WO2005092909A1 - Processes for producing ribonucleotide analogue with high stereoregularity and deoxyribonucleotide analogue - Google Patents

Abstract

A process for producing a ribonucleotide analogue having high stereoregularity. The process, which is for producing a ribonucleotide analogue having high stereoregularity represented by the formula (IV) or (V), is characterized by condensing an optically active nucleoside 3'-phosphoroamidite with a nucleoside with the aid of an activator and then subjecting the condensate to sulfurization and protective-group elimination. [In the formulae, Y+ represents C1-3 linear or branched alkyl, etc.; and Bs represents urasil, etc., provided that the two Bs's in each formula may be the same or different.]

Description

Method for producing ribonucleotide analogs and deoxyribonucleotide analogs with high stereoregularity

 The present invention relates to a method for producing a ribonucleotide analog having high stereoregularity and an oligodoxyribonucleotide analog. Background art

 Phosphate-site modified dinucleotides have recently attracted attention as an important antisense drug (antisense method), and I. Clinical trials are also being conducted on many diseases. The antisense method is a method in which a nucleic acid having a nucleotide sequence complementary to a target mRNA is selectively bound to the mRNA to inhibit protein translation. The properties required as an antisense molecule include (1) the ability to recognize and specifically bind to the base sequence of the target mRNA, (2) the ability to form a stable duplex, and (3) the nuclease. (4) high cell membrane permeability.

 Phosphate moiety-modified dinucleotides have an asymmetric center on the phosphorus atom and differ in their antisense effects due to differences in their absolute configuration. In recent studies in vitro, the properties of phosphate-modified dinucleotides, such as their ability to form duplexes with DNA and RNA, nuclease resistance, and RNase H activity, are affected by chirality on the phosphorus atom. (Med. Chem. Lett.

2000, 8, 275-284), there is a need for an efficient method for producing phosphate-modified oligonucleotides with controlled stereochemistry on the phosphorus atom.

 Conventionally, phosphate site-modified dinucleotides have been produced by the phosphoramidite method or the like (Beaucage,

S 丄.; Iyer, RP Tetrahedron, 1992, 48, 2223- 2311). Since it was difficult to control the stereochemistry on the thiol atom, the produced phosphate-modified oligonucleotide was a mixture of R and S diastereomers. In addition, when natural type DNA is used as an antisense molecule, it is easily hydrolyzed by nucleases and has a fatal problem as an antisense molecule having low cell membrane permeability. Attempts have been made to overcome these problems by making various modifications to the native DMA. Among them, DNA analogs in which two non-bridging oxygen atoms on the phosphorus atom of natural DMA internucleotides are variously substituted, that is, inter-nucleotide modified analogs, have both nuclease resistance and cell membrane permeability. It is known to increase (Lev in, AA B i ochem.

Biophys. Acta., 1 999, 1489 (1), 69-84.).

 However, if the introduced substituents are different, the DNA analog will have chirality on the phosphorus atom. It is known that the properties and functions of these DNA relatives differ depending on the chirality (Yu, D .; Kanduma lla, ER; Rosky, A .; Zhao, Q .; Chen, J .; Agrawal , S. Bioorg. Med. Ghem., 2000, 8, 275-284.)). For example, phosphorothioate DNA, an internucleotide-modified DNA analog in which one of two non-bridging oxygen atoms has been replaced with a sulfur atom, has a double-stranded structure that forms with complementary RNA, a nuclease. It is known that the resistance to ze hydrolysis differs between the Sp-oligomers and the Rp-oligomers (see the above literature). For this reason, it is extremely important to obtain an internucleotide-modified DNA analog with a controlled steric phosphorus atom in order to enhance its efficacy as a drug.

H-phosphonate DNA is a DNA analog in which one of the two non-bridging oxygen atoms on the phosphorus atom of the internucleotide of natural DNA is replaced with a hydrogen atom. Having. In addition, it can be converted into various inter-nucleotide-modified DMA analogs by a stereospecific conversion reaction. Thus, if stereochemically pure H-phosphonate DNA is obtained, it will be possible to obtain an internucleotide-modified DMA analog whose stereochemistry is controlled as it is, using the DNA as it is. As described above, H-phosphonate DNA has various three-dimensionally controlled proteins. It is a useful synthetic intermediate that can be converted into a single nucleotide modified DNA analog. To date, there has been no other method of obtaining stereochemically pure H-phosphonate DMA except by optically resolving the diastereomer by silica gel column chromatography. Therefore, stereochemically pure H-phosphonate DMA has only been reported at the dimer level ((a) See la, F .; Kretschner, UJ Org. Chem. 1991, 56, 3861-3869. (B) Loshmer, T .; Engels, JW Nucleic Acids Res. 1990, 18, 5143). Moreover, even when the dimer of H-phosphonate DNA is optically resolved, H-phosphonate DNA is unstable on silica gel column chromatography, and there is a polar difference between the two diastereomers. Since there is no significant difference, the optical division is extremely inefficient. Considering stereochemically pure H-phosphonate DNA as a synthetic intermediate that can be applied to nucleic acid medicine, H-phosphonate DNA with a steric control at the oligomer level is required. In that case, the number of diastereomers increases exponentially, making optical resolution of H-phosphonate DMA oligomers virtually impossible. Therefore, if a stereoselective synthesis reaction of H-phosphonate DMA can be developed, it will be possible to obtain H-phosphonate DMA with steric control at the oligomer level.

