオロ-2',5'-ジデオキシ-5'-N-モノエトキシトリチルヌクレオシドが得られる。 Next, 5 ′ represented by the formula [2] is obtained by a method known per se described in S. Obika et al., Angew. Chem. Int. Ed., 2005, vol.44, pp.1944-1947, etc. -Amino-2'-fluoro-2 ', 5'-dideoxynucleoside is tritylated by reacting with a tritylating agent such as 4-methoxytriphenylmethyl chloride (MMTrCl) in the presence of a catalyst such as DMAP. The following formula [3] of the invention

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group.)
5′-amino-2′-fluoro-2 ′, 5′-dideoxy-5′-N-monoethoxytrityl nucleoside represented by the following formula is obtained.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。i-Prはイソプロピル基を表す。）で表される、5'-アミノ-N-モノメトキシトリチル-2'-フルオロ-2',5'-ジデオキシヌクレオシド・アミダイトユニットが得られる。 Further, the obtained 5′-amino-2′-fluoro-2 ′, 5′-dideoxy-5′-N-monomethoxytrityl nucleoside was converted to 2-cyanoethyldiisopropylchloro-phosphoramidite in the presence of a base such as diisopropylethylamine (DIPEA). When phosphorylated by reacting with a phosphorylating agent such as (i-Pr ₂ NP (Cl) OCE), the following formula [5]

(Wherein R ₁ represents a nucleobase optionally having a protecting group; R ₂ represents a hydrogen atom or an amino group protecting group; i-Pr represents an isopropyl group); A 5′-amino-N-monomethoxytrityl-2′-fluoro-2 ′, 5′-dideoxynucleoside amidite unit is obtained.

Next, the method for synthesizing the dideoxynucleoside derivative of the present invention will be described more specifically by taking as an example the case where R ₁ in formula [1] is uracil. The synthesis scheme is described below (Synthesis scheme A).

The formal names of the abbreviations used in the following synthesis scheme A are as follows.
(Ph) ₂ CO: Diphenyl carbonate
DMF: N, N-dimethylformamide
DMTrCl: 4,4'-dimethoxytrityl chloride
DMAP: 4-dimethylaminopyridine
MeOH: methanol
DAST: (diethylamino) sulfur trifluoride
AcOH: Acetic acid
(Ph) ₃ P: Triphenylphosphine
Pd / C: Palladium carbon
MMTrCl: 4-Methoxytriphenylmethyl chloride
i-Pr ₂ NP (Cl) OCE: 2-cyanoethyldiisopropylchloro-phosphoramidite
i-Pr ₂ Net: diisopropylethylamine

[Reaction Scheme A]

First, the material uridine is cyclized using diphenyl carbonate ((PhO) ₂ CO) as a condensing agent to give 2,2'-anhydro-1-β-D-arabinofuranosyluridine (1) (in reaction scheme A above). (1) represents the compound represented by (1), the same applies hereinafter).

  Next, in the presence of 4-dimethylaminopyridine (DMAP) as a catalyst, it was reacted with 4,4'-dimethoxytrityl chloride (DMTrCl) in an Ar atmosphere to give 2,2'-anhydro-1-β-D-arabinofuranosyluridine ( Tritylate the 3 ', 5'-hydroxyl group of 1) to obtain 2,2'-anhydro-3', 5'-di-O- (4,4'-dimethoxytrityl) -1-β-D-arabinofuranosyluridine (Not shown).

  This is then reacted with NaOH (40 ml) to open the ring, yielding 3 ′, 5′-di-O- (4,4′-dimethoxytrityl) -uridine (2).

  Furthermore, (2) -position of the sugar part of 3 ', 5'-di-O- (4,4'-dimethoxytrityl) -uridine (2) is reacted with (diethylamino) sulfur trifluoride (DAST) under Ar atmosphere. Fluorination yields 2′-deoxy-3 ′, 5′-di-O- (4,4′-dimethoxytrityl) -2′-fluorouridine (not shown).

  This is then reacted with acetic acid to detritylate the 3′-position of the sugar moiety to obtain 2′-fluoro-2 ′, 5′-dideoxyuridine (3).

Under an Ar atmosphere, the obtained 2'-fluoro-2 ', 5'-dideoxyuridine (3) is reacted with sodium azide, triphenylphosphine ((Ph) ₃ P), and CBr ₄ together to form the sugar 5 The '-hydroxyl group is azidated to give 5'-azide-2'-fluoro-2', 5'-dideoxyuridine (4).

If the obtained 5'-azide-2'-fluoro-2 ', 5'-dideoxyuridine (4) is reduced by reacting in the presence of palladium carbon (Pd-C) catalyst in H ₂ atmosphere, 5'-amino-2'-fluoro-2 ', 5'-dideoxyuridine (5) is obtained.

  Next, the obtained 5'-amino-2'-fluoro-2 ', 5'-dideoxyuridine (5) is tritylated by reacting 4-Methoxytriphenylmethyl chloride (MMTrCl) in the presence of DMAP to produce 5'- Amino-2'-fluoro-2 ', 5'-dideoxy-5'-N-monomethoxytrityluridine (6) is obtained.

Furthermore, the obtained 5′-amino-2′-fluoro-2 ′, 5′-dideoxy-5′-N-monomethoxytrityluridine was converted to 2-cyanoethyldiisopropylchloro-phosphoramidite (i-Pr ₂ NP) in the presence of diisopropylethylamine (DIPEA). (Cl) OCE) and phosphorylation, 5'-amino-N-monomethoxytrityl-2'-fluoro-2 ', 5'-dideoxyuridine -3'-O-[( 2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (7) is obtained.

