JP2006248949A - Nucleoside derivative, nucleotide derivative and manufacturing method thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a nucleoside derivative and a nucleotide derivative having controlled stereostructures. <P>SOLUTION: The nucleoside derivative and the nucleotide derivative represented by each general formula (B, B<SB>1</SB>and B<SB>2</SB>are each selected from hydrogen, an OH group, a nucleobase and its derivative; Z is S or Se; R<SB>1</SB>and R<SB>2</SB>are mutually different and each selected from hydrocarbon groups in which hydrogen atom may be partially substituted; Q is an OH-protecting group) have controlled configurations derived from an asymmetric phosphorus atom. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a nucleoside derivative whose steric configuration is controlled with high selectivity and a method for producing the same, and a nucleotide derivative and a method for producing the same.

  In recent years, artificially modified DNA that inhibits the translation of genes and suppresses the production of proteins, such as anti-sense DNA, has been gaining widespread attention as a pharmaceutical product for genes that want to suppress expression, such as viral disease genes and cancer genes. Yes.

  One such molecule is an oligodeoxyribonucleotide phosphorothioate (all-PS-DNA oligomer) in which the phosphodiester (phosphate) sites of a 20-mer DNA oligomer are all replaced with thiophosphate diester (phosphorothioate). High pharmacological effects have attracted much attention in recent years.

  However, although all-PS-DNA oligomers are superior in terms of nuclease resistance and cell membrane permeability, they are inferior to ordinary oligodeoxyribonucleotides in terms of double-stranded binding ability to DNA and RNA. It has the disadvantages that the interaction with is strong and toxic.

  Therefore, a phosphate / phosphorothioate mixed oligomer (PO / PS-hybridized DNA oligomer) in which only a part of the internucleotide portion is substituted with phosphorothioate for the purpose of alleviating this drawback has recently been created and attracting attention.

  By the way, since the phosphorus atom of the phosphorothioate part of these modified internucleotide parts becomes an asymmetric atom, it may cause a difference in the biological properties (bioactivity) of the compounds. Therefore, when a diastereomer mixture is used, unexpected side effects may occur, and clinically, it is important to use a single stereochemistry of the phosphorothioate moiety.

  A method for synthesizing PO / PS-hybridized DNA oligomers with controlled stereochemistry was developed by Stec et al. In 1998 using a stereochemically single compound as a structural unit for the thioate moiety ( Non-patent document 1: See the following reaction formula).

However, in this method, since it is obtained as a mixture with another diastereomer by ordinary synthesis, complicated operations such as purification are required, and the yield of the long-chain oligomer is low. Furthermore, in order to synthesize two kinds of phosphorothioate forms with different stereochemistry (R form and S form), it is said that it is a perfect method because two kinds of precursors (synthesis intermediates) corresponding to each must be prepared. hard.
Deoxyribonucleoside 3'-O- (2-Thio- and 2-Oxo- "spiro" -4,4-pentamethylene-1,3,2-oxathiaphospholane) s: Monomers for Stereocontrolled Synthesis of Oligo (deoxyribonucleoside phosphorothioate) s and Chimeric PS / PO Oligonucleotides Stec, WJ; Karwowski, B .; Boczkowska, M .; Guga, P .; Koziolkiewicz, M .; Sochacki, M .; Wicczorek, MW; Blaszczyk, JJ Am. Chem. Soc. 1998, 120, 7156 -7167.

  The present invention has been made in view of the above circumstances, and for the purpose of obtaining a PO / PS-hybridized DNA oligomer having a controlled three-dimensional structure, a compound that can be a constituent element and a precursor of a PO / PS-hybridized DNA oligomer It is an object to be solved to provide a nucleoside derivative and a nucleotide derivative, and methods for producing them.

  In order to solve the above problems, the present inventors have conducted intensive research and obtained the following findings. In other words, stereoselective synthesis of stereochemically single thiophosphoric acid triesters, which are the building blocks of PO / PS-hybridized DNA oligomers, is carried out by different treatment from one intermediate and the absolute configuration of the thioate moiety. We found that two different diastereomers were obtained. After several steps, we succeeded in creating a PO / PS-hybridized DNA oligomer with more efficient stereochemistry.

(1) Method for Producing Nucleoside Derivatives and Nucleotide Derivatives (1-1) A method for producing a nucleoside derivative of the present invention comprises a deoxyribonucleoside derivative represented by the following general formula (1) and a general formula (2): R ₁ OPXY (R ₁ is a hydrocarbon group which may be partially substituted with hydrogen; X and Y are Cl, Br and dialkyl-substituted amines —NRR ′ (R and R ′ are partially substituted with hydrogen; And R and R ′ may be combined together to form a ring.) Are each independently selected from phosphine derivatives represented by And a nucleoside cyclic phosphite triester derivative represented by the following general formula (3a) and / or (3b) by reacting in the presence of an acid compound selected from an acid azole complex and a carboxylic acid A first step that,
A second step of obtaining a nucleoside cyclic phosphite triester derivative represented by the following general formula (4a) and / or (4b) by reacting the nucleoside cyclic phosphite triester derivative with a S agent or Se agent: It is characterized by having.

R ₁ in R ₁ of the general formula (2); (Formula (1), (3a), (3b), (4a) and (4b) in, B is selected from hydrogen, OH group, a nucleobase or a derivative thereof And Z is O, S or Se.)
By reacting the deoxyribonucleoside derivative represented by the general formula (1) with the phosphine derivative represented by the general formula (2) in the presence of an acid compound selected from azole, acid azole complex and carboxylic acid, the general formula An equivalent amount of the nucleoside derivative shown in (3a) or (3b) is synthesized.

  Thereafter, by reacting with a S agent or a Se agent, the nucleoside derivative represented by the general formula (4a) is converted from the compound represented by the general formula (3a), and the compound represented by the general formula (3b) is represented by the general formula (4b). The nucleoside derivatives shown in FIG. Here, the nucleoside derivative represented by the general formula (3b) is represented by the general formula (3a) by maintaining the mixture at a high temperature as necessary in the state of the mixture of the nucleoside derivatives represented by the general formulas (3a) and (3b). Since it can be isomerized to the nucleoside derivative shown, the nucleoside derivative shown in the general formula (4a) can be used as the main product. Therefore, the three-dimensional structure of the obtained compound can be controlled.

Then, starting from the nucleoside cyclic phosphite triester derivative represented by the general formula (4a) and / or (4b) in the R ₂ OH solution (R ₂ is a hydrocarbon which may be partially substituted with hydrogen) A nucleoside derivative represented by the following general formula (5a) and / or (5b) can be obtained by reacting an alcoholate of R ₂ OH with a group different from R ₁ . From the compound represented by the general formula (4a), the nucleoside derivative represented by the general formula (5a) is obtained, and from the compound represented by the general formula (4b), the nucleoside derivative represented by the general formula (5b) is obtained. This reaction has a very high stereoselectivity, and a compound having the desired stereostructure can be synthesized.

(In the formulas (5a) and (5b), B is the same group as B explained in the general formula (1); R ₁ is the same group as R ₁ explained in the general formula (2). R ₂ is a hydrocarbon group which may be partially substituted with hydrogen and is a group different from R ₁ ; Z in the formula (5a) is the same as Z in the general formula (4a) Z is the same group as Z in the general formula (4b).
Note that the description of B, R ₁ , X, Y, etc. described here is applicable to other descriptions described later unless otherwise specified. That is, the symbols in the general formulas are used consistently unless otherwise specified in this specification.

(1-2) Another method for producing a nucleoside derivative according to the present invention includes a de-R ₁ agent or a de-R ₂ in a nucleoside derivative represented by the following general formula (5) in which the configuration derived from an asymmetric phosphorus atom is controlled. It is characterized in that a nucleoside derivative represented by the following general formula (6) or (7) in which the configuration derived from an asymmetric phosphorus atom is controlled by reacting an agent is obtained.

That is, the present invention has been completed by the discovery that the configuration derived from the asymmetric phosphorus atom changes depending on which group of R ₁ and R ₂ is eliminated. When R ₁ is eliminated, the configuration derived from the asymmetric phosphorus atom when R ₁ and R ₂ are equivalent is opposite to that when R ₂ is eliminated. Therefore, the configuration derived from the asymmetric phosphorus atom can be arbitrarily controlled.

(In the formulas (5) to (7), B is selected from hydrogen, OH group, nucleobase or derivatives thereof; Z is O, S or Se; R ₁ and R ₂ are partially substituted with different hydrogens. Selected from hydrocarbon groups which may be substituted; Q is a protecting group for the OH group.)
The compound represented by the general formula (5) is a compound corresponding to the above general formula (5a) or (5b) except that a protective group is introduced into the OH group. It selects from the compound corresponding to general formula (5a) or (5b) according to the three-dimensional structure of the target compound of general formula (6) or (7).

R ₁ and R ₂ in the general formulas (5) to (6) are preferably selected from a methyl group, an allyl group (—CH ₂ CH═CH ₂ ), and a cyanoethyl group. In that case, the demethylating agent can be selected from monoalkylamine, the deallylating agent can be selected from zerovalent palladium or NaI, and the decyanoethylating agent can be selected from trialkylamine, ammonia or diazabicycloundecene.

  (1-3) The method for producing a nucleotide derivative of the present invention includes a nucleoside derivative represented by the following general formula (9) in a nucleoside derivative represented by the following general formula (8) in which the configuration derived from an asymmetric phosphorus atom is controlled. And a step of synthesizing a nucleotide derivative represented by the following general formula (10).