RNAi was first reported in 1998 in a study using nematodes by Fire and Mel lo (Fire, A .; Xu, S .; Montgomery, Μ. Κ .; Kostas, SA; Driver, SE; Mel lo , CG Nature. 1998, 391, 806-811.), It has been revealed that it is a gene silencing system that is conventionally provided among various species such as insects, plants, and fungi. . In 2001, Tuschl et al. Showed that RNAi was applicable to mammalian cells (Elbashir, SM; Harborth, J .; Lendeckel,.; Yale in, A .; Weber, K .; Tuschl, T. Nature. 2001, 411, 494-498.). As a result, RNAi has been attracting attention as a powerful method for gene therapy and gene function analysis, as an excellent gene suppression method with high gene suppression effect. RNAi is characterized by its sequence specificity, which can knock out the target gene accurately, and the use of gene silencing mechanisms inherent in living organisms. This makes RNAi a promising new gene therapy with fewer side effects. ing. However, the effect of RNAi cannot be maintained for a long time at present, because RNA strands of about 21 bases such as siRNA are gradually degraded by nucleases in the living body. Disclosure of the invention

 An object of the present invention is to provide a method for producing a ribonucleotide analog and a deoxysilipnucleotide analog having a high stereoregularity, which can be used for the antisense method or RNA interference and has a controlled stereo structure on a phosphorus atom. Is to do.

 The present invention provides a compound represented by the following general formula (I):

[Wherein, R ¹ and R ′ may be the same or different and represent a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or an aryl group having 6 to 14 carbon atoms,

R ² and R "may be the same or different and represent a hydrogen atom, an alkyl group having 1 to 3 carbon atoms or an aryl group having 6 to 14 carbon atoms;

R ³ represents an alkyl group having 1 to 3 carbon atoms,

R ⁴ is a hydroxyl-protecting group, is OR ⁵ (where R ⁵ is a hydroxyl-protecting group), a hydroxyl group or a hydrogen atom,

 B s is

Ο

Represents a group derived from peracyl, adenine, cytosine, guanine, thymine or a derivative thereof represented by. However, R ² and R ³ may form a monocyclo structure or a bicyclo structure together with the nitrogen atom. ]

An optically active nucleoside 3'-phosphoramidite represented by

 General formula (II)

[Wherein, R ^e and B s have the same meaning as described above. ]

And a nucleoside represented by

 General formula (III)

[Wherein, X— is BF ₄ —, PF ₆ —, T f O— (T f is CF ₃ S 0 ₂ —; the same applies hereinafter), T f ₂ N \ A s F ₆ — or S b F ₆ — is indicated. The cyclic structure A represents a monocyclo or bicyclo structure having 3 to 16 carbon atoms formed together with a nitrogen atom. ]

After condensation with an activator represented by the formula (1), followed by reaction with an electrophile and deprotection, characterized by high stereoregularity represented by the formula (IV) or (V): A method for producing a nucleotide analog, and a method for producing an oligoribonucleotide analog and an oligodoxyribonucleotide analog having high stereoregularity are provided.

[In each formula, Y is a straight-chain or branched-chain alkyl group having 1 to 10 carbon atoms, a straight-chain or branched-chain alkoxy group having 1 to 10 carbon atoms, a straight-chain or branched-chain alkoxy group having 1 to 10 carbon atoms, Branched hydroxyalkyl group, aryl group having 6 to 14 carbon atoms, alkylthio group having 1 to 10 carbon atoms, acyl group having 1 to 10 carbon atoms, amino group, alkyler having 1 to 10 carbon atoms amino group, a dialkylamino group having 1 to 1 0 carbon atoms, or Upsilon = Upsilon 'shows the Zeta + (Upsilon' is S one, S e-, BH ₃ - a, Z + is ammonium Niu-ion, primary, secondary Represents quaternary lower alkyl ammonium ions or monovalent metal ions). B s has the same meaning as described above, and two B s in each formula may be the same or different. D ₂ and E ₂ represent a hydroxyl group or a hydrogen atom. Detailed description of the invention

Hereinafter, the production method of the present invention will be described by dividing into a condensation reaction (first reaction step), a reaction with an electrophile and a deprotection reaction (second reaction step). The first and second divisions are for convenience of explanation only and are not limiting. If necessary, known processing steps such as purification processing can be added.

 (First reaction step)

 An optically active nucleoside 3'-phosphoramidite represented by the general formula (I) [hereinafter referred to as "phosphoramidite (I)"] and a nucleoside represented by the general formula (II) [hereinafter referred to as "nucleoside (II)"] Is condensed in the presence of an activating agent represented by the general formula (III) [hereinafter referred to as “activating agent (III)”].

 The phosphoramidite (I) can be produced from an appropriate 1,2-amino alcohol by a known method as described below (for example, see Tetrahedron: Asy et al. 1995, 6, 1051-1054).

 That is, the general formula (VII) obtained by reacting an optically active 1,2-amino alcohol (hereinafter referred to as “amino alcohol (VI)”) represented by the general formula (VI) with phosphorus trichloride is used. It can be obtained by reacting the optically active phosphitylating agent represented by [hereinafter referred to as “phosphitylating agent (VII) J”] with the nucleoside represented by the general formula (VIII).

[In the formula, R ¹ , R ² , R ³ , RD, and B s have the same meaning as in the general formula (1). ] The amino alcohols (VI) include (S)-and (R) -2-methylamino-1-phenylethanol, (1R, 2S) -ephedrine, (1R, 2S) 1-2-methylamino-1 , 2-diph Xnylethanol and the like.