  The insoluble carrier used in the dideoxynucleoside-insoluble carrier conjugate of the present invention is not particularly limited as long as it is generally used in this field, and examples thereof include polystyrene and glass beads. In particular, CPG (controlled pore glass), which is a porous powdery glass bead, is commonly used.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基、Ｒ₂は水素原子又は水酸基の保護基を表す。）
で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシド誘導体が、その５単糖の3'-Ｏ-部分が、コハク酸等の有機分子のスペーサーを介してエステル型で結合しているものが挙げられる。 Specific examples of the dideoxynucleoside-insoluble carrier conjugate of the present invention include, for example, a nucleoside derivative of the present invention, that is, the following formula [1] on the surface of CPG.

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group, and R ₂ represents a hydrogen atom or a hydroxyl group protecting group.)
5'-amino-2'-fluoro-2 ', 5'-dideoxynucleoside derivative represented by the formula, wherein the 5'-saccharide 3'-O- moiety is esterified via a spacer of an organic molecule such as succinic acid. Examples include those linked by type.

（式中、Ｒ₁は保護基を有していてもよい核酸塩基を表す。Ｒ_２は水素原子又はアミノ基の保護基を表す。）
で表される5'−アミノ−2'−フルオロ−2',5'−ジデオキシヌクレオシド誘導体が結合したものである。式[６]において、Ｒ₁で表される置換基を有していてもよい核酸塩基の置換基及び核酸塩基の具体例、及びＲ_２におけるアミノ基の保護基の具体例は前記したとおりである。 That is, the dideoxynucleoside-insoluble carrier conjugate of the present invention has the following formula [6] via an insoluble carrier and, if necessary, an appropriate spacer.

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group.)
And a 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula: In the formula [6], specific examples of the nucleobase substituent and nucleobase which may have a substituent represented by R ₁ , and specific examples of the amino group protecting group in R ₂ are as described above. is there.

  The dideoxynucleoside-insoluble carrier conjugate obtained by binding the dideoxynucleoside derivative of the present invention to an insoluble carrier can be used as a starting material used in the solid-phase synthesis method of nucleic acids known per se.

  The method for binding the dideoxynucleoside derivative of the present invention to an insoluble carrier (for example, CPG) is known per se as described in, for example, R. Kierzek et al., Biochemistry, 1986, vol.25, pp.7840-7846. A method is mentioned.

  The dideoxynucleoside derivative of the present invention can also be used as a raw material for producing an oligonucleotide analog.

  In order to produce an oligonucleotide analog into which the dideoxynucleoside derivative of the present invention has been introduced, synthesis may be carried out by a chemical synthesis method known per se, except that the dideoxynucleoside derivative of the present invention is used as a raw material. For example, using a DNA synthesizer, which is commonly used for DNA synthesis, the usual phosphoramidite method or solid phase phosphoramidite method (R. Kierzek et al., Biochemistry, 1986, vol.25, pp.7840-7846 ) And is purified by a conventional method using anion exchange column chromatography, the target oligonucleotide analog into which the dideoxynucleoside derivative of the present invention is introduced can be obtained.

  In addition, double-stranded nucleic acids (DNA / DNA, DNA / RNA, RNA / RNA) containing an oligonucleotide analog into which the dideoxynucleoside derivative of the present invention has been introduced are usually synthesized by methods known per se that are carried out in this field. do it. It can also be obtained using a commercially available kit.

  Since the dideoxynucleoside derivative of the present invention introduces a fluorine atom at the 2′-position of the sugar moiety and makes the sugar moiety conformation N-type as described above, the oligonucleotide analog into which the dideoxynucleoside derivative is introduced In the double-stranded nucleic acid using the double-stranded nucleic acid, the double-stranded nucleic acid is thermally stabilized and satisfies the binding affinity. Further, the dideoxynucleoside derivative of the present invention has an —NH— group introduced at the 5′-position of the sugar moiety. Thus, when an oligonucleoside analog is synthesized using this, nucleotides derived from the dideoxynucleoside derivative are bound to the previous nucleotides by phosphate amidate linkages. Therefore, in the oligonucleotide analog into which the dideoxynucleoside derivative is introduced, the phosphodiester bond at the part into which the dideoxynucleoside derivative is introduced is a phosphate amidate bond. From this, double-stranded nucleic acid using this oligonucleotide analog is expected to have improved nuclease resistance.

  Furthermore, since it has the above features, the oligonucleotide analog introduced with the dideoxynucleoside derivative of the present invention is a double-stranded nucleic acid (DNA) obtained by using this as an excellent antisense nucleic acid in the antisense method. / DNA, DNA / RNA, RNA / RNA) can be used as siRNA or dsRNA in the RNAi method. Furthermore, application to the pharmaceutical field is also expected.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

(1) Experimental equipment In each example and comparative example, the following experimental equipment was used.
DNA synthesis and RNA synthesis were performed using 3400 DNA Synthesizer (Applied Biosystems, nucleic acid synthesizer).
The NMR spectrum was measured using a JEOL JNM AL400 spectrometer (manufactured by JEOL Datum Co., Ltd.).

(2) In each Example and Comparative Example, the following reagents were used.