(In formulas (8), (9) and (10), B ₁ and B ₂ are independently selected from nucleobases or derivatives thereof; Z is O, S or Se; R ₁ is partially hydrogenated. It is selected from an optionally substituted hydrocarbon group, Q is a protecting group for the OH group and can be independently selected.) The compound represented by the general formula (8) includes the aforementioned general formula (6) or ( By adopting the compound shown in 7), the configuration can be controlled.

  As a method for reacting the compound represented by the general formula (9) with the compound represented by the general formula (8), a known method such as a so-called Mitsunobu reaction can be employed.

(1-4) The method for producing the nucleotide derivative of the present invention comprises:
(A) (a-1) a step of producing a nucleoside derivative represented by the general formula (5a) and / or (5b) by the production method described in (1-1) above;
(A-2) Using the nucleoside derivative represented by the general formula (5a) and / or (5b) as the nucleoside derivative represented by the general formula (5), the production method according to the above (1-2), A step of producing a nucleoside derivative represented by the general formula (6) and / or (7);
(A-3) Using the nucleoside derivative represented by the general formula (6) and / or (7) as the nucleoside derivative represented by the general formula (8), the production method according to the above (1-3) Producing a nucleotide derivative represented by the general formula (10);
A step of synthesizing a nucleotide derivative with controlled steric configuration,
(B) (b-1) A nucleotide derivative represented by the general formula (10) obtained by reacting the nucleotide derivative represented by the general formula (10) synthesized in the nucleotide derivative synthesis step with a phosphoramidite derivative; Condensing between the phosphoramidite derivatives to produce amidated nucleotide derivatives;
(B-2) Nucleoside, nucleoside derivative (general formulas (1), (2), (3a), (3b), (4a), (4b), (5), (5a), (5b), (6 ), (7), (8) and (9) compound) or a nucleotide derivative addition step of reacting the nucleotide derivative synthesized in the nucleotide derivative synthesis step with the amidated nucleotide derivative,
A nucleotide derivative extending step to obtain a nucleotide derivative of a necessary length by repeating
It is characterized by having.

  In other words, by using the compound represented by the general formula (10) with controlled steric configuration as a starting material and applying the so-called amidite method proposed by the present inventors, it has the necessary length and steric structure. Nucleotide derivatives can be extended.

(2) Nucleoside Derivatives and Nucleotide Derivatives The present inventors have been able to obtain a number of useful novel compounds in developing the above-described methods for producing nucleoside derivatives and nucleotide derivatives.

  (2-1) The nucleoside derivative of the present invention is a nucleoside cyclic phosphite triester derivative represented by the following general formula (4a) and / or (4b).

(In formulas (4a) and (4b), B is selected from hydrogen, OH group, nucleobase or derivatives thereof; Z is S or Se.)
(2-2) The nucleoside derivative of the present invention is a compound represented by the following general formula (5a) and / or (5b).

(In the formulas (5a) and (5b), B is selected from hydrogen, OH group, nucleobase or derivative thereof; Z is S or Se; R ₁ and R ₂ are partially substituted with different hydrogens. Selected from hydrocarbon groups that may be present.)
(2-3) The nucleoside derivative of the present invention is a compound represented by the following general formula (11) in which the configuration derived from an asymmetric phosphorus atom is controlled.

(In the formula (11), B is selected from hydrogen, OH group, nucleobase or derivatives thereof; Z is S or Se; R ₁ is a hydrocarbon group which may be partially substituted with different hydrogens. Q is a protecting group for the OH group.)
(2-4) The nucleotide derivative of the present invention is a compound represented by the following general formula (10) in which the configuration derived from an asymmetric phosphorus atom is controlled.

(In formula (10), B ₁ and B ₂ are independently selected from nucleobases or derivatives thereof; Z is S or Se; R ₁ is a hydrocarbon group that may be partially substituted with hydrogen. Q is a protecting group for the OH group and can be independently selected.)

  According to the method for producing a nucleoside derivative and a nucleotide derivative of the present invention, a compound having a steric structure necessary as a configuration derived from an asymmetric phosphorus atom can be efficiently obtained. And since the nucleoside derivative and nucleotide derivative of the present invention have a controlled configuration derived from an asymmetric phosphorus atom, the resulting reaction product has high stereoselectivity when applied to asymmetric synthesis. Since these compounds themselves are also nucleoside (nucleotide) derivatives, it can be expected to have some pharmacological activity.

(1) Nucleoside derivative and nucleotide derivative and method for producing the same The nucleoside derivative and nucleotide derivative of the present invention and the method for producing the same will be described in detail below. The production method will be mainly described, and the nucleoside derivative and the nucleotide derivative will be described together with the production method.

  (1-1) The method for producing a nucleoside derivative of the present invention includes a first step and a second step.

  (A) In the first step, a deoxyribonucleoside derivative represented by the above general formula (1) and a phosphine derivative represented by the above general formula (2) are converted into an acid compound selected from an azole, an acid azole complex, and a carboxylic acid. This is a step of reacting in the presence to produce a nucleoside cyclic phosphite triester derivative represented by the above general formula (3a) and / or (3b).

  The atmosphere in which the reaction in the first step is performed is not particularly limited, but it is desirable to use an aprotic solvent. Acetonitrile and the like can be suitably employed from the viewpoint of cost and the like. In particular, it is preferable to employ anhydrous conditions, and it is desirable to employ some means (such as a dry box) for allowing the reaction to proceed in the presence of a dehydrating agent such as molecular sieves or preventing moisture from entering from the outside.

  The mixing ratio of each compound is not particularly limited, but the compound represented by the general formula (1) can be used with respect to the compound represented by the general formula (1) from the viewpoints of allowing the reaction to proceed more reliably and separating the impurities after the completion of the reaction. It is desirable that the addition amount of the compound and acid compound shown in 2) is slightly excessive.

  The reaction temperature is not particularly limited. It can be presumed that the reaction proceeds sufficiently even under mild conditions such as room temperature. In particular, a high temperature can be selected as long as inconveniences such as by-product formation do not become a problem. Here, after the completion of the first step, it has been found that the compound represented by the general formula (3b) is isomerized into the compound represented by the general formula (3a) by maintaining at a high temperature state, and the asymmetric The configuration derived from the phosphorus atom can be controlled. Although it changes with compounds to be used as high temperature, about 40 to 80 degreeC is preferable and about 50 to 60 degreeC is more preferable.

  In addition, when hold | maintaining in a high temperature state, it is possible to use the reaction product in a 1st process as it is, without performing separation, such as refinement | purification. In particular, it is presumed that isomerization is promoted by maintaining a high temperature in the presence of an acid compound.

  The compound represented by the general formula (1) is a compound having deoxyribose as a skeleton. In the above general formula (1), since it becomes complicated, it is not described, but each hydrogen atom of deoxyribose which is a skeleton can be substituted with any substituent. For example, the compound of the general formula (1) includes a compound represented by the following general formula (1 ′). Although omitted in the following structural formulas, a chemical structure represented by the general formula (1 ') can be introduced for the portion corresponding to the general formula (1).

(In the formula (1 ′), B is selected from hydrogen, OH group, nucleobase or derivative thereof; X ¹ is selected from hydrogen, halogen atom, OH group substituted with a protecting group, and alkoxy group having 1 to 6 carbon atoms. X ² is selected from a hydrogen atom, an alkyl group having 1 to 6 carbon atoms and an aromatic substituent, and the above alkoxy group, alkyl group, alkenyl group and C _n H _2n are In addition, those having a chain and a branch, and those in which a part of hydrogen atoms are substituted with halogen, and when X ¹ is an alkoxy group and X ² is an alkyl group, X ¹ and X ² are It may be bonded to form a ring.)
The substituent bonded as B is hydrogen, OH group, nucleobase or derivative thereof. Nucleobase includes naturally occurring bases and derivatives thereof (purine bases (adenine, guanine, etc.), pyrimidine bases (cytosine, thymine, uracil, etc.) and derivatives thereof. Of course, other bases that do not generally exist in nature can also be employed. In addition, since it is predicted that the same reaction will proceed even if deoxyribose having no base is employed, B may be a hydrogen or OH group. When used as a derivative), it is generally considered that a nucleobase is inserted at the position B.

The compound represented by the general formula (2) is a compound represented by R ₁ OPXY. R ₁ is a hydrocarbon group which may be partially substituted with hydrogen. For example, it is a hydrocarbon group that may be linear or branched, and includes an alkyl group, an alkenyl group, a phenyl group, a cyanoalkylene group, and the like. Specific examples include a methyl group, an allyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a phenyl group, and a cyanoethyl group.

X and Y are each independently selected from Cl, Br and -NRR 'which is a dialkyl-substituted amine. Here, R and R ′ are each independently selected from hydrocarbon groups that may be partially substituted with hydrogen. The hydrocarbon group can be selected from the same groups as R ₁ described above. Further, R and R ′ may be combined to form a ring.