 In addition, prolinol derivatives, for example, (Of R, 2S)-(pyrrolidine-12-yl) benzyl alcohol, (aS, 2R)-(pyrrolidine-12-yl) benzyl alcohol Amino alcohols that can be converted to H-phosphonates, such as (S) -a, of-diphenyl (pyrrolidine-1-yl) methanol and (2S) -monomethyl (pyrrolidine-l-2-yl) ethanol Ethyl, (2R) -bimethyl (pyrrolidine-1-yl) ethanol, (oiR, 2S)-monomethyl (pyrrolidine-12-yl) benzyl alcohol, (S, 2R)-monomethyl (Pyrrolidine-1-yl) In nucleosides (VIII) including benzyl alcohol, Bs represents a group derived from peracyl, adenine, cytosine, guanine or thymine or a derivative thereof. The body, adenine, also, and the like of protected with a protecting group to the amino group of cytosine and Guanin, and specific examples thereof include compounds represented by the following formula.

[Wherein, R ⁷ has the same meaning as above, and R ⁸ represents an alkyl group having 1 to 15 carbon atoms, an aryl group, an aralkyl group, an aryloxyalkyl group, among which a methyl group, an isopropyl group, A phenyl group, a benzyl group and a phenoxymethyl group are preferred, and a phenyl group is particularly preferred. R ⁹ and R ^1C) each represent an alkyl group having 1 to 4 carbon atoms, and a methyl group is particularly preferable. R "represents a protecting group at position 06 of guanine, preferably 2-cyanoethyl group, p-nitrophenylethyl group, phenylsulfonylethyl group, benzyl group, 2-trimethylsilylethyl group or the like.

Nucleoside (VIII) is Urashiru, adenosine, which was protected cytidine, guanosine, the hydroxyl group of thymine or 5 'position of their derivatives, the protecting group (R ^4), tert - butyl diphenyl silyl group (TBDPS) Alkylsilyl groups such as tert-butyldimethylsilyl group (TBDMS), trityl groups such as 4,4'-dimethyoxytrityl group (DMT r) and 4-methoxytrityl group (MMT r), and a protecting group represented by the following formula: And the like.

When is in the nucleoside (VIII) is a hydrogen atom, Bs is preferably thymine or a derivative thereof. When using a nucleoside other than thymine or a derivative thereof, it is desirable to introduce a protecting group into the base, since side reactions to the base may be feared. Adenine and guanine can use the phenoxyacetyl (Pac) group, and cytosine can use the isobutyl (iBu) group.

In the phosphoramidite (I), the meanings of R ¹ , R,, R ² and R "are as described above.

As R ¹ and R ² , ^one of R ¹ and R ² is a hydrogen atom and the other is a phenyl group, ^one of R ¹ and R ² is a methyl group and the other is a phenyl group, or R ¹ and R ² R ² is preferably a combination of phenyl groups, R ¹ is a phenyl group, and R ² is more preferably a combination of hydrogen atoms. R ³ is preferably a methyl group. Further, it is preferable that R ¹ forms a phenyl group and R ² and R ³ form a pyrrolidine skeleton together with a nitrogen atom. When R ⁴ and R ⁵ are OR ⁵ , R ⁵ is preferably TBDP S or TBDMS, and more preferably TBD PS.

When R ¹ and R ² are the above combination, R ′ and R ″ can be selected from a hydrogen atom, an alkyl group having 1 to 3 carbon atoms or an aryl group having 6 to 14 carbon atoms. In the optically active nucleoside 3′-phosphoroamidite represented by (I), R ¹ and R ′ may be the same or different and each may be an alkyl group having 1 to 3 carbon atoms or an alkyl group having 6 to 14 carbon atoms. When an H-phosphonate (Y-H in the general formula (XII)) is obtained, both R ¹ and R, are not hydrogen atoms (that is, R ¹ and R ′ are The carbon atom to be bonded is a tertiary carbon), and the substituent of the tertiary carbon does not include an aryl group.

 Nucleoside (II) protects the hydroxyl groups at the 2- and 3-positions of peridine, adenosine, cytidine, guanosine or their derivatives, and peracyl, adenine, cytosine, guanine, thymine or their derivatives represented by Bs The group derived from is exemplified by the nucleoside (VIII).

 The Bs of the nucleoside (II) and the nucleoside (VI) may be the same or different.

R ⁶ is the same as above, and when E, is 10 R ⁷ , the protecting group for the hydroxyl group represented by R ⁷ is TBDPS, TBDMS, acetyl group (Ac), phenoxy Asechiru group (PA c), benzyl group (B z), DMT r, MM Ding r etc. mentioned et been, R ^6, R ⁷ is PA c is preferred.

 The activator (III) has a capability of supplying a proton to the nitrogen atom of the phosphoramidite (I) and does not act as a nucleophile.

In the activator (III), X— is preferably BF ₄ —, PF ₆ —, T f O—, or T f ₂ N—. The cyclic structure A represents a monocyclo or bicyclo structure having 3 to 16 carbon atoms formed with a nitrogen atom, and particularly preferably has a monocyclo structure represented by the formula (IU-1).

(Wherein, X— has the same meaning as described above. N represents a number of 3 to 7, preferably 4 or 5.)

 The activator (III) has the formula (IX)

(In the formula, the cyclic structure A has the same meaning as described above.)

And the following formula (X):

 HX (X) (wherein, X has the above meaning.)

Can be easily obtained by reacting with the compound represented by

 Since the activator (III) exhibits particularly good solubility in acetonitrile, the reaction between the phosphoamidite (I) and the nucleoside (M) is preferably carried out in a solvent such as acetonitrile.