Uridine: made by SIGMA
Thymidine: SIGMA Palladium carbon: Nacalai Tesque
4-methoxytriphenylmethyl chloride: manufactured by Tokyo Chemical Industry Co., Ltd. diisopropylethylamine: manufactured by Wako Pure Chemical Industries, Ltd. diphenyl carbonate, dichloromethane (dehydrated), pyridine, manufactured by Wako Pure Chemical Industries, Ltd.
N, N-dimethylformamide, sodium hydroxide, palladium carbon (Pd-C), acetic anhydride: manufactured by Nacalai Tesque Co., Ltd. chloroform: manufactured by Tokuyama Co., Ltd. chloroform for isolation and purification: manufactured by ALDRICH, chloroform-d, 99.8 atom% D
Ethyl acetate, n-hexane, methanol: Sankyo Chemical Industry Co., Ltd.
4-Dimethylaminopyridine: manufactured by Tokyo Chemical Industry Co., Ltd.
4,4'-dimethoxytrityl chloride, (diethylamino) sulfur trifluoride: manufactured by SIGMA-ALDRICH Corp. tetrahydrofuran: manufactured by Kanto Chemical Co., Ltd. silica gel column chromatography: silica gel 60N manufactured by Kanto Chemical Co., Ltd. (spherical, neutral)
2-cyanoethyldiisopropylchloro-phosphoramidite: Lancaster

(3) The following abbreviations were used in each example and comparative example.

(PhO) ₂ CO: Diphenyl carbonate
DMF: N, N-dimethylformamide
MeOH: methanol
DMAP: 4-dimethylaminopyridine
DMTrCl: 4,4'-dimethoxytrityl chloride
THF: tetrahydrofuran
DAST: (diethylamino) sulfur trifluoride
(Ph) ₃ P: Triphenylphosphine
Pd-C: Palladium carbon
MMTrCl: 4-methoxytriphenylmethyl chloride
DIPEA: Diisopropylethylamine
i-Pr ₂ NP (Cl) OCE: 2-cyanoethyldiisopropylchloro-phosphoramidite
i-Pr ₂ Net: diisopropylethylamine

Example 1. Synthesis of 5′-amino-2′-deoxy-2′-fluoriuridine amidite unit (i) 2,2′-anhydro-1-β-D-arabinofuranosyluridine (1) ((1 in the above reaction scheme A) ), The same shall apply hereinafter))
Uridine (5.00 g) and (PhO) ₂ CO (5.7 g) were dissolved in DMF (17 ml), NaHCO ₃ (120 mg) was added and reacted at 130 ° C. for 4 hours, and then the reaction was stopped. The extract was washed 3 times with chloroform (30 ml), and the aqueous layer was evaporated. The residue was dissolved in MeOH (400 ml) and recrystallized to obtain white crystals of compound (1) (yield 3.58 g, 15.84 mmol, 77%).

¹ H NMR (400 MHz) (DMSO) δ: 3.16-3.28 (m, 2H, 5'-H), 4.06 (s, 3'-H), 4.37 (d, 1H, 4'-H), 4.98 (t , 1H, J = 4.80Hz, 3'-OH), 5.18 (d, 1H, J = 6.00Hz, 2'-H), 5.83 (d, 1H, J = 7.20Hz, 5-H), 5.87 (d , 1H, J = 4.40Hz, 5'-OH), 6.29 (d, 1H, J = 6.00Hz, 1'-H), 7.83 (d, 1H, J = 7.20Hz, 6-H).

(Ii) Synthesis of 2,2'-anhydro-3 ', 5'-di-O- (4,4'-dimethoxytrityl) -1-β-D-arabinofuranosyluridine Compound (1) obtained in (i) above (3 g) and DMAP (0.4 g) were dissolved in pyridine (68 ml), DMTrCl (13.76 g) was added, and the mixture was stirred under an argon (Ar) atmosphere. After 144 hours, sat. NaHCO ₃ (sat .: saturated aqueous solution, the same applies hereinafter) was added to stop the reaction. Dilute with ethyl acetate, extract and wash 3 times with H ₂ O (200 ml), once with sat. NaHCO ₃ (200 ml) and once with sat. NaCl (200 ml). It was dried with sodium sulfate and the solvent was distilled off. The residue was dissolved in ethyl acetate (15 ml), followed by isolation and purification by silica gel column chromatography (hexane: ethyl acetate = 9: 1 to 0: 1). White crystals were obtained by distilling off the solvent.

(iii) Synthesis of 3 ', 5'-di-O- (4,4'-dimethoxytrityl) -uridine (2) The white crystal residue obtained in (ii) above was dissolved in THF (150 ml), 1N NaOH (40 ml) was added dropwise and reacted at 95 ° C in an oil bath for 3 hours, diluted with ethyl acetate (80 ml), 3 times with H ₂ O (120 ml), sat NaHCO ₃ (120 ml). And once with sat NaCl (120 ml), and washed, and the organic layer was dried over sodium sulfate and evaporated. The residue was dissolved in ethyl acetate (15 ml), and then isolated and purified by silica gel column chromatography (hexane: ethyl acetate = 4: 1 to 0: 1). The solvent was distilled off to obtain white crystals of compound (2) (yield 10.7 g, 43.1 mmol, 95%).