  The acid compound is a compound selected from azoles, acid azole complexes and carboxylic acids. As azoles, 1H-tetrazole, benzimidazole, imidazole, 5- (p-nitrophenyl) -1H-tetrazole, 5- (ethylthio) -1H-tetrazole, 4,5-dicyanoimidazole, 2-bromo-4,5-dicyano Imidazolepyridine, 1-methylimidazole, 1-phenylimidazole, 1- (4-acetoxyphenyl) imidazole, 2-phenylimidazole, 4-phenylimidazole, 2-methylbenzimidazole, 2-phenylbenzimidazole, N-methylaniline, etc. Examples include alkylanilines and triazoles. Examples of acid azole complexes include benzimidazole-trifluoromethanesulfonic acid complex, benzimidazole-perchloric acid complex, benzimidazole-trifluoroacetic acid complex, benzimidazole-tetrafluoroboric acid complex, benzimidazole-hexafluoroline. Acid complex, imidazole-trifluoromethanesulfonic acid complex, imidazole-perchloric acid complex, imidazole-trifluoroacetic acid complex, N-methylaniline-trifluoroacetic acid complex, 1-phenylimidazole-trifluoromethanesulfonic acid complex And 1-phenylimidazole-perchloric acid complex, 1-methylimidazole-trifluoromethanesulfonic acid complex, 1-methylimidazole-perchloric acid complex, and pyridine-hydrochloric acid complex. Examples of carboxylic acids include monocarboxylic acids such as acetic acid, formic acid, propionic acid, butyric acid, and benzoic acid, and dicarboxylic acids such as maleic acid, phthalic acid, malonic acid, succinic acid, glutaric acid, phthalic acid, tartaric acid, and cyclohexanedicarboxylic acid. Can be illustrated. Particularly preferred acid compounds include 1H tetrazole, benzimidazole-trifluoromethanesulfonic acid complex, and imidazole-trifluoromethanesulfonic acid complex.

  (B) In the second step, the nucleoside cyclic phosphite triester derivative, which is a compound represented by the general formula (3a) and / or (3b), is reacted with a S agent or a Se agent, and the above general formula (4a) And / or a step of obtaining a nucleoside cyclic phosphite triester derivative shown in (4b).

  Here, the S agent (or Se agent) attacks the electron pair possessed by the phosphorus atom to form S (Se). Although it does not specifically limit as an S agent, bis [3- (triethoxysilyl) propyl] tetrasulfide (TEST), 3H-1,2-benzodithiol-3-one 1,1-dioxide, tetraethylthiuram disulfide, 2 4, bis (4-methoxyphenyl) -1,3-dithia-2,4-diphosphetane-2,4-disulfide, bisbenzenesulfonyl disulfide. Examples of the Se agent include selenium, diphenyl diselenide, and dibenzyl diselenide. The amount of the S agent or Se agent added is not particularly limited, and the reaction proceeds smoothly by adding a slightly excessive amount to the compound represented by the general formula (1).

  (C) The reaction is allowed to proceed using the nucleoside cyclic phosphite triester derivative represented by the general formula (4a) and / or (4b) as a starting material, so that the above general formula (5a) and / or (5b) Can be obtained.

As reaction conditions, R ₂ OH alcoholate was dissolved in a solution of R ₂ OH. Here, a compound represented by the general formula (4a) or (4b) is generated by introducing a substituent corresponding to R ₂ . As R ₂ , a substituent basically similar to R ₁ described above can be adopted, but a functional group different from R ₁ is selected from a request for identification to be performed later. Examples of the R ₂ OH alcoholate include those containing an alkali metal (such as sodium or potassium) as the metal.

Here, in the compounds represented by the general formulas (5a) and (5b), the corresponding groups in the general formulas (4a) and (4b) remain as they are for B, Z, and R ₁ , respectively.

  This reaction has very high stereoselectivity. From the compound represented by the general formula (4a), the nucleoside derivative represented by the general formula (5a) is represented by the general formula (5b). A nucleoside derivative is obtained. In addition, since the compound shown in the following general formula (12) is by-produced as a side reaction, these compounds are separated and purified.

(In the formula (12), B, Z, R ₁ and R ₂ are the substituents present in the nucleoside cyclic phosphite triester derivative represented by the general formula (4a) or (4b) as a starting material).
(1-2) The method for producing a nucleoside derivative of the present invention includes a de-R ₁ agent or a de-R ₂ agent in addition to the nucleoside derivative represented by the above general formula (5) in which the configuration derived from an asymmetric phosphorus atom is controlled. To obtain a compound of the above general formula (6) or (7) in which the configuration derived from the asymmetric phosphorus atom is controlled.

That is, the configuration derived from the asymmetric phosphorus atom is controlled by selecting the substituent to be eliminated. Therefore, it is desirable to employ a combination of R ₁ and R ₂ that can be desorbed freely without affecting each other.

For example, it is preferable to employ a combination of selecting any two combinations from a methyl group, an allyl group, a cyanoethyl group, and the like, and it is more preferable to employ a combination of a methyl group and an allyl group. In particular, it is desirable to employ an allyl group for R ₁ and a methyl group for R ₂ .

Examples of the demethylating agent include t-butylamine, methylamine, diethylamine, n-butylamine, and alkylamines such as phenylenediamine as preferable compounds. Desirable deallylating agents are zero-valent palladium such as Pd ₂ (dibenzylideneacetone) ₃ .CHCl ₃ and tetrakistriphenylphosphine palladium, and iodide ions such as NaI, potassium iodide and calcium iodide. Examples of the decyanoethyl agent include diazabicycloundecene, triethylamine, methylamine, diethylamine, dialkylethylamine and other trialkylamines, and aqueous ammonia.

For example, a compound represented by the formula (5 ′) in which an allyl group is employed as R _{1 and a} methyl group is employed as R ₂ , DMTr (dimethoxytrityl group) is employed as Z as S and Q (dextrorotation: this compound is the above reaction) Is a compound synthesized by the series following (4a) and (5a) from the general formula (3a) in (1), the compound (left-handed) shown in the formula (6 ′) is obtained by dearylation. When demethylation is performed, the compound (dextrorotatory) represented by the formula (7 ′) can be obtained quantitatively.

The compound represented by the general formula (B) (Z, R ₁ and R ₂ are the same groups as in the general formula (5); the portion represented by C can have any chemical structure). by reacting any of the de-R ₁ agents and de R ₂ agent, it is possible to control the configuration derived from the asymmetric phosphorus atom. For example, it can be applied to compounds (such as pharmaceuticals and agricultural chemicals) that contain an asymmetric phosphorus atom and the configuration is problematic, and the steric structure can be controlled.

  (1-3) The method for producing a nucleotide derivative of the present invention is represented by the above-described general formula (9) in the nucleoside derivative represented by the above general formula (8) in which the configuration derived from the asymmetric phosphorus atom is controlled. It comprises a step of reacting a nucleoside derivative to synthesize a nucleotide derivative represented by the above general formula (10).

  It is desirable to employ the so-called Mitsunobu reaction as this reaction. For example, an aprotic solvent such as THF is used as a solvent, and each is independently selected from diethyl azodicarboxylate and PRR′R ″ (R, R ′ and R ″ alkyl groups, alkenyl groups, phenyl groups, etc. The reaction is allowed to proceed in the presence of all phenyl groups. In this reaction, the Mitsunobu reaction is a reaction that preserves the configuration derived from the asymmetric phosphorus atom.

  Here, as the compound represented by the general formula (8), it is desirable to employ the compound represented by the above general formula (6) or (7) and maintain the steric configuration.

  In this reaction, as a side reaction, the position at which the compound represented by the general formula (9) is bonded to the compound represented by the general formula (8) was mediated by an oxygen atom in the compound represented by the general formula (10). , Which is bonded via a sulfur atom. Therefore, this by-product is removed as necessary.

(1-4) The method for producing the nucleotide derivative of the present invention comprises:
(A) A compound represented by general formula (10) having a target steric structure from a compound represented by general formula (1) by combining the production methods described in (1-1) to (1-3) above. After producing only the required amount,
(B) (b-1) reacting a phosphoramidite derivative with a nucleotide derivative in which the configuration shown in the general formula (10) is controlled to produce an amidated nucleotide derivative;
(B-2) Nucleoside, nucleoside derivative (general formulas (1), (2), (3a), (3b), (4a), (4b), (5), (5a), (5b), ( 6), a nucleotide derivative addition step of reacting the nucleotide derivative produced in (a) with the compound described in any of (7), (8) and (9), or the amidated nucleotide derivative;
And a nucleotide derivative extension step of obtaining a nucleotide derivative having a necessary length by repeating the above.

  Here, it does not specifically limit as a phosphoramidite derivative in (b-1), A general compound is employable. For example, the compound shown to the following general formula (13) is mentioned.

(In the formula (13), X ³ ~ ⁷ are each independently a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, a phenyl _{group, -C n H 2n CN, -Si} ( _{^{X 9) 3, -C n H}} 2n Si (X 9) 3, -C n H 2n OSi (X 9) 3 and _{-C n H 2n -S-Si (} X 9) is selected from _3; X ⁸ is Selected from —O—, —S— and —N (X ⁹ ) —; each of the aforementioned X ⁹ is independently selected from an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms and a phenyl group. In addition, the above-mentioned alkoxy group, alkyl group, alkenyl group and C _n H _2n include those having linear and branched groups and those in which a part of hydrogen atoms are substituted with halogen.
Here, X ⁸ is preferably an oxygen atom. X ⁵ is preferably selected in consideration of remaining in the nucleotide derivative finally produced.