Phosphoramidite (I) and nucleoside (||) are reacted with nucleoside (II) at a ratio of 5 to 1.0 equivalent times to phosphoramidite (I). Preferably. Activator (III) reacts with phosphoramidite (I)

It is preferable to use 1 to 5 equivalent times. The reaction temperature is preferably 0 to 40 ° C, and the reaction pressure is preferably 1 atm.

 By the above first reaction step, the following general formula (XI)

[Wherein, RR ² , R ³ , RR ⁶ , D ,, E and B s have the same meaning as described above. ]

[Hereinafter referred to as “phosphite (XI)”].

 (Second reaction step)

 First, the phosphite (XI) obtained in the first reaction step is acylated with acetic anhydride, trifluoroacetic anhydride or the like, and then reacted with an electrophilic reagent such as a sulfurizing agent, a selenating agent, or a boranolating agent. Then, the asymmetric auxiliary group of the compound of the general formula (XII) is treated with 1,8-diazabicyclo [5.4.0] pendecar 7-ene (DBU) and the like to remove the compound.

E _1¾ B s and Y have the same meaning as described above. ] To obtain a protected diphosphate site-modified dinucleotide represented by Depending on the type of electrophile used (for example, 1,2,4-dithiazolidine-1,3,5-dione, 3-ethoxy-1,2,4-dithiazoline-5-one, 3-methyl In the case of using 1,2,4-dithiazoline-15-one or the like), the preceding acylation step may be omitted.

Finally, it is possible to obtain a highly stereoregular liponucleotide analog represented by the general formula (IV) or (V) by removing the hydroxyl-protecting group with (CH ₃ CH ₂ ) ₃ N 3 HF or the like. it can.

 Further, in the present invention, by repeating the first reaction step and the second reaction step, an oligomer represented by the general formula (XIII) [hereinafter referred to as “oligomer”

 (XI I I) ").

The carbon to which R ^{1 of the} monomer represented by the general formula (I) is bonded is a tertiary carbon (both R ¹ and R ′ are not hydrogen atoms), and the substituent of the tertiary carbon is When the phosphite (XI) obtained in the first reaction step does not contain an aryl group, the phosphite (XI) obtained in the first reaction step is acylated with anhydrous acetic acid, trifluoroacetic anhydride, or the like, and then is acidified with an acid such as a 1% trifluoroacetic acid dichloromethane solution of methane. Upon treatment, the chiral auxiliary is removed to give the corresponding H-phosphonate (XI then Y = H). When one or more of the tertiary carbon substituents is an aryl group, the acylation step can be omitted in the second reaction step, but in order to reduce the carbocation formed, It is necessary to add a reducing agent such as triethylsilane or borane-pyridine complex.

When a dimer is synthesized by this method, a phosphorothioate (XI and Y = S ") is obtained by reacting the obtained H-phosphonate (XI and Y = H) with a sulfurizing agent. By reacting the carbon tetrachloride solution, phosphoramidate (XI then Y = NR ₂ ) can be obtained.

When an oligomer is synthesized by this method, a monomer having a DMTr group as a protecting group for the 5 ′ hydroxyl group is used, and the phosphite intermediate obtained by the above method is subjected to an acid treatment to form an asymmetric auxiliary group and a 5 ′ hydroxyl group. The protecting group, DMTr, is removed at the same time, and the resulting dimer having a hydroxyl group at the 5'-position is condensed with a monomer. By repeating the above steps, an oligomer having a 3 H-phosphonate bond can be synthesized.

 Next, an oligomer having an H-phosphonate bond is subjected to a conversion reaction in the same manner as in the case of a dimer, and is guided to a desired phosphorus atom-modified DNA, followed by deprotection, whereby a target nucleic acid analog is obtained. Obtainable.

In the general formula (XIII), n represents an integer of 1 to 150, a preferred range is 5 to 50, a more preferred range is 10 to 30, and a still more preferred range is 15 to 22. D ₂ and E ₂ represent a hydroxyl group or a hydrogen atom.

 The oligomer (XII I) can be produced by applying an oligomer synthesis method by a solid phase method. Specifically, a commercially available automatic synthesizer (Expedite, manufactured by ABI, or ABI Model 394, DNA / RNA Synthesizer ABI) Or a manual method using a solid-phase synthesis vessel equipped with a glass filter. Solid-phase carriers used in the solid-phase method include aminoalkylated porous glass (control led pore glass: CPG) and aminoalkylated highly cross-linked polystyrene (HCP). ) Is preferred, and a polymer carrier that is as swellable as possible and can easily remove excess reagents by washing.

Either the 3 'or 2' hydroxyl groups of the ribonucleoside and the solid support are bound via a linker such as succinate, oxalate, or phthalate. May be. Protecting groups for 2 'or 3' hydroxyl groups to which the solid phase carrier is not bound include acetyl, benzoyl, 2- (cyanoethoxy) ethyl, t-butyldimethylsilyl, and other RNA and DNA synthesis groups. The protecting groups used can be mentioned.

 The highly stereoregular ribonucleotide analogs and deoxyribonucleotide derivatives obtained by the production method of the present invention can be used for antisense and RNA interference, which are one of the methods that have attracted attention in the field of gene therapy. Can be used.

 According to the present invention, ribonucleotide analogs and deoxyribonucleotide analogs having high stereoregularity and effective as antisense molecules can be obtained in high yield. Example

 The percentages in the examples are mass% unless otherwise specified.

 In the formulas and tables, dr and d. Indicate diastereomeric ratios, rt indicates room temperature, equiv and eq indicate equivalents, TFA indicates trifluoroacetic acid, and Py indicates pyridine.