¹ H NMR (400 MHz) (CDCl ₃ ) δ: 3.51 (dd, 1H, J = 14.40Hz, 5'-H), 3.33 (d, 1H, J = 9.28Hz, 2'-OH), 3.43 (dd, 1H, J = 13.44Hz, 5'-H), 3.66 (dd, 1H, J = 12.2Hz, 2'-H), 3.93 (s, 1H, 3'-H), 4.04 (s, 1H, 4 ' -H), 5.58 (dd, J = 10.28Hz, 5-H), 6.09 (d, J = 2.96, 1'-H), 7.14-7.35 (m, 26H, DMTr), 7.61 (d, J = 8.32 Hz, 6-H), 8.35 (s, 1H, 5-NH).

(iv) Synthesis of 2'-deoxy-3 ', 5'-di-O- (4,4'-dimethoxytrityl) -2'-fluorouridine
Under an Ar atmosphere, the compound (2) obtained in (iii) above was dissolved in CH ₃ CN (185 ml), and then DMF (14 ml) was added. DAST (14 ml) was dripped here under cold ice, and reaction was started. After 24 hours, sat NaHCO ₃ (180 ml) was added under cold ice to quench the reaction. The reaction solution was diluted with ethyl acetate, extracted and washed ₃ times with H ₂ O (180 ml), 1 time with sat NaHCO ₃ (180 ml), 1 time with sat NaCl (180 ml), and organic The layer was dried over sodium sulfate and then evaporated to give a residue.

(v) Synthesis of 2'-fluoro-2 ', 5'-dideoxyuridine (3) The residue obtained in (iv) above was dissolved in MeOH, 80% acetic acid (100 ml) was added, and the mixture was heated at 80 ° C for 6 hours. After stirring, the reaction mixture was concentrated and dissolved in H ₂ O (150 ml). Subsequently, extraction and washing were performed 3 times with CHCl ₃ (150 ml), and the aqueous layer was evaporated. The residue was dissolved in chloroform (15 ml), and then isolated and purified by silica gel column chromatography (chloroform: methanol = 1: 0 to 7: 1). The solvent was distilled off to obtain white crystals of compound (3) (yield 1.68 g, 6.8 mmol, 58%).

¹ H NMR (400 MHz) (DMSO) δ: 3.57 (dd, 2H, 5'-H), 3.75 (dd, J = 8.00Hz, 3'-H), 3.85-3.87 (m, 1H, 4'-H ), 4.09-4.18 (m, 1H, 5-H), 4.94-5.09 (dq, 1H, 2'-H), 5.61 (dd, 1H, 5'-OH), 5.89 (dd, 1H, 1'- H), 7.90 (d, 1H, J = 8.08Hz, 6-H), 11.38 (s, 1H, 3-NH).

(vi) Synthesis of 5'-azide-2'-fluoro-2 ', 5'-dideoxyuridine (4)
In an Ar atmosphere, the compound (3) obtained in (v) above was dissolved in (600 mg), (Ph) ₃ P (908 mg), and NaN ₃ (793 mg) in DMF (12 ml), then CBr ₄ ( 972 mg) was added, the mixture was reacted at 85 ° C. in an oil bath, and the reaction was stopped after 48 hours. The reaction product was concentrated and isolated and purified by silica gel column chromatography (chloroform: methanol = 1: 0 to 19: 1). The solvent was distilled off to obtain white crystals of compound (4) (yield 0.48 g, 1.8 mmol, 73%).

¹ H NMR (400 MHz) (DMSO) δ: 3.52 (dd, 1H, 5′-H), 3.73 (dd, 1H, 5′-H), 3.91-3.95 (m, 1H, 3′-H), 4.17 -4.28 (m, 1H, 4'-H), 5.19 (dq, 1H, 2'-H), 5.65 (d, 1H, J = 8.00Hz, 3'-OH), 5.73 (d, 1H, J = 6.4, 5-H), 5.85 (dd, 1H, 1'-H), 7.66 (d, 1H, J = 8.4, 6-H), 11.43 (s, 1H, 3-NH).

(vii) Synthesis of 5'-amino-2'-fluoro-2 ', 5'-dideoxyuridine (5) Compound (4) (507 mg) obtained in (vi) above was dissolved in MeOH (10 ml). , Pd-C (123 mg) was added, and the mixture was reacted under H ₂ atmosphere. After 15 hours, the reaction product was filtered through Celite to remove Pd—C, and the solvent was distilled off to obtain white crystals of compound (5) (yield 0.39 mg, 1.6 mmol, 85%).

¹ H NMR (400 MHz) (DMSO) δ: 2.82 (ddd, 2H, J = 3.60Hz J = 3.20Hz J = 4.80Hz J = 5.20Hz, 5'-H), 3.76 (m, 1H, 3'-H ), 4.11 (ddd, 1H, J = 7.20Hz J = 7.20Hz J = 7.20 Hz J = 7.20Hz, 4'-H), 5.06 (ddd, 1H, J = 2.00Hz J = 2.00Hz J = 2.40Hz J = 2.00Hz, 2'-H), 5.60 (d, 1H, J = 8.00, 5-H), 5.86 (dd, 1H, J = 2.00Hz J = 2.00Hz, 1'-H), 7.91 (d, 1H, J = 8.00, 6-H).