  An amidite nucleotide derivative can be obtained by reacting a phosphoramidite derivative in the presence of an accelerator such as (1H-) tetrazole or a benzimidazole trifluoromethanesulfonic acid complex. In addition, the compound represented by the general formula (10) is reacted in a state in which Q (protecting group) of the lower OQ group (position 3 (position 5 may be higher if necessary)) is eliminated to form an OH group. Is.

Here, a compound represented by the following general formula (14) can be obtained by employing —N (X ⁹ ) — for X ⁸ .

(In the formula (14), X ¹⁰ to ¹³ correspond to X ³ to ⁷ and 9 of the compound represented by the general formula (13) which remain in the group remaining without leaving; the portion represented by A Represents a compound obtained by extending the length of the compound represented by the general formula (10) or the compound represented by the general formula (10))
By adopting the compound represented by the general formula (14) in place of the compound represented by the general formula (2) used in (1-1) and performing the subsequent reaction in the same manner, The configuration of the chiral phosphorus atom can also be controlled.

  Hereinafter, the nucleoside derivative and nucleotide derivative of the present invention and the production method thereof will be described based on experiments conducted in detail. In addition, the number given to each compound in the (Example) column is not related to the number given to each general formula.

(Stereoselective synthesis of deoxyribonucleoside-3 ', 5'-cyclic thiophosphate triester)
-The reaction of the stereoselective synthesis of deoxyribonucleoside-3 ', 5'-cyclic thiophosphate triester was carried out as follows (Scheme 2).

In the presence of molecular sieves 3A in acetonitrile, deoxyribonucleoside 1a and allyloxybis (diisopropylamino) phosphine are reacted with 1H-tetrazole as an accelerator to give nucleoside-3'-5'-cyclic phosphite triester 2a And a mixture of trans isomer 3a. The spectrum analysis of ³¹ P-NMR showed that the cis and trans isomers were almost 1: 1. After confirming the disappearance of allyloxybis (diisopropylamino) phosphine, the mixture was heated to 55 ° C. to cause thermal equilibrium isomerization between the cis isomer 2a and the trans isomer 3a. As a result, cis isomer 3a was obtained as the main product except for deoxyribonucleoside 1d in which no progress of the reaction was observed. Thereafter, it was sulfurized using bis [3- (triethoxysilyl) propyl] tetrasulfide (TEST) to obtain the desired deoxyribonucleoside-3 ′, 5′-cyclic thiophosphate triester 4a in a yield of 70% ( Table 1). Similarly, from deoxyribonucleoside 1b, 1c and 1e, the target deoxyribonucleoside-3 ′, 5′-cyclic thiophosphate triester 4b is obtained in 23% yield, 4c through cyclic phosphorylation, thermal equilibrium isomerization and sulfurization. Was obtained in a yield of 79%, and 4e in a yield of 22%.

Detailed Experimental Method Molecular Sieves 3A was heat-dried under vacuum at 250 ° C. for 3 hours and left to cool to room temperature. 1c (4.84 g, 20.0 mmol), 1H-tetrazole (4.20 g, 60.0 mmol) and acetonitrile (250 mL) were added thereto, and the mixture was stirred at room temperature for 30 minutes. A solution of allyloxybis (N, N-diisopropylamino) phosphine (7.67 mL, 24.0 mmol) in dichloromethane (25.0 mL) was added dropwise to the solution at 0 ° C., and the mixture was stirred for 30 minutes.

  After confirming disappearance of the raw materials, the reaction solution was stirred at 55 ° C. for 24 hours to carry out an isomerization reaction. Furthermore, it returned to room temperature, bis [3- (triethoxysilyl) propyl] tetrasulfide (11.8 mL, 24.0 mmol) was added, and it stirred at room temperature for 3 hours. After completion of the reaction, the reaction solution was filtered and concentrated.

  The residue was dissolved in ethyl acetate (200 mL), washed with a saturated aqueous sodium hydrogen carbonate solution (200 mL), and the aqueous layer was further washed twice with ethyl acetate (200 mL). The obtained organic layer was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered.

  The residue was subjected to silica gel column chromatography (developing solvent; ethyl acetate: hexane = 1: 2), and the fraction containing the target product was concentrated to give a colorless amorphous substance, yielding 5.68 g (79%) of 4c. It was.

4c; TLC: R _f = 0.41 (developing solvent; ethyl acetate: hexane = 1: 2); ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.96 (s, 3H), 2.53-2.64 (m, 2H) , 3.94 (dt, J = 4.8 and 10.0Hz, 1H), 4.45 (dt, J = 2.0 and 10.8Hz, 1H), 4.53-4.72 (m, 3H), 4.77-4.84 (m, 1H), 5.34 (dd , J = 0.8 and 10.4Hz, 1H), 5.45 (dd, J = 1.2 and 17.2Hz, 1H), 5.97-6.10 (m, 2H), 6.98 (d, J = 1.2Hz, 1H), 9.05 (br, 1H) ppm; ¹³ C-NMR (100 MHz, CDCl ₃ ): δ 12.5, 35.2 (d, J = 8.2 Hz), 69.2 (d, J = 4.2 Hz), 70.0 (d, J = 11.5 Hz), 73.9 (d, J = 7.4Hz), 77.2 (d, J = 8.2Hz), 86.1, 112.0, 119.4, 132.0 (d, J = 7.4Hz), 136.2, 149.8, 163.4ppm; ³¹ P-NMR (162MHz, CDCl ₃ ): δ 61.0ppm; HRMS (ESI ⁺ ): m / z calcd for C ₁₃ H ₁₇ N ₂ O ₆ PS (M + Na) ⁺ 383.04, found383.07.
4a, 4b and 4e were obtained in the same manner.

(2.2. Regioselective and stereoselective methanolysis of deoxyribonucleoside-3 ', 5'-cyclic thiophosphate triester)
Deoxyribonucleoside-3 ', 5'-cyclic thiophosphoric acid triester 4a was decomposed in methanol with sodium methoxide for 24 hours at room temperature, resulting in two mixtures (Scheme 3) .

  As a result of spectral analysis by NMR, it was found that the main product was 6a ring-opened on the 5 ′ side (yield 70%) and the by-product 7a ring-opened on the 3 ′ side (position) Selectivity 6a: 7a = 92: 8). Furthermore, 6a was found to be a stereochemically single compound (Table 2). Similarly, from deoxyribonucleoside-3 ′, 5′-cyclic thiophosphoric acid triesters 4b and 4c, 6b was obtained in a yield of 80% and 6c was obtained in a yield of 83% (regioselectivity 6b: 7b = 90: 10 6c: 7c = 90:10).

Detailed experimental method
4c (1.80 g, 5.00 mmol) was dissolved in methanol (50.0 mL), and a separately prepared solution of sodium (345 mg, 15.0 mmol) in methanol (50.0 mL) was added dropwise at 0 ° C. Then, it returned to room temperature and stirred for 24 hours. After completion of the reaction, ethyl acetate (500 mL) was added to the reaction solution, and after washing with a saturated aqueous ammonium chloride solution (100 mL), the aqueous layer was further washed twice with ethyl acetate (200 mL).

  Thereafter, the obtained organic layer was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, and filtered. The residue was subjected to silica gel column chromatography (developing solvent; ethyl acetate: hexane = 1: 2), and the fraction containing the target product was concentrated to give a colorless amorphous substance to obtain 1.63 g (83%) of 6c. It was.

6c; TLC: R _f = 0.37 (developing solvent; ethyl acetate: hexane = 1: 2); ¹ H-NMR (400 MHz, CDCl ₃ ): δ1.85 (s, 3H), 2.49 (dd, J = 4.4 and 7.2Hz, 1H), 2.58 (br, 1H), 3.78 (d, J = 13.6Hz, 3H), 3.89-3.97 (m, 1H), 4.24 (dd, J = 2.4 and 5.2Hz, 1H), 4.58 ( ddt, J = 1.2,5.6 and 10.4Hz, 1H), 5.18-5.23 (m, 1H), 5.29 (dd, J = 0.4 and 11.2Hz, 1H), 5.39 (ddd, J = 1.6,2.8 and 17.2Hz, 1H), 5.90-5.99 (m, 1H ), 6.20 (t, J = 7.2Hz, 1H), 7.37 (s, 1H), 8.61 (br, 1H) ppm; 13 C-NMR (100MHz, CDCl 3): δ12.5, 38.2 (d, J = 5.7Hz), 54.7 (d, J = 5.7Hz), 62.1, 69.1 (d, J = 4.9Hz), 78.2 (d, J = 4.9Hz), 85.5 (d, J = 5.7 Hz), 86.5, 111.3, 118.8, 132.1 (d, J = 7.4 Hz), 136.6, 150.4, 163.7 ppm; ³¹ P-NMR (162 MHz, CDCl ₃ ): δ 66.2 ppm; HRMS (ESI ⁺ ): m / z calcd for C ₁₄ H ₂₁ N ₂ O ₇ PS (M + Na) ⁺ 415.07, found415.09.
6a and 6b were obtained in the same way.

(Protection of the 5'-hydroxyl moiety of deoxyribonucleoside-3'-phosphorothioate)
Deoxyribonucleoside-3'-phosphorothioate 6a was stirred with 4,4'-dimethoxytrityl chloride in N, N-dimethylformamide, pyridine for 7 hours at room temperature (Scheme 4). As a result, the target dimethoxytritylated compound 8a was obtained in a yield of 58% (Table 3). In the same manner, 8c was obtained from deoxyribonucleoside-3'-phosphorothioate 6c in a yield of 91%. On the other hand, deoxyribonucleoside-3′-phosphorothioate 6b was stirred at 0 ° C. for 3 hours with 4,4′-dimethoxytrityl chloride, dichloroacetic acid and triethylamine in pyridine to obtain 8b in a yield of 52%.