 <Production example of activator (III)>

 Production Example 1-1: Production of N-cyanomethylpyrrolidinium tetrafluoroborate

 Under an argon atmosphere, a solution of 0.551 g (5.00 mmol) of N-cyanomethylpyrrolidine in ethyl ether (5.OO ml) was cooled to 178 ° C and stirred while stirring to give a 54% ethyl ether solution of boron tetrafluoride. 0.689 ml (5.0 Ommol) was added dropwise. After returning the solution to room temperature, it was concentrated under reduced pressure and dried.

(5 ml) was added and stirred vigorously, and the solvent was removed using a syringe. After repeating this washing operation 5 times, vacuum drying is carried out, and the target substance [activator of general formula (III), _n = 4, X— = BF ₄ —] 0.990 g (5.00 ol) Got. Yield quantitative. White powder. Large deliquescence.

 -Melting point: 1 13.0 ~ 11.4 ° C

, IR (KB r) _x : 2988, 2950, 2825, 2527, 2445, 1451, 1407, 1374, 1298, 1119, 929 cm " ¹

^ H—NMR (300MHz, CD ₃ CN) δ: 7.17 (br, 1H), 4.30 (s, 2H), 3.51 (br, 4H), 2.13 to 2.08 (m, 4H)

■ ¹³ C—NMR (75 MHz, CD ₃ CN) δ: 112.0, 55.9, 41.8, 23.2.

 Production Example 1-2: Production of Ν-cyanomethylpyrrolidinium hexafluorophosphate

6 To 1% hexafluorophosphoric acid aqueous solution (1.20 g, 5.0 O mmol) was added 5.0 Oml of water, and while stirring, 0.51 g (5.00 ol) of N-cyanomethylpyrrolidine was added dropwise. Later, the solution was lyophilized. Ethyl ether (1 Oml) was added to the residue, vigorously <stirred, and the solvent was removed using a syringe. After repeating this washing operation three times, the product is dried under vacuum, and the desired product (in the general formula (III), n = 4, an activator of X-12 PF ₆ —) 1.28 g (5. Obtained. Yield quantitative. White powder. Large deliquescence.

 • Melting point: 56.0-5 ° C

-IR (KBr): 2988, 2828, 2532, 2448, 1626, 1457, 1296, 1082, 987, 834 cm- ¹

' ¹ Η— NMR (300MHz, CD ₃ CN) δ:, 8.27 (br, 1H), 4.24 (s, 2H), 3.48 (br, 4H), 2.12〜2.08 (m, 4H)

■ ¹³ C—NMR (75 MHz, CD ₃ CN) δ: 112.1, 56.0, 42.1, 23.5

■ ³¹ P-NMR (121 MHz, CD ₃ CN) δ: -146.0 (septet, ¹ J _PF = 707 Hz) ₀ Production example 1-3: N-cyanomethylpyrrolidinium trifluoromethanesulfone Production of birds

A solution of 0.51 g (5.0 Omtnol) of N-cyanomethylpyrrolidine in 5.0 ml of dichloromethane is cooled to 0 ° C, and 0.442 ml of trifluoromethanesulphonic acid is added with stirring. 5. O 2 mmol) was added dropwise, and then ethyl ether (10 ml) was added. The resulting solid was collected by suction filtration, washed with ethyl ether (1 ml X 3), dried under reduced pressure, and the target compound (in general formula (III), n = 4, X- = T f O— activation Agent] 1.1 1 _g (4.27 mmol) was obtained. Yield 85. Roh _0. White powder. Deliquescence small.

 -Melting point: 67.0 ~ 67.5 ° C

■ IR (KBr): 2996, 2841, 2651, 2477, 2347, 2282, 1637, 1462, 1437, 1269, 1228, 1168, 1033, 985, 911, 849, 761, 641 cm— ¹

■ ¹ H-NMR (300 MHz, CD ₃ CN) δ: 8.16 (br, 1H), 4.30 (s, 2H), 3.50 (br, 4H), 2.14 to 209 (m, 4H)

■ ¹³ C—NMR (75 MHz, CD ₃ CN) δ: 121.2 (q, ¹ J _CF = 320 Hz), 55.9, 42.0, 23.5.

 Production Examples 1-4: Production of Ν-cyanomethylbiperidinium tetrafluoroborate

To a solution of 1.24 g (10.Ommol) of Ν-cyanomethylbiperidine in dichloromethane (10.Oml), with stirring, a 54% solution of ethyl tetrafluoroboronate ether solution 1.38ml (10.Ommol) ) Was added dropwise. The solution was diluted with ethyl ether (20 ml), and the resulting solid was collected by suction filtration, washed with ethyl ether (10 mix 2), dried under reduced pressure, and dried under reduced pressure. = 5, X— = BF ₄ —activator] 2.01 g (9.48 mmol) was obtained. Yield 95%. White powder. No deliquescence. .

 'Melting point: 103.0 ~ 103.5 ° C

■ IR (KB r) y _mx 3149, 2997, 2952, .2876, 2591, 2570, 2491, 2372, 1457, 1422, 1296, 1074, 980, 935, 850, 641 cm " ¹

^-1 H-NMR (300MHz, CD ₃ CN) δ 6.74 (br, 1H), 4.22 (s, 2H), 3.58 (br, 2H), 3.15 (br, 2H), 1.97 ~ 1.51 (m, 6H)

^{- 13 C-NMR (75 MHz} , CD 3 CN) δ: 111.2, 54.6, 44.0, 23.0, 20.5. Production Examples 1-5: Production of di-cyanomethylbiperidinium hexafluorophosphate

6 Add 1 5.20 g (5.0 Ommol) of 1% aqueous solution of hexafluorophosphoric acid to 5.0 Oml of water, and add 0.621 g of N-cyanomethylbiperidine (5.00 ol) with stirring. After dropwise addition, the solution was freeze-dried. Dichloromethane (5mi) Ethyl ether (10 ml) was added, and the mixture was cooled to 178 ° C and vigorously stirred to produce a solid. After the temperature was raised to room temperature, the solvent was removed using a syringe. Ethyl ether (5 ml) was added to the residue, stirred vigorously, and the solvent was removed using a syringe. After repeating this washing operation three times, vacuum drying is performed, and the target substance (general formula

(III), n = 5, X— = PF ₆ —Activator] 1.31 g (4.85 mmol) was obtained. 97% yield. White powder. Large deliquescence.