(viii) Synthesis of 5'-amino-2'-fluoro-2 ', 5'-dideoxy-5'-N-monomethoxytrityluridine (6) Compound (5) (555 mg) obtained in (vii) above is pyridine (12 ml) was dissolved, and MMTrCl (1 g) and DMAP (28 mg) were added and reacted. After 210 hours, dilute with ethyl acetate (40 ml), extract three times with H ₂ O (40 ml), once with sat NaHCO ₃ (40 ml), once with sat NaCl (40 ml) and washed. And the organic layer was dried over sodium sulfate and evaporated. The residue was dissolved in ethyl acetate (5 ml) and isolated and purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1 to 0: 1) to give white crystals (6) (yield 0.69). g, 1.3 mmol, 59%).

¹ H NMR (400 MHz) (DMSO) δ: 2.43-2.70 (ddd, 2H, J = 3.40Hz J = 3.40Hz J = 6.08Hz J = 6.08Hz, 5'-H), 4.06 (m, 1H, 3 ' -H), 5.10 (ddd, 1H, 2'-H), 5.69 (d, 1H, J = 8.04Hz, 5-H), 5.79 (dd, 1H, 1'-H), 6.80-7.46 (17H, m, MMTr).

(ix) 5'-amino-N-monomethoxytrityl-2'-fluoro-2 ', 5'-dideoxyuridine -3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (7) Compound (6) (400 mg) obtained in (viii) above was dissolved in dichloromethane (dehydrated, 3.9 ml), DIPEA (0.41 ml) was added and stirred, and then i-Pr ₂ NP (Cl ) OCE was added dropwise (0.26 ml). After 1 hour, dilute with ethyl acetate (30 ml), extract three times with H ₂ O (40 ml), once with sat NaHCO ₃ (40 ml), once with sat NaCl (40 ml) and washed. And the organic layer was dried over sodium sulfate and evaporated. The residue was dissolved in ethyl acetate (5 ml), and then isolated and purified by silica gel column chromatography (ethyl acetate) to obtain white foam crystals of compound (7) (yield 0.51 mg, 0.70 mol, 91 %).
³¹ P NMR (400 MHz) (DMSO) δ: (151.14, 151.58)

Comparative Example 1. Synthesis of 5'-aminothymidine amidite unit
A synthesis scheme of 5′-aminothymidine amidite unit is shown below (Synthesis scheme B). The formal names of the abbreviations in the following synthesis scheme B are as described above. The nucleoside derivative and amidite unit obtained by this method are those in which the 2′-position of the sugar moiety is not fluorinated.

[Synthesis Scheme B]

(I) Synthesis of 5′-azidethymidine (8) (compound represented by (8) in the above synthesis scheme B, the same shall apply hereinafter)
Thymidine (3 g) and (Ph) ₃ P (4.6 g) were dissolved in DMF (62 ml), and CBr ₃ (4.9 g) was added and stirred. After 4 hours, NaN ₃ (4.0 g) was added and reacted at 60 ° C. in an oil bath. After 23 hours, the mixture was concentrated by an evaporator. After the concentrate was dissolved in chloroform (15 ml), it was isolated and purified by silica gel column chromatography (chloroform: methanol = 1: 0 to 9: 1). The solvent was distilled off to obtain white crystals of compound (8) (yield 2.26 g, 8.46 mmol, 68%).

¹ H NMR (400 MHz) (DMSO) δ: 1.78 (3H, s, 5-CH ₃ ), 2.05-2.28 (2H, m, 2'-H), 3.54-3.55 (2H, m, 5'-H) , 3.82-3.83 (1H, m, 3'-OH), 4.18 (1H, m, 4'-H), 5.40-5.41 (1H, m, 3'-H), 6.20 (1H, t, J = 7.00 Hz, 1'-H), 7.48 (1H, s, 6-H), 11.3 (1H, s, 3-NH).

(Ii) Synthesis of 5'-aminothymidine (9) Compound (8) obtained in (i) above was dissolved in (2.26 g) in methanol (84 ml), and Pd-C (558 mg) was added. The reaction was carried out under H ₂ atmosphere. After 13 hours, Pd—C was removed by celite filtration, and the solvent was distilled off to obtain white crystals of compound (9) (yield 2.02 g, 8.35 mmol, 99%).

¹ H NMR (400 MHz) (DMSO) δ: 1.77 (s, 3H, 5-CH ₃ ), 1.99-2.15 (m, 1H, 2'-H), 2.71 (d, 1H, J = 5.6 Hz, 5 ' -H), 3.15 (s, 1H, 3'-OH), 3.63 (m, 1H, 4'-H), 4.16-4.19 (m, 1H, 3'-H), 6.27 (t, 1H, 1 ' -H), 7.64 (s, 1H, 6-H).

(iii) Synthesis of 5'-amino-5'-N-monomethoxytritylthymidine (10) Compound (9) (400 mg) obtained in (ii) above was dissolved in pyridine (8.7 ml) and MMTrCl (709 mg ), DMAP (20 mg) was added and reacted. After 48 hours, dilute with ethyl acetate (30 ml), extract three times with H ₂ O (30 ml), once with sat NaHCO ₃ (30 ml), once with sat NaCl (30 ml) and washed. And the organic layer was dried over sodium sulfate and evaporated. The residue was dissolved in chloroform (5 ml) and isolated and purified by silica gel column chromatography (chloroform: methanol = 1: 0 to 23: 2) to obtain white crystals of compound (10) (yield 715 mg, 1.39 mmol, 84%).

¹ H NMR (400 MHz) (DMSO) δ: 1.84 (s, 3H, 5-CH ₃ ), 2.05-2.15 (m, 1H, 2'-H), 2.33-2.60 (m, 1H, 5'-H) , 3.97-3.99 (m, 1H, 3'-OH), 3.62 (q, 1H, 4'-H), 4.31-4.33 (m, 1H, 3'-H), 6.80-7.46 (m, 17H, MMTr ), 8.43 (s, 1H, 6-H).