Detailed experimental method Method 1: 6b (1.14 g, 3.02 mmol) was dissolved in pyridine (15.0 mL) and kept at 0 ° C., and dichloroacetic acid (0.27 mL, 3.32 mmol) and triethylamine (0.46 mL, 3.32 mmol) were added. In addition, 4,4′-dimethoxytrityl chloride (1.23 g, 3.62 mmol) was added in three portions. The reaction solution was allowed to stir at 0 ° C. for 3 hours.After completion of the reaction, dichloromethane (100 mL) was added to the reaction solution, and after washing with a saturated aqueous sodium hydrogen carbonate solution (30 mL), the aqueous layer was further washed twice with dichloromethane (50 mL). did.

  Thereafter, the obtained organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The residue was subjected to silica gel column chromatography (developing solvent; dichloromethane: methanol = 15: 1), and the fraction containing the target product was concentrated to give a colorless amorphous substance, yielding 1.06 g (52%) of 8b. .

8b; TLC: R _f = 0.50 (developing solvent; dichloromethane: methanol = 15: 1); ¹ H-NMR (400 MHz, CDCl ₃ ): δ2.26-2.32 (m, 1H), 2.77 (ddd, J = 3.2 , 5.6 and 14.0Hz, 1H), 3.46 (d, J = 3.2Hz, 2H), 3.66 (d, J = 14.0Hz, 3H), 3.80 (s, 6H), 4.28 (d, J = 3.2Hz, 1H ), 4.53-4.56 (m, 2H), 5.24-5.41 (m, 3H), 5.87-5.97 (m, 1H), 6.36 (t, J = 6.0Hz, 1H), 6.85 (d, J = 8.8Hz, 4H), 7.22-7.41 (m, 10H ), 7.87 (d, J = 7.6Hz, 1H) ppm; 13 C-NMR (100MHz, CDCl 3): δ40.1 (d, J = 4.9Hz), 54.5 ( d, J = 5.8Hz), 55.2, 62.6, 69.0 (d, J = 4.9Hz), 77.9 (d, J = 5.0Hz), 84.5 (d, J = 5.7Hz), 86.0, 87.0, 93.8, 113.3, 118.8, 127.0, 128.0, 128.2, 130.1, 132.1 (d, J = 7.4Hz), 135.2, 135.3, 141.3, 144.2, 158.7, 165.4ppm; 31 P-NMR (162MHz, CDCl 3): δ68.4ppm; HRMS ( ESI ⁺ ): m / z calcd for C ₃₄ H ₃₈ N ₃ O ₈ PS (M + Na) ⁺ 702.20, found 702.23.
Method 2: 6c (834 mg, 2.12 mmol) was dissolved in N, N-dimethylformamide (20.0 mL) and pyridine (4.00 mL), kept at 0 ° C., and 4,4′-dimethoxytrityl chloride (1.01 g, 2.97 mmol). ) Was added in three portions. The reaction solution was stirred at 0 ° C. for 30 minutes, then returned to room temperature and reacted for 7 hours. After completion of the reaction, ethyl acetate (100 mL) was added to the reaction solution and washed with a saturated aqueous sodium hydrogen carbonate solution (30 mL), and then the aqueous layer was further washed twice with ethyl acetate (100 mL).

  Thereafter, the obtained organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The residue was subjected to silica gel column chromatography (developing solvent; ethyl acetate: hexane = 1: 1), and the fraction containing the target product was concentrated to give a colorless amorphous substance, yielding 1.34 g (91%) of 8c. It was.

8c; TLC: R _f = 0.43 (developing solvent; ethyl acetate: hexane = 1: 1); ¹ H-NMR (400 MHz, CDCl ₃ ): δ1.42 (s, 3H), 2.37-2.43 (m, 1H) , 2.59-2.63 (m, 1H), 3.42-3.48 (m, 2H), 3.69 (d, J = 13.6Hz, 3H), 3.80 (s, 6H), 4.27 (d, J = 2.0Hz, 1H), 4.57 (dd, J = 5.6 and 10.4Hz, 2H), 5.27-5.40 (m, 3H), 5.88-5.97 (m, 1H), 6.44 (dd, J = 5.2 and 8.4Hz, 1H), 6.81-6.84 ( m, 4H), 7.22-7.38 (m, 9H), 7.56 (s, 1H), 8.25 (br, 1H) ppm; ¹³ C-NMR (100 MHz, CDCl ₃ ): δ11.7, 39.2 (d, J = 5.7Hz), 54.6 (d, J = 5.8Hz), 55.3, 63.3, 69.0 (d, J = 5.0Hz), 78.8 (d, J = 4.2Hz), 84.4, 84.7 (d, J = 5.0Hz), 87.2, 111.5, 113.4, 118.8, 127.2, 128.1 (x2), 130.1, 132.1 (d, J = 7.4Hz), 135.2 (x2), 144.2, 150.1, 163.3ppm; 31 P-NMR (162MHz, CDCl 3): δ68.5ppm; HRMS (ESI ⁺ ): m / z calcd for C ₃₅ H ₃₉ N ₂ O ₉ PS (M + Na) ⁺ 717.20, found 717.24.
8a was obtained in a similar manner.

(Synthesis of deoxyribonucleoside-3'-phosphorothioate diester by deallylation and demethylation of deoxyribonucleoside-3'-phosphorothioate)
First, demethylation of deoxyribonucleoside-3′-phosphorothioate 8a by maintaining the stereochemistry was examined (Scheme 5). Demethylribonucleoside-3′-phosphorothioate 8a was stirred in tert-butylamine at 35 ° C. for 24 hours to obtain demethylated deoxyribonucleoside-3′-phosphorothioate diester 9a in a yield of 95%. In the same manner, demethylation was carried out by retaining the stereochemistry of deoxyribonucleoside-3′-phosphorothioate 8b and 8c. As a result, 9b was obtained in 93% yield and 9c was obtained in 92% yield.

  Next, deallylation of deoxyribonucleoside-3′-phosphorothioate 8a by maintaining the stereochemistry was investigated (Scheme 5). Add deoxyribonucleoside-3'-phosphorothioate 8a in tetrahydrofuran with a catalytic amount of tris (dibenzylideneacetone) dipalladium / chloroform adduct and triphenylphosphine, stir, then allow n-butylamine and formic acid to act, stir at room temperature for 2 hours As a result, deallylated deoxyribonucleoside-3′-phosphorothioate diester 10a was obtained in a yield of 80%. In the same manner, deallylation of deoxyribonucleoside-3′-phosphorothioate 8b and 8c by retaining the stereochemistry was carried out. As a result, 10b was obtained with a yield of 62% and 10c with a yield of 88%. As a result, we succeeded in synthesizing two nucleoside-3'-phosphorothioate diesters with pure stereochemistry.

Detailed Experimental Method Demethylation: 8c (1.00 g, 1.44 mmol) was dissolved in tert-butylamine (25.0 mL) and reacted at 35 ° C. for 24 hours. After completion of the reaction, the reaction solution was concentrated, and the residue was dissolved in dichloromethane (20 mL) and washed twice with 0.1 M aqueous triethylammonium hydrogencarbonate (10 mL).

  Thereafter, the obtained organic layer was dried over anhydrous magnesium sulfate and filtered. The residue was subjected to silica gel column chromatography (developing solvent; dichloromethane: methanol = 9: 1), and the fraction containing the target product was concentrated, further dissolved in dichloromethane (20 mL), and 0.1 M aqueous triethylammonium hydrogen carbonate solution (10 mL). And washed twice.

  Thereafter, the obtained organic layer was dried over anhydrous magnesium sulfate and filtered. The residue was concentrated, dissolved in dichloromethane (1.0 mL), and pulverized with petroleum ether (200 mL) to give a colorless amorphous substance to obtain 1.04 g (92%) of 9c.

9c; TLC: R _f = 0.37 (developing solvent; dichloromethane: methanol = 9: 1); ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 1.16 (t, J = 7.2 Hz, 9H), 1.34 ( s, 3H), 2.24-2.40 (m, 2H), 3.05-3.15 (m, 7H), 3.27-3.30 (m, 1H), 3.74 (s, 6H), 4.16-4.28 (m, 3H), 5.03 ( dd, J = 1.6 and 10.4Hz, 2H), 5.19 (ddd, J = 2.0,4.0 and 17.2Hz, 1H), 5.82-5.91 (m, 1H), 6.20 (dd, J = 5.6 and 8.4Hz, 1H) , 6.87-6.89 (m, 4H), 7.22-7.39 (m, 9H), 9.23 (br, 1H), 11.31 (s, 1H) ppm; ¹³ C-NMR (100 MHz, DMSO-d ₆ ): δ 8.56 , 11.5, 39.5 (d, J = 3.3Hz), 45.6, 55.2, 64.0, 67.0 (d, J = 5.8Hz), 77.2 (d, J = 4.2Hz), 84.7, 85.6 (d, J = 5.8Hz) , 87.0, 111.1, 113.3, 116.2, 127.0, 128.0, 128.2, 130.2, 134.6 (d, J = 9.0 Hz), 135.4, 135.6, 135.9, 144.4, 150.4, 163.7 ppm; ³¹ P-NMR (162 MHz, DMSO-d _{6): δ52.8ppm; HRMS (ESI} -): m / z calcd for C 40 H 52 N 3 O 9 PS (MH-Et 3 N) - 679.19, found679.27.
9a and 9b were obtained in the same way.