 • Insect point: 54.0-55.0 ° C

-IR (KBr) v _mm : 2997, 2953, 2876, 2589, 2570, 2490, 2372, 1655, 1455, 1422, 1297, 1192. 1142, 1084, 1037, 981, 953, 837, 746 cm ^-1

^-1 H-NMR (30 OMHz, CD ₃ CN) δ: 7.94 (br, 1H), 4.15 (s, 2H), 3.31 (br, 4H), 1.92 to 1.83 (m, 4H), 1.63 (br, 2H )

^{- 13 C- NMR (75MHz, CD} 3 CN) δ 111.5, 54.5, 44.2, 23.1, 20.8

■ ³¹ P-NMR (121 MHz, CD ₃ CN) δ: -145.9 (septet, ¹ J _PF = 707 Hz). Production Examples 1-6: N-Cyanomethylbiperidinium trifluoromethanesulfone

— Production of birds

 A solution of 0.621 g (5.0 圆 ol) of N-cyanomethylbiperidine in dichloromethane (5.0 Oml) was cooled to 0 ° C and stirred with 0.1 ml of trifluoromethanesulfonate. 442 ml (5.0 O mmol) were added dropwise. After the solution was warmed to room temperature, ethyl ether (1 Oml) was added, the solid was collected by suction filtration, washed with ethyl ether (1 ml x 3), dried under reduced pressure, and dried under reduced pressure. In the formula (III), n = 5, X— = T f O—activator] T. 37 g (5.0 O mmol) was obtained. Yield quantitative. White powder. Deliquescence small.

 -Melting point: 1 10.0 ~ 1 10.5 ° C

_{■ IR (KB r) v mx} : 2999, 2723, 1460, 1289, 1226, 1168, 1083, 1027, 978, 936, 762, 641 cm one ¹

' ¹ Η-NMR (300MHz, CD ₃ CN) δ 8.12 (br, 1H), 4.19 (s, 2H), 3.58 (br, 2H), 3.09 (br, 2H), 2.21 (br, 4H), 1.50 ( br, 1H)

, ¹³ C—NMR (75 MHz, CD ₃ CN) 6: 120.9 (q, ¹ J _CF = 319 Hz), 111.4, 54.5, 44.2, 23.0, 20.7.

 <Production of phosphitylating agent (VII)>

 Preparation Example 2-1: Preparation of (5S) —2-chloro-3-methyl-5-phenyl-1,3,2-oxazaphospholidine

 A solution of 3.02 g (15.0 mmol) of (S) -2-methylamino-1-phenylethanol and 5.58 ml of triethylamine (40.Ommol) in tetrahydrofuran (THF) (20.Oml) was brought to 0 ° C. To a cooled solution of 1.75 ml (20. Ommol) of phosphorus trichloride in THF (20. Oml) was added dropwise with stirring, and the mixture was stirred at room temperature for 30 minutes. The resulting salt was filtered through a glass filter under an argon atmosphere, and the salt was washed with THF (1 Oml X 3).

The filtrate is concentrated, and the residue is distilled under reduced pressure to give the desired product [5S-form compound of the general formula (VII) in which R ¹ -phenyl group, R ² = H, R ³ = methyl group] 2 59 g (1 2. Ommol) were obtained. Yield 60%. 89 ~ 90 ° CZ0.2 stroke H go Colorless transparent liquid.

■ ¹ H-NMR (300 MHz, CDC I ₃ ) δ: 7.54-7.34 (m, 5Η), 5.83,

5.44 (br.br, 1H), 3.60-3,42 (m, 1H), 3.22-312 (m, 1H),

• ³¹ P-NMR (121 MHz, CDC I ₃ ) δ ■■ 172.4 (br), 171.3 (br).

 Production Example 2-2: Production of (5 R) —2-chloro-1--3-methyl-1-phenyl-1,1,3,2-oxazaphospholidine

(R)-2-methylamino-1-phenylpropyl ethanol 2. using 27 g (1 5. 0 kingdom ol), the desired product in the same manner as in Production Example 2-1 [Te general formula (VII) smell, R ¹ 5R form of a compound having a phenyl group, R ² = H and R ³ = methyl group] was produced. Yield 65%. 81-82 ° CZ 0.2mmHg. Colorless transparent liquid.

' ¹ H-NMR (300 MHz, CDC I ₃ ) δ: 7 · 55 to 7.35 (m, 5Η), 5.84,

5.46 (br.br, 1Η),

3.58 to 3.43 (m, 1Η), 3.22 to 13 (m, 1H), 2.78 (d, ³ J _HP = 16.5Hz, 3Η)

■ ³¹ P-NMR (1 21 MHz, CDC I ₃ ) δ: 172.4 (br), 171.4 (br) Production Example 2-3: Production of (2R, 4S, 5R) —2-chloro-1,3-methyl-1,4,5-diphenyl 1,3,2-oxazazaphospholidine

 (1R, 2S) -2-Methylamino-1,2-diphenylethanol 2.27 g (10. Ommol), triethylamine 2.79ml (20. Ommol) solution in THF (10. Oml) Was added dropwise to a THF (10. Oml) solution of 0.872 ml (10.0 mmol) of phosphorus trichloride cooled to 0 ° C, and the mixture was refluxed with heating for 1 hour.