(iv) Synthesis of 5'-amino-N-monomethoxytritylthymidine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (11) Compound (10) obtained in (iii) above ) (295 mg) was dissolved in dichloromethane (for dehydration, 2.87 ml), DIPEA (0.3 ml) was added and stirred, and i-Pr ₂ NP (Cl) OCE (0.19 ml) was added dropwise. After 1 hour, dilute with ethyl acetate (20 ml), extract three times with H ₂ O (20 ml), once with sat NaHCO ₃ (20 ml), once with sat NaCl (20 ml) and washed. And the organic layer was dried over sodium sulfate and evaporated. The residue was dissolved in ethyl acetate (5 ml), and isolated and purified by silica gel column chromatography (ethyl acetate) to obtain white foam crystals of compound (11) (yield 357 mg, 0.752 mmol, 87 %).
³¹ P NMR (400 MHz) (DMSO) δ (149.58, 149.83).

Example 2 Synthesis of Oligonucleotide by Solid Phase Phosphoramidite Method An oligonucleotide analog into which 5′-amino-2′-fluoro-2 ′, 5′-dideoxyuridine of the present invention was introduced was synthesized by the following method.

  First, 5'-amino-N-monomethoxytrityl-2'-fluoro-2 ', 5'-dideoxyuridine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl) synthesized in Example 1 was used as a material. )]-phosphoramidite (7) and commercially available dA-CE phosphoramidite [chemical name: (5'-Dimethoxytrityl-N-benzoyl-2'-deoxyAdenosine, 3 '-[(2-cyanoethyl)-(N, N-diisopropyl )]-phosphoramidite)], dG-CE phosphoramidite [Chemical name: 5'-Dimethoxytrityl-N-isobutyryl-2'-deoxyGuanosine, 3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite] (Both manufactured by Glen research Corp.) were used. In addition, 3'-dA-CPG (CPG means Controlled Pore Glass, manufactured by Glen research Corp.) and 1 μmol packed in column were synthesized as starting materials. A 3400 DNA Synthesizer (Applied Biosystems, nucleic acid synthesizer) was used as a synthesizer.

  The structure of the two obtained oligonucleotide analogs was confirmed by MALDI TOF / MAS using AXIMA (manufactured by Shimadzu Corporation).

The resulting two oligonucleotide analogues were named UNF-1 and UNF-2. Table 1 shows the base sequence of UNF-1 (SEQ ID NO: 1), the base sequence of UND-2 (SEQ ID NO: 2), the theoretical value (calculated) and the observed value (observed) of each molecular weight. In Table 1, the position where 5′-amino-2′-fluoro-2 ′, 5′-dideoxyuridine was introduced in the base sequence is indicated by “U ^NF ”.

Comparative Example 2
5'-amino-N-monomethoxytrityl-2'-fluoro-2 ', 5'-dideoxy-uridine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)] synthesized in Example 1 Instead of -phosphoramidite (7), 5'-amino-N-monomethoxytritylthymidine-3'-O-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (11) synthesized in Comparative Example 1 was used. Two kinds of oligonucleotide analogs into which 5′-aminothymidine was introduced were synthesized in the same manner as in Example 2 except that they were used as materials.

The obtained two types of oligonucleotide analogs were named TN-1 and TN-2. Further, the structure was confirmed by MALDI TOF / MAS as in Example 2. Table 1 shows the base sequence of TN-1 (SEQ ID NO: 3) and the base sequence of TN-2 (SEQ ID NO: 4), the theoretical value (calculated) and the actually measured value (observed) of each molecular weight. In Table 1, the introduction position of 5′-aminothymidine is indicated by “T ^N ” in the base sequence.

In addition, commercially available dT-CE phosphoramidite [chemical name: 5'-Dimethoxytrityl-2'-deoxyThymidine, 3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite], dA-CE phosphoramidite , DG-CE phosphoramidite
(Both from Glen research Corp.) and 3′-dA-CPG 500 1 μmol column (manufactured by Glen research Corp.) filled with 3′-dA-CPG as the starting material. The oligonucleotide was synthesized by the method described above to obtain an oligonucleotide consisting only of natural nucleosides. The obtained two types of oligonucleotides were named T-1 (control) and T-2 (control). Further, the structure was confirmed by MALDI TOF / MAS as in Example 2. The base sequence of T-1 (control) (SEQ ID NO: 5) and the base sequence of T-2 (control) (SEQ ID NO: 6) are also shown in Table 1.

Example 3 Measurement of Tm value (melting temperature) (DNA / DNA match duplex)
(I) Synthesis of complementary oligonucleotides Commercially available dT-CE phosphoramidite and dA-CE phosphoramidite (both manufactured by Glen research Corp.) were used as materials, and 3'-dT-CPG (Glen research Corp.) was used as a starting material. 1 μmol was packed in a column and oligonucleotides complementary to UNF-1 and UNF-2 synthesized in Example 2 were synthesized in the same manner as in Example 2. Table 2 below shows the nucleotide sequence of the oligonucleotide complementary to UNF-1 (SEQ ID NO: 7) and the nucleotide sequence of the oligonucleotide complementary to UNF-2 (SEQ ID NO: 8).