  Deallylation: 8c (69.5 mg, 100 mmol) dissolved in tetrahydrofuran (1.00 mL), tris (dibenzylideneacetone) dipalladium / chloroform adduct (5.18 mg, 5.00 mmol), triphenylphosphine (6.56 mg, 25.0 mmol) ) And stirred at room temperature for 30 minutes. Thereafter, the reaction solution was kept at 0 ° C., n-butylamine (19.0 mL, 200 mmol) and formic acid (7.50 mL, 200 mmol) were added dropwise, and the mixture was returned to room temperature and stirred for 2 hours.

  After completion of the reaction, the reaction solution was concentrated, and the residue was dissolved in dichloromethane (5.0 mL) and washed twice with 0.1 M aqueous triethylammonium hydrogencarbonate (3.0 mL). Thereafter, the obtained organic layer was dried over anhydrous magnesium sulfate and filtered. The residue was subjected to silica gel column chromatography (developing solvent; dichloromethane: methanol = 9: 1), and the fraction containing the target product was concentrated, further dissolved in dichloromethane (5.0 mL), and 0.1 M aqueous triethylammonium hydrogencarbonate (3.0%). 2) mL).

  Thereafter, the obtained organic layer was dried over anhydrous magnesium sulfate and filtered. The residue was concentrated, dissolved in dichloromethane (1.0 mL), and pulverized with petroleum ether (100 mL) to give a colorless amorphous substance to obtain 66.5 mg (88%) of 10c.

10c; TLC: R _f = 0.21 (developing solvent; dichloromethane: methanol = 9: 1); ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 1.16 (t, J = 7.2 Hz, 9H), 1.36 ( s, 3H), 2.24-2.38 (m, 2H), 3.06 (q, J = 7.2Hz, 6H), 3.14 (dd, J = 2.8 and 10.4Hz, 1H), 3.28-3.30 (m, 1H), 3.73 (s, 6H), 4.09-4.11 (m, 1H), 4.97-5.04 (m, 1H), 6.17 (dd, J = 6.0 and 8.4Hz, 1H), 6.87-6.89 (m, 4H), 7.21-7.47 (m, 9H), 9.47 (br, 1H), 11.31 (s, 1H) ppm; ¹³ C-NMR (100 MHz, DMSO-d ₆ ): δ 8.62, 11.6, 39.8 (d, J = 4.1 Hz), 45.7, 52.9 (d, J = 6.5Hz), 55.2, 63.9, 76.5 (d, J = 4.9Hz), 84.7, 85.1 (d, J = 5.8Hz), 87.0, 111.1, 113.3, 127.0, 127.9, 128.2, 130.1, 135.4, 135.6, 135.8, 144.4, 150.4, 163.8ppm; 31 P-NMR (162MHz, DMSO-d 6): δ54.3ppm; HRMS (ESI -): m / z calcd for C 38 H 50 N 3 O _{9 PS (MH-Et 3 N} ) - 653.17, found653.26.
10a and 10b were obtained in the same way.

(Stereoselective synthesis of dinucleoside phosphorothioate based on Mitsunobu reaction of nucleoside-3'-phosphorothioate diester and 5'-O-unprotected nucleoside)
A stereoselective synthesis of dinucleoside phosphorothioate based on Mitsunobu reaction of nucleoside-3'-phosphorothioate diester and 5'-O-unprotected nucleoside was investigated (Scheme 6).

  The Mitsunobu reagent was prepared by stirring diethyl azocarboxylate (DEAD) and triphenylphosphine in tetrahydrofuran at 0 ° C. After that, nucleoside-3'-phosphorothioate diester 9a and 5'-O-unprotected nucleoside 11a were added and stirred at 55 ° C for 3 hours. The nucleoside phosphorothioate 12a was obtained with a yield of 32%. Although the reaction proceeded with high stereoselectivity, the functional group selectivity (O / S selectivity) was low (Table 4). As a result of examining the stereoselective synthesis of dinucleoside phosphorothioates using the same method, 12b yield 59%, 12c yield 27%, 12d yield 25%, 12e yield 53%, and 12f yield. Yield was 50%.

Furthermore, 9c is a nucleoside-3'-phosphorothioate diester 10a having a different stereochemistry,
A stereoselective synthesis of dinucleoside phosphorothioates based on the Mitsunobu reaction of 5'-O-unprotected nucleoside 11a was investigated (Scheme 7).

  Although the reaction proceeded with high stereoselectivity, the functional group selectivity (O / S selectivity) was low (Table 5). As a result of examining the stereoselective synthesis of dinucleoside phosphorothioates by the same method, 14b was obtained in a yield of 20%, 14c was obtained in a yield of 51%, and 14d was obtained in a yield of 15%. However, for 14d, the yield could be improved to 62% by changing the number of equivalents of the condensing agent.

Detailed experimental method Triphenylphosphine (202 mg, 770 mmol) was dissolved in tetrahydrofuran (12.0 mL), the reaction solution was kept at 0 ° C., diethyl azocarboxylate 40% toluene solution (350 mL, 770 mmol) was added dropwise, 30 Stir for minutes.

9c (460 mg, 590 mmol) and 11d (335 mg, 1.18 mmol) were added to the solution and stirred for a while, and then the reaction solution was kept at 60 ° C. and stirred for 3 hours. After completion of the reaction, yields of 12f, 13f and other compounds were converted by ³¹ P-NMR spectral analysis. Then, ethyl acetate (50 mL) was added to the reaction solution, and after washing with a saturated aqueous ammonium chloride solution (20 mL), the aqueous layer was further washed twice with ethyl acetate (50 mL).

  Thereafter, the obtained organic layer was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and filtered. The residue was subjected to silica gel column chromatography (developing solvent; dichloromethane: methanol = 9: 1, ethyl acetate: hexane: methanol = 3: 1: 0.1), and the fraction containing the target product was concentrated to give a colorless amorphous substance. As a result, 279 mg (50%) of 12f was obtained.

12f; TLC: R _f = 0.43 (developing solvent; ethyl acetate: hexane: methanol = 3: 1: 0.1); ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.47 (s, 3H), 1.94 (s, 3H), 2.10-2.19 (m, 4H), 2.35-2.44 (m, 2H), 2.60 (dd, J = 5.6 and 12.8Hz, 1H), 3.46 (d, J = 2.4Hz, 2H), 3.80 (s , 6H), 4.13-4.35 (m, 4H), 4.60 (dd, J = 5.6 and 10.8Hz, 2H), 5.21-5.41 (m, 4H), 5.88-5.97 (m, 1H), 6.36 (dd, J = 5.6 and 9.2Hz, 1H), 6.41 (dd, J = 5.2 and 8.4Hz, 1H), 6.85 (d, J = 8.8Hz, 4H), 7.22-7.40 (m, 10H), 7.58 (s, 1H) , 8.81 (s, 1H), 8.85 (s, 1H) ppm; ¹³ C-NMR (100 MHz, CDCl ₃ ): δ 11.7, 12.5, 21.0, 37.0, 39.0 (d, J = 5.8 Hz), 55.2, 63.2 , 67.4 (d, J = 6.6Hz), 69.3 (d, J = 4.9Hz), 74.3, 79.5 (d, J = 4.1Hz), 82.5 (d, J = 9.0Hz), 84.4, 84.5, 84.6 (d , J = 5.8Hz), 87.2, 111.6, 111.8, 113.3, 119.4, 127.1, 128.0 (x2), 130.0, 131.6 (d, J = 7.4Hz), 133.0, 134.8, 135.0, 135.1, 144.1, 150.4, 163.5, 163.7, 170.3 ppm; ³¹ P-NMR (162 MHz, CDCl ₃ ): Δ67.0 ppm; 12a, 12b, 12c, 12d, 12e, 14a, 14b, 14c and 14d were obtained in the same manner.

(Removal and phosphoramidite formation of dinucleoside phosphorothioate-3'-hydroxyl protecting group)
The removal (deacetylation) of the dinucleoside phosphorothioate-3'-hydroxyl protecting group was investigated (Scheme 8 and 9).

  Dinucleoside phosphorothioate 12f was stirred with sodium methoxide in methanol at room temperature for 4 hours. As a result, deacetylated dinucleoside phosphorothioate 16 was obtained in a yield of 88%. In the same manner, 17 was obtained in a yield of 84%.

  Next, phosphoramidite formation of the obtained dinucleoside phosphorothioates 16 and 17 was examined (Scheme 10 and 11).

  In the presence of Molecular Sieves 3A in acetonitrile, dinucleoside phosphorothioate 16 and allyloxybis (diisopropylamino) phosphine were reacted with diisopropylammonium tetrazolide as an accelerator and stirred for 3 hours at room temperature, resulting in the desired phosphoramidite. 18 was obtained in 71% yield. Further, 19 was obtained in a similar manner using methyloxybis (diisopropylamino) phosphine in a yield of 75%.