The solution was allowed to cool to room temperature, and the resulting salt was filtered through a glass filter under an argon atmosphere.The salt was washed with THF (1 Oml X 2), and the filtrate was concentrated under reduced pressure. [2R, 4S, 5R form of a compound of the general formula (VII) wherein R ¹ -phenyl group, R ² = phenyl group, R ³ = methyl group] 3.17 g (10. ). Yield quantitative (purity 92%). Milky white solid.

■ ¹ H-NMR (30 O Hz, CDC I ₃ ) δ 7.08-7, 05 (m, 6Η), 6.91-6.81 (m, 4H), 6.15 (d, ³ J = 8.3 Hz, 1H), 4.64 ( dd, ³ J _HH = 8.3Hz, ³ J _HP = 4.2Hz, 1 H) '

2.64 (d, ³ J _HP = 15.3Hz, 3H)

^{■ 31 P - NMR (1 21} MHz, CDC I 3) δ 171.7 0

 <Production of phosphoramidite (I)>

 Production Example 3-1

 5'-0- [Bis (4-methoxyphenyl) phenylmethyl] -3'-0-[(2S, 5S) -3-Methyl-5-phenyl-1,3,2-year-old xazaphospholidine- 2-yl]-Production of 2'-0- (tert-butyldimethylsilyl) peridine [(Sp) -19b]

 5'-0- [Bis (4-methoxyphenyl) phenylmethyl] -2'-0- (tert-butyldimethylsilyl) pyridine (4) (0.820 g, 1.5 mmol) is repeated with pyridine and toluene. Dried by azeotrope to give a THF (7.50 ml) solution.

To this was added triethylamine (1.05 ml, 7.5 I), and -78. After cooling to G, a 0.22 M THF solution of (5S) -18b shown in the following formula and in Table 1 was added dropwise under an argon atmosphere. After the reaction mixture was stirred at room temperature for 30 minutes, a saturated aqueous solution of sodium hydrogencarbonate (75 ml) and chloroform (75 ml) were added. After separating the organic phase, saturated carbonated water After washing with aqueous sodium hydrogen chloride solution (75 ml × 2), the collected washings were extracted with black form (75 ml 2).

 The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography [2.5 × 14 cm, silica gel 40 g, toluene-ethyl acetate triethylamine (10: 1: 0.2, v / v / v)]. The fraction containing the target compound is collected, washed with a saturated aqueous solution of sodium hydrogencarbonate (100 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to dryness. (Sp) -19b shown in the following formula and Table 1 Was obtained in a yield of 70%. Colorless amorphous.

1H NMR (300 MHz, CDC I ₃ ) <5 8.80 (br, 1H), 8.20 (d, ³ J _HH = 8.1Hz, 1H), 7.42-7.18 (m, 13H), 6.84 (m, 4H), 5.80 (s, 1H), 5.58 (t, ³ J _HH = 6.6Hz 1H), 4.60 (m, 1H), 4.18 (br, 2H), 3.80 (s, 6H), 3.40 (m, 2H), 1.40- 1.00 (m, 6H), 0.91 (t, ³ J _HH = 13.5Hz 9H), 2.22 (d, ³ J _HH = 13.5Hz 6H).

 Production Example 4-2

 5'-0- [Bis (4-methylphenyl) phenylmethyl] -3'-0-[(2R.4S.5R) 1-5-phenyl-tetrahydro-1H.3H-pyro mouth [1,2-G]- 1,3,2-year-old oxazaphospholidine-2-yl] -2'-0- (tert-butyldimethylsilyl) peridine [(Sp) -19d]

 5'-0- [Bis (4-methoxyphenyl) phenylmethyl] -2'-0- (tert-butyldimethylsilyl) peridine (4) (0.820 g, 1.5 mmo I) is repeated with pyridine and toluene Dried by azeotrope to give a THF (7.50 ml) solution.

 Triethylamine (1.05 ml, 7.5 inmol) was added to the mixture, and the mixture was cooled to 78 ° G. Then, under an argon atmosphere, a 0.22 M THF solution of (5S) -18d shown in the following formula and Table 1 was added dropwise. After the reaction mixture was stirred at room temperature for 30 minutes, a saturated aqueous solution of sodium hydrogencarbonate (75 ml) and chloroform (75 ml) were added.

After separating the organic phase, it was washed with a saturated aqueous solution of sodium hydrogencarbonate (75 ml x 2), and the collected washings were extracted with black hole form (75 ml x 2). The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (2.5 x 14 cm, silica gel 40 g, toluene monoacetate ethyl acetate). Lumin (10: 1: 0.2, v / v / v)].

 Collect the fractions containing the desired product and use a saturated aqueous sodium hydrogen carbonate solution

 (100 ml), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure to obtain (Sp) -19d shown in the following formula and Table 1 in a yield of 46%. Colorless amorphous.