(Ii) Thermal Stability Test Next, double-stranded DNA / DNA was usually used using the UNF-1, UNF-2 synthesized in Example 2 and the oligonucleotides complementary to these obtained in Table 2 as materials. (R. Kierzek et al., Biochemistry, 1986, vol. 25, pp. 7840-7846).

  The obtained double-stranded DNA / DNA was subjected to a thermal stability test by a conventional method.

  That is, using a UV-2450 SPECTROPHOTOMETER (manufactured by SHIMADZU) equipped with a variable temperature device in a 10 mm 8-series cell under a nitrogen atmosphere, so that the oligonucleotide is 3 μM, sodium phosphate buffer 10 mM, chloride Sodium 100 mM (pH 7.0) was added for adjustment, the temperature was raised from 20 ° C. to 90 ° C. at a rate of 0.5 ° C. per minute, and the absorbance at 260 nm was measured.

The results are shown in FIG.
In FIG. 1, ● represents the results obtained for double-stranded DNA / DNA using UNF-1, and ○ represents the results obtained for double-stranded DNA / DNA using UNF-2.
Table 3 shows the Tm value and ΔTm value of each double-stranded DNA / DNA obtained from the results of FIG.

Comparative Example 3 Measurement of Tm value (DNA / DNA match duplex)
In the same manner as in Example 3, double-stranded DNA / DNA of TN-1 synthesized in Comparative Example 2 and an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 7 complementary thereto, and TN-2 and the same Double-stranded DNA / DNA was synthesized with a complementary oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 8, and the thermal stability of the obtained double-stranded DNA was examined in the same manner as in Example 3. The results are also shown in FIG.
In FIG. 1, ▪ represents the results obtained for double-stranded DNA / DNA using TN-1, and □ represents the results obtained for double-stranded DNA / DNA using TN-2.
In addition, Table 3 shows the Tm value and ΔTm value of each double-stranded DNA / DNA obtained from the results of FIG.

Further, in the same manner as in Example 3, double-stranded DNA comprising T-1 (control) synthesized in Comparative Example 2 and an oligonucleotide having a base sequence represented by SEQ ID NO: 7 complementary thereto, and T- 2 (control) and a complementary double-stranded DNA with an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 8 were synthesized, and the resulting double-stranded thermal stability was determined in the same manner as in Example 3. We investigated by. The results are also shown in FIG.
In Fig. 1, ▲ indicates the results for double-stranded DNA / DNA using T-1 (control), and △ indicates the results for double-stranded DNA / DNA using T-2 (control). Show.
In addition, Table 3 shows the Tm value of each double-stranded DNA / DNA obtained from the results of FIG.

  As is apparent from the results in FIG. 1 and Table 3, two oligonucleotides (UNF-1 and UNF-2) containing 5′-amino-2′-fluoro-2 ′, 5′-dideoxyuridine of the present invention were introduced. The Tm value of double-stranded DNA / DNA is equivalent to the Tm value of double-stranded DNA / DNA containing oligonucleotides (T-1 (control) and T-2 (control)) consisting only of natural nucleosides, It can be seen that the thermal stability of the double-stranded nucleic acids is equivalent to each other. In contrast, the Tm value of double-stranded DNA / DNA containing oligonucleotide analogs (TN-1 and TN-2) introduced with 5'-aminothymidine that is not fluorinated at the 2'-position of the sugar moiety is Since it is lower than the Tm value of double-stranded DNA / DNA containing oligonucleotides (T-1 (control) and T-2 (control)) consisting only of a type of nucleoside, it can be seen that the thermal stability is low.

Example 4 Measurement of Tm value (DNA / RNA match duplex)
(I) Synthesis of complementary oligonucleotide Using commercially available rA-CE phosphoramidite, rU-CE phosphoramidite, rC-CE phosphoramidite, and 3'-dU-CPG (all manufactured by Glen research Corp.) as starting materials In the same manner as in Example 2, oligonucleotides complementary to each of UNF-1 and UNF-2 synthesized in Example 2 were synthesized.

  Table 4 below shows the nucleotide sequence of the oligonucleotide complementary to UNF-1 (SEQ ID NO: 9) and the nucleotide sequence of the oligonucleotide complementary to UNF-2 (SEQ ID NO: 10).

(Ii) Thermal Stability Test Next, double-stranded DNA / RNA using UNF-1, UNF-2 synthesized in Example 2 and oligonucleotides complementary to them listed in Table 4 obtained above as materials. Was obtained by a conventional method (R. Kierzek et al., Biochemistry, 1986, vol. 25, pp. 7840-7846).

  Using the obtained double-stranded DNA / RNA, a thermal stability test was conducted in the same manner as in Example 3.

The results are shown in FIG.
In FIG. 2, ● represents the results obtained for double-stranded DNA / RNA using UNF-1, and ○ represents the results obtained for double-stranded DNA / RNA using UNF-2.
Table 5 shows the Tm value and ΔTm of each double-stranded DNA / RNA obtained from the results of FIG.

Comparative Example 4 Measurement of Tm value (DNA / RNA match duplex)
In the same manner as in Example 4, double-stranded DNA / RNA of TN-1 synthesized in Comparative Example 2 and the oligonucleotide having the base sequence represented by SEQ ID NO: 9 complementary thereto, and TN-2 and the same A double-stranded DNA / RNA was synthesized with a complementary oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 10, and the thermal stability of the resulting double-stranded was examined by the same method as in Example 4. The results are also shown in FIG.
In FIG. 2, ▪ represents the results obtained for double-stranded DNA / RNA using TN-1, and □ represents the results obtained for double-stranded DNA / RNA / using TN-2.
In addition, Table 5 shows the Tm value and ΔTm value of each double-stranded DNA / RNA obtained from the results of FIG.