Detailed Experimental Method Removal of protecting group: 12f (103 mg, 109 mmol) was dissolved in methanol (4.00 mL), and 1.0 M sodium methoxide methanol solution (55.0 mL, 55.0 mmol) prepared separately was added dropwise at 0 ° C. Then, it returned to room temperature and stirred for 4 hours. After completion of the reaction, ethyl acetate (20 mL) was added to the reaction solution, and after washing with a saturated aqueous ammonium chloride solution (5.0 mL), the aqueous layer was further washed twice with ethyl acetate (20 mL).

  Thereafter, the obtained organic layer was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The residue was subjected to silica gel column chromatography (developing solvent; dichloromethane: methanol = 9: 1), and the fraction containing the target product was concentrated to give a colorless amorphous substance to obtain 86.8 mg (88%) of 16. .

16; TLC: R _f = 0.53 (developing solvent; dichloromethane: methanol = 9: 1); ¹ H-NMR (400 MHz, CDCl ₃ ): δ1.48 (s, 3H), 1.92 (s, 3H), 2.10- 2.17 (m, 1H), 2.35-2.45 (m, 2H), 2.60 (dd, J = 5.6 and 13.2Hz, 1H), 3.46 (d, J = 2.4Hz, 2H), 3.80 (s, 6H), 4.08 -4.32 (m, 4H), 4.46 (t, J = 2.8Hz, 1H), 4.59 (dd, J = 5.6 and 10.8Hz, 2H), 5.26-5.40 (m, 3H), 5.87-5.97 (m, 1H ), 6.32 (t, J = 6.4Hz, 1H), 6.41 (dd, J = 5.2 and 8.8Hz, 1H), 6.85 (d, J = 9.2Hz, 4H), 7.22-7.40 (m, 10H), 7.56 (d, J = 1.2Hz, 1H), 9.36 (s, 1H), 9.53 (s, 1H) ppm; ¹³ C-NMR (100 MHz, CDCl ₃ ): δ11.8, 12.6, 39.0 (d, J = 4.9 Hz), 40.1, 55.3, 63.4, 67.6 (d, J = 6.6 Hz), 69.3 (d, J = 4.1 Hz), 71.3, 79.6 (d, J = 4.9 Hz), 84.5, 84.6 (d, J = 8.2 Hz), 84.7 (d, J = 4.1 Hz), 85.1, 87.3, 111.5, 111.9, 113.4, 119.3, 127.2, 128.0, 128.1, 130.1, 131.8 (d, J = 7.4 Hz), 132.1, 135.1, 135.2, 135.6 , 144.2, 150.7, 150.9, 164.0, 164.2 ppm; ³¹ P-NMR (162 MHz, CDCl ₃ ): δ 65.7 ppm;
17 was obtained in a similar manner.

  Phosphoramididation: Molecular sieves 3A was heat-dried under vacuum at 250 ° C. for 3 hours and left to cool to room temperature. 16 (49.0 mg, 54.1 mmol), diisopropylammonium-1H-tetrazolide (18.5 mg, 108 mmol) and acetonitrile (1.00 mL) were added thereto, and the mixture was stirred at room temperature for 30 minutes. To the solution, allyloxybis (N, N-diisopropylamino) phosphine (25.9 mL, 81.1 mmol) was added dropwise and allowed to stir for 2 hours. After completion of the reaction, the reaction solution was filtered and concentrated. The residue was dissolved in ethyl acetate (10 mL), washed with saturated aqueous sodium hydrogen carbonate solution (5.0 mL), and the aqueous layer was further washed twice with ethyl acetate (10 mL). Thereafter, the obtained organic layer was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and filtered. The residue was concentrated, dissolved in dichloromethane (1.0 mL), and pulverized with petroleum ether (100 mL) to give a colorless amorphous substance to obtain 18 (42.0 mg, 71%).

18; TLC: R _f = 0.39 (developing solvent; ethyl acetate: hexane = 2: 1); ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.16 (d, J = 6.8 Hz, 12H), 1.43 (s , 3H), 1.91 (s, 3H), 2.05-2.14 (m, 1H), 2.36-2.49 (m, 2H), 2.56-2.61 (m, 1H), 3.40-3.47 (m, 2H), 3.55-3.63 (m, 2H), 3.79 (s, 6H), 4.06-4.24 (m, 6H), 4.45-4.61 (m, 3H), 5.12 (d, J = 10.4Hz, 1H), 5.23-5.39 (m, 4H ), 5.87-5.96 (m, 2H), 6.31 (t, J = 6.4Hz, 1H), 6.41 (t, J = 7.2Hz, 1H), 6.84 (d, J = 8.8Hz, 4H), 7.22-7.40 (m, 10H), 7.58 (s, 1H), 8.92 (br, 2H) ppm; ¹³ C-NMR (100 MHz, CDCl ₃ ): δ 11.7, 12.6, 24.5 (d, J = 6.6 Hz), 24.6 ( d, J = 8.3Hz), 39.2 (d, J = 5.0Hz), 39.5, 43.2 (d, J = 12.3Hz), 55.3, 63.4, 64.3 (d, J = 17.3Hz), 67.3 (d, J = 6.0Hz), 69.3 (d, J = 6.6Hz), 72.8 (d, J = 1.7Hz), 73.0, 79.5 (d, J = 3.3Hz), 84.4, 84.8 (d, J = 5.8Hz), 85.0 ( d, J = 7.4Hz), 87.3, 111.4, 111.7, 113.4, 115.9 (d, J = 13.2Hz), 119.3, 127.2, 128.0, 128.1, 130.0, 131.7 (d, J = 2.5Hz), 131.8 (d, J = 3.3Hz), 135.1, 135.2, 135.3, 135.4, 144.4, 150.4, 150.5, 150.9, 163.9 (x2) ppm; ³¹ P-NMR (162 MHz, CDCl ₃ ): δ 67.4, 147.5, 147.9 ppm.
19 was obtained in a similar manner.

(Stereoselective synthesis of phosphorothioate / phosphate mixed oligonucleotides using dinucleoside phosphorothioate-3'-phosphoramidite as a building block (liquid phase synthesis))
A phosphorothioate / phosphate mixed oligonucleotide was synthesized using dinucleoside phosphorothioate-3′-phosphoramidite 18 as a building block (Scheme 12).

Dinucleoside phosphorothioate-3′-phosphoramidite 18 and 5′-O-unprotected nucleoside 11d were reacted in acetonitrile in the presence of molecular sieves 3A for 1 hour at room temperature with 1H-tetrazole as a promoter. The disappearance of the starting amidite was confirmed by ³¹ P-NMR spectral analysis, and then oxidized with a t-butylhydroxyperoxide-toluene solution to obtain the target 20 in a yield of 70%. In a similar manner, dinucleoside phosphorothioate-3′-phosphoramidite 19 to 21 was obtained in 65% yield (Scheme 13).

Detailed Experimental Method Molecular Sieves 3A was heat-dried under vacuum at 250 ° C. for 3 hours and left to cool to room temperature. 18 (70.5 mg, 64.5 mmol), 1H-tetrazole (9.00 mg, 129 mmol) and acetonitrile (1.30 mL) were added thereto, and the mixture was stirred at room temperature for 30 minutes. 11d (27.5 mg, 96.8 mmol) was added to the solution and allowed to stir at room temperature for 1 hour. After confirming disappearance of the starting amidite, it was oxidized with 4.0Mt-butylhydroxyperoxide-toluene solution (47.8 mL, 194 mmol) to obtain 58.4 mg (70%) of the target 20.

20; TLC: R _f = 0.60 (developing solvent; ethyl acetate: hexane: methanol = 3: 1: 0.5); ¹ H-NMR (400 MHz, CDCl ₃ ): δ1.44-1.46 (m, 3H), 1.87- 1.94 (m, 6H), 2.10 (s, 3H), 2.21-2.31 (m, 2H), 2.37-2.45 (m, 2H), 2.51-2.64 (m, 2H), 3.38-3.79 (m, 2H), 3.80 (s, 6H), 4.15-4.37 (m, 6H), 4.58-4.61 (m, 4H), 5.06 (t, J = 6.0Hz, 1H), 5.24-5.41 (m, 7H), 5.88-5.99 ( m, 2H), 6.16-6.43 (m, 3H), 6.85 (d, J = 8.8Hz, 4H), 7.22-7.42 (m, 11H), 7.56-7.58 (m, 1H), 9.11-9.37 (m, 3H) ppm; ¹³ C-NMR (100 MHz, CDCl ₃ ): δ 11.7 (x2), 12.5 (x2), 36.9, 38.3 (x2), 39.0 (x3), 55.2, 63.4 (x2), 67.0, 67.1, 67.5 (x2), 68.9, 69.0 (x2), 69.4, 69.5, 73.9, 77.2, 77.9, 79.8 (x2), 79.9, 82.5, 82.6, 83.1, 83.2, 84.4, 84.7, 84.8, 84.9 (x2), 85.5, 87.3, 111.7 (x2), 111.8 (x2), 113.4, 119.4, 119.5, 119.6, 127.2, 128.0, 128.1, 130.0, 131.6, 131.7 (x2), 131.8, 135.0, 135.1 (x2), 135.3, 144.1, 150.3, 150.4, 150.5, 150.6 (x2), 150.7, 158.7, 158.8, 163.7 (x2), 163.8, 170.5, 170.6 ppm; ³¹ P-NMR (162 MHz, CDCl ₃ ): δ-2.64, -2.27, 66.7, 66.9 ppm;
21 was obtained in a similar manner.