In addition, double-stranded DNA / RNA of T-1 (control) synthesized in Comparative Example 2 and the oligonucleotide of the base sequence represented by SEQ ID NO: 9 complementary thereto, and T-2 (control) and complement thereof A double-stranded DNA / RNA was synthesized with an oligonucleotide having the base sequence represented by SEQ ID NO: 10, and the thermal stability of the resulting double-stranded was examined by the same method as in Example 4. The results are also shown in FIG.
In Fig. 2, ▲ indicates the results for double-stranded DNA / RNA using T-1 (control), and △ indicates the results for double-stranded DNA / RNA using T-2 (control). Show.
In addition, Table 5 shows the Tm values of each double-stranded DNA / RNA obtained from the results of FIG.

  As is apparent from the results of FIG. 2 and Table 5, two oligonucleotides (UNF-1 and UNF-2) containing 5′-amino-2′-fluoro-2 ′, 5′-dideoxyuridine of the present invention were used. The Tm value of double-stranded DNA / RNA is equivalent to the Tm value of double-stranded DNA / DNA containing oligonucleotides (T-1 (control) and T-2 (control)) consisting only of natural nucleosides, It can be seen that the thermal stability of the double-stranded nucleic acids is equivalent to each other. In contrast, the Tm value of double-stranded DNA / RNA containing oligonucleotide analogs (TN-1 and TN-2) introduced with 5'-aminothymidine that is not fluorinated at the 2'-position of the sugar moiety is Since it is lower than the Tm value of double-stranded DNA / DNA containing oligonucleotides (T-1 (control) and T-2 (control)) consisting only of a type of nucleoside, it can be seen that the thermal stability is low.

  Further, from the results of Tables 3 and 5, it can be seen that UNF-2 including two UNFs has a higher Tm value than UNF-1 including one UNF. From this, it is expected that the double-stranded nucleic acid is further stabilized by increasing the number of introduction of the dideoxynucleotide derivative of the present invention in the oligonucleotide.

  The 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative of the present invention is used as a phosphoramidite reagent in nucleic acid synthesis and by solid-phase synthesis of nucleic acid by binding the dideoxynucleoside derivative to a solid phase. It can be used as a starting material used in the method. In addition, the oligonucleotide analog into which the derivative is introduced is expected to have excellent thermal stability and excellent nuclease resistance. Therefore, the oligonucleotide analog can be used as an excellent antisense nucleic acid having nuclease resistance used in the antisense method. It can also be used as dsRNA and siRNA introduced into cells in the RNAi method. Furthermore, it can be used as an antisense oligonucleotide analog used in a method of knocking out a target gene. Furthermore, it is also expected to be used in the pharmaceutical field and as a probe for gene polymorphism analysis.

The results obtained by conducting the thermal stability test on the double-stranded DNA / DNA obtained in Example 3 and the thermal stability test on the double-stranded DNA / DNA obtained in Comparative Example 3 It is the obtained result. The results obtained by conducting the thermal stability test on the double-stranded DNA / RNA obtained in Example 4 and the thermal stability test on the double-stranded DNA / RNA obtained in Comparative Example 4 It is the obtained result.

Explanation of symbols

  In FIG. 1, ▲ indicates the results for double-stranded DNA / DNA using T-1 (control), and △ indicates the results for double-stranded DNA / DNA using T-2 (control). ● shows results for double-stranded DNA / DNA using UNF-1, ○ shows results for double-stranded DNA / DNA using UNF-2, ■ shows results for double-stranded DNA / DNA using UNF-2 The results obtained for the stranded DNA / DNA and the squares the results obtained for the double stranded DNA / DNA using TN-2, respectively.

  In FIG. 2, ▲ indicates the results obtained for double-stranded DNA / RNA using T-1 (control), and △ indicates the results obtained for double-stranded DNA / RNA using T-2 (Control). ● shows results for double-stranded DNA / RNA using UNF-1, ○ shows results for double-stranded DNA / RNA using UNF-2, ■ ■ shows results for double-stranded DNA / RNA using UNF-2 The results obtained for the stranded DNA / RNA and the squares the results obtained for the double stranded DNA / RNA / using TN-2, respectively.

Claims (5)

Following formula [1]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a protecting group for a hydrogen atom or an amino group, and R ₃ represents a protecting group for a hydrogen atom or a hydroxyl group.)
A 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by: The 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula [1] is represented by the following formula [3].

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group.)
The derivative | guide_body of Claim 1 which is represented by these. The 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula [1] is represented by the following formula [5].

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group. I-Pr represents an isopropyl group.) The derivative according to claim 1, wherein Following formula [6]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group.)
A dideoxynucleoside-insoluble carrier conjugate obtained by binding a 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula: Following formula [1]

(In the formula, R ₁ represents a nucleobase which may have a protecting group. R ₂ represents a hydrogen atom or an amino group protecting group, and R ₃ represents a hydrogen atom or a hydroxyl protecting group.)
An oligonucleotide analog into which a 5′-amino-2′-fluoro-2 ′, 5′-dideoxynucleoside derivative represented by the formula:

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