(Stereoselective synthesis of phosphorothioate / phosphate mixed oligonucleotides using dinucleoside phosphorothioate-3'-phosphoramidite as a building block (solid phase synthesis))
Stereoselective synthesis of phosphorothioate / phosphate mixed oligonucleotides in a solid phase reaction was performed (Scheme 14). Synthesis of thymidine phosphorothioate 15-mer 22 using dinucleoside phosphorothioate-3 '-(S) -phosphoramidite 18 as a building block on a solid support (CPG (Controlled Pore Glass)) benzimidazolium trifluoromethanesulfonate The result using (BIT) as an accelerator is shown. The chain length extension was performed by a series of reaction operations shown in Table 6 on a model 392 DNA / RNA Synthesizer automatic solid phase synthesizer manufactured by Applied Biosystems.

  After the end of chain extension, deallylation (catalytic amount of tris (dibenzylideneacetone) dipalladium / chloroform adduct and triphenylphosphine in tetrahydrofuran was added, stirred, and then reacted with n-butylamine and formic acid at 50 ° C. After removing the protecting group of the phosphoric acid moiety under the conditions of stirring for a long time, treatment with concentrated aqueous ammonia (25 ° C, 2 hours) gave phosphorothioate 15mer 22 in 97% yield (overall). It was. By purification by HPLC, stereochemically pure phosphorothioate 15-mer 22 with the purity shown in Fig. 1 was obtained.

Detailed Experimental Method Solid phase synthesis was performed using an Applied Biosystems model 392DNA / RNASynthesizer automatic solid phase synthesizer. Use 0.1M dinucleoside phosphorothioate-3'-phosphoramidite / acetonitrile solution, 0.1MBIT / acetonitrile solution, 1.0MTBHP / toluene solution, capping agent (acetic anhydride / tetrahydrofuran solution, N-methylimidazole / tetrahydrofuran solution) Then, the solid phase carrier was 0.5 mmol. Deprotection was carried out by adding a catalytic amount of tris (dibenzylideneacetone) dipalladium / chloroform adduct and triphenylphosphine in tetrahydrofuran, followed by reaction with n-butylamine and formic acid, and stirring at 50 ° C. for 12 hours. The cutting was performed using concentrated aqueous ammonia at room temperature for 2 hours, and then concentrated aqueous ammonia was distilled off by freeze-centrifugation to obtain thymidine phosphorothioate 15-mer 22 as a white solid.

^{22; HRMS (ESI -):} m / z calcd for C 150 H 196 N 30 O 96 P 14 S 7 (M-3H) - 1536.20, found1536.19.

  The nucleoside derivatives and nucleotide derivatives of the present invention are expected to exhibit various pharmacological activities on living organisms. In particular, since the three-dimensional structure is controlled, expression of a specific pharmacological activity can be expected.

  Therefore, the method for producing nucleoside derivatives and nucleotide derivatives is also a method capable of obtaining useful compounds with high selectivity, and thus contributes to the production and search for novel pharmacologically active substances.

Claims (11)

Deoxyribonucleoside derivative represented by the following general formula (1) and general formula (2): R ₁ OPXY (R ₁ is a hydrocarbon group which may be partially substituted with hydrogen. X and Y are Cl, Br, a dialkyl-substituted amine —NRR ′ (R and R ′ are each independently selected from a hydrocarbon group which may be partially substituted with hydrogen. R and R ′ together form a ring. And a phosphine derivative represented by the following formula) is reacted in the presence of an acid compound selected from an azole, an acid azole complex and a carboxylic acid. A first step of producing the nucleoside cyclic phosphite triester derivative shown in 3a) and / or (3b);
A second step of obtaining a nucleoside cyclic phosphite triester derivative represented by the following general formula (4a) and / or (4b) by reacting the nucleoside cyclic phosphite triester derivative with a S agent or Se agent: A process for producing a nucleoside derivative characterized by comprising:

(In the formulas (1), (3a), (3b), (4a) and (4b), B is selected from hydrogen, an OH group, a nucleobase or a derivative thereof; R ₁ is R in the general formula (2) ₁ is the same group as Z; Z is O, S or Se.)   Between the first step and the second step, a mixture of the nucleoside cyclic phosphite triester derivative represented by the general formulas (3a) and (3b) is maintained at a high temperature for a predetermined time, and the general formula (3b The method for producing a nucleoside derivative according to claim 1, further comprising a thermal equilibrium isomerization step of isomerizing the nucleoside cyclic phosphite triester derivative represented by formula (3) into the nucleoside cyclic phosphite triester derivative represented by formula (3a). In the nucleoside cyclic phosphite triester derivative represented by the general formula (4a) and / or (4b), in an R ₂ OH solution (R ₂ is a hydrocarbon group which may be partially substituted with hydrogen, The nucleoside derivative represented by the following general formula (5a) and / or (5b) is obtained by reacting an alcoholate of R ₂ OH with a group different from R ₁ . A method for producing a nucleoside derivative.

(In the formulas (5a) and (5b), B is the same group as B explained in the general formula (1); R ₁ is the same group as R ₁ explained in the general formula (2). R ₂ is a hydrocarbon group which may be partially substituted with hydrogen and is a group different from R ₁ ; Z in the formula (5a) is the same as Z in the general formula (4a) Z in formula (5b) is the same group as Z in formula (4b).) A nucleoside derivative represented by the following general formula configuration is controlled (5) derived from the asymmetric phosphorus atom, configuration derived from the asymmetric phosphorus atom by reacting the de-R _1, or de-R ₂ agent is controlled A method for producing a nucleoside derivative, comprising obtaining a nucleoside derivative represented by the following general formula (6) or (7):

(In the formulas (5) to (7), B is selected from hydrogen, OH group, nucleobase or derivatives thereof; Z is O, S or Se; R ₁ and R ₂ are partially substituted with different hydrogens. Selected from hydrocarbon groups which may be substituted; Q is a protecting group for the OH group.) R ₁ and R ₂ in the general formulas (5) to (6) are selected from a methyl group, an allyl group, and a cyanoethyl group,
The nucleoside derivative according to claim 4, wherein the demethylating agent is a monoalkylamine, the deallylating agent is zero-valent palladium or NaI, and the decyanoethylating agent is a trialkylamine, ammonia or diazabicycloundecene. Production method. The nucleoside derivative represented by the following general formula (8) in which the configuration derived from the asymmetric phosphorus atom is controlled is reacted with the nucleoside derivative represented by the following general formula (9) to obtain a nucleotide derivative represented by the following general formula (10). A method for producing a nucleotide derivative, comprising a step of synthesizing.

(In formulas (8), (9) and (10), B ₁ and B ₂ are independently selected from nucleobases or derivatives thereof; Z is O, S or Se; R ₁ is partially hydrogenated. (Selected from an optionally substituted hydrocarbon group, Q is a protecting group for the OH group and can be independently selected.) A step of producing a nucleoside derivative represented by the general formula (5a) and / or (5b) by the production method according to claim 3;
Using the nucleoside derivative represented by the general formula (5a) and / or (5b) as the nucleoside derivative represented by the general formula (5), the production method according to claim 4 or 5, wherein the general formula (6) and / Or the step of producing the nucleoside derivative shown in (7),
Using the nucleoside derivative represented by the general formula (6) and / or (7) as the nucleoside derivative represented by the general formula (8), the production method according to claim 6 shows the formula (10). Producing a nucleotide derivative;
A step of synthesizing a nucleotide derivative with controlled steric configuration,
The nucleotide derivative represented by the general formula (10) synthesized in the nucleotide derivative synthesizing step is reacted with a phosphoramidite derivative so that the gap between the nucleotide derivative represented by the general formula (10) and the phosphoramidite derivative is obtained. Condensing to produce an amidated nucleotide derivative;
Nucleosides, nucleoside derivatives (general formulas (1), (2), (3a), (3b), (4a), (4b), (5), (5a), (5b), (6), (7 ), A compound according to any one of (8) and (9)) or a nucleotide derivative addition step in which the nucleotide derivative synthesized in the nucleotide derivative synthesis step is reacted with the amidated nucleotide derivative;
A nucleotide derivative extending step to obtain a nucleotide derivative of a necessary length by repeating
A method for producing a nucleotide derivative having A nucleoside derivative which is a nucleoside cyclic phosphite triester derivative represented by the following general formula (4a) and / or (4b).

(In formulas (4a) and (4b), B is selected from hydrogen, OH group, nucleobase or derivatives thereof; Z is S or Se.) A nucleoside derivative represented by the following general formula (5a) and / or (5b).

(In the formulas (5a) and (5b), B is selected from hydrogen, OH group, nucleobase or derivative thereof; Z is S or Se; R ₁ and R ₂ are partially substituted with different hydrogens. Selected from hydrocarbon groups that may be present.) A nucleoside derivative represented by the following general formula (11) in which the configuration derived from an asymmetric phosphorus atom is controlled.

(In the formula (11), B is selected from hydrogen, OH group, nucleobase or derivatives thereof; Z is S or Se; R ₁ is a hydrocarbon group which may be partially substituted with different hydrogens. Q is a protecting group for the OH group.) A nucleotide derivative represented by the following general formula (10) in which the configuration derived from an asymmetric phosphorus atom is controlled.

(In formula (10), B ₁ and B ₂ are independently selected from nucleobases or derivatives thereof; Z is S or Se; R ₁ is a hydrocarbon group that may be partially substituted with hydrogen. Q is a protecting group for the OH group and can be independently selected.)