1H NMR (300 MHz, CDC \ _ζ ) δ 9.82 (br, 1Η), 8.17 (d, ³ J _HH = 8.1Hz, 1H), 7.42-7.18 (m, 14H), 6.81 (m, 4H), 5.88 ( s, 1H), 5.71 (d, ³ J _HH = 6.6 Hz, 1H), 5.18 (d, ³ J _HH = 8.1 Hz, 1H), 2.62 (br, 1H), 4.40 (s, 1H), 4.28 (d , 1H), 3.83 (br, 1H), 3.8 (s, 6H), 3.60 (m, 3H), 3.20 (br, 1H), 2.39 (s 1H), '2.64 (br, 2H), 1.21 ( br, 1H), 0.97 (s, 9H), 0.24 (s, 6H).

table 1

 Bird

R ³ R ⁴ R ⁵ 19a-d ^a trans: cis

1 18a iPr H P 19a 8713

2 18b CH ₃ H Ph 19b 94: 6

3 18c (CH2) ₃ H 19c

4 18d (CH ₂ ) ₃ Ph 19d> 99: 1 a: ³ ? Diastereomeric ratio measured by NMR

Example 1

According to the following reaction formula, a phosphoramidite (I) and a nucleoside (II) were condensed using an activator (III), and then a sulfuration reaction was performed. ^{Pyridine (1} ° ^eqUiV)

Ac ₂ 0 (2 equi)

 (Sp.

 d.r.> 99: 1 *

 (Sp) -7 (Sp) -9 d.r.> 99: 1 'd.> 99: 1 * d.r.> 99: 1 *

Jiasutereoma one ratio measured by the * ³¹ P NMR

Thereafter, deprotection was performed according to the following reaction formula to obtain a target ribonucleotide analog.

 (Sp) -11 (fip) -11

 Sp: flp => 99: 1 Sp: Rp => 1:99

 Measured by reversed phase HPLC. Measured by reversed phase HPLC.

(flp) -3c to 37%, 6 steps (Sp) -3c to 32%, 6 steps The detailed reaction operation in the above reaction is as follows. The reaction tracking of the condensation reaction and the measurement of the diastereomer ratio of the product were all performed in the following manner. During Tsuyoshi R sample tube, trans - 19b (50jumol) and 2 ', - 3' -0 - Fuenoki Xia cetyl © lysine (50 mol), and vacuum-dried for 12 hours over P ₂ 0 _5, 8 hours MS 3A 0.25 M solution of N- (cyanomethyl) pyrrolidinium trifluorofluorone methanesulfonate (27a) (400 1, 100 mol) in acetonitrile

CD ₃ CN (100ju I) was added under an argon atmosphere. Three minutes later, NMR integration was started and the diastereomeric ratio of the reaction was determined by the integral ratio of the NMR signals.

 (Compound 3 → 6)

During NMR sample tube, trans- 19b (0.0520 g, 50 mo I) and 2 ', 3' - 0 - off enoki Xia cetyl © lysine (0.0256 £, 50 ^ υηιοΙ) 12 hours in a vacuum drying at [rho ₂ 0 ₅ on Α— (cyanomethyl) pyrrolidinium trifluorofluoroester dried for 8 hours with MS 3Α A 0.25 M solution of methanesulfonate (27a) (400, 100 mol) in acetonitrile and CD ₃ CN (100 ju I) were added under an argon atmosphere.

 (Compound 6 → 7)

 After stirring this well for 15 minutes, pyridine (43 I, 0.5 瞧 01) and acetic anhydride (10 jU I, 0.1 country ol) were added with a microsyringe to convert compound 6 to 7.

 (Compound 7 → 8)

 Further, Beaucage reagent (0.0120 g, 0.06 mmo I) was added to this solution to sulfide the compound 7.

 (Compound 8 → 9)

 Here, the reaction solution was transferred from the NMR sample tube to a 50 ml narrow-necked eggplant flask, washed with 3 ml of pyridine, and then added with 20 ml of a mixed solution of ammonia water / ethanol (3: 1, v / v). In addition, it was sealed and heat-treated at 60 ° C for 4 hours.

 After heating, the solvent was distilled off under reduced pressure, and 5 ml of 0.1 M TEAA buffer and 5 ml of dichloromethane were added.Compound 9 was recovered in the organic phase, dried over anhydrous sodium sulfate, filtered, and concentrated and dried under reduced pressure. Was.

 (Compound 9 → 10)

To thoroughly dried Compound 9, 3HF- ξ1: added neat to ₃ iota \ iota 1.5 ml, after 2 hours撹拌added 0.1 M AA buffer one 3ml of methanol 3ml, washed with ether 3ml Thereafter, the aqueous phase was collected, concentrated and dried under reduced pressure, and further freeze-dried to desalinate.

 (Compound 10 → 11)

 To the compound 10 after freeze-drying, 20 ml of an 80% aqueous acetic acid solution was added, and the mixture was stirred at room temperature for 30 minutes, and then concentrated and dried under reduced pressure. After 80% acetic acid was distilled off, the residue was washed with 3 ml of getyl ether, and extracted with distilled water. The collected aqueous phase was concentrated and dried under reduced pressure, freeze-dried repeatedly, desalted, and analyzed by reversed-phase HPLG and UV. As a result, (Sp) -11 was obtained with a total yield of 37% from the condensation.

Example 2 Oligomer (XIII) was produced by the following reactions (1) to (4) and (5) (the following reaction formula).

 (1) Condensation reaction

 Ribonucleic acid side bound to solid support [highly cross-linked polystyrene (HCP)] [General formula (I)] 20 equivalents of monomer per immol [Unit of general formula (II), (III)] ( 0.2 Ml) and 50 equivalents of an activator (N-cyanomethylammonium salt, 0.5 M) were reacted in acetonitrile for 90 seconds. After the completion of the reaction, the resultant was washed with acetonitrile.

 (2) Capping reaction (acetylation reaction)

 The ribonucleotide bound to the solid support is treated with a mixed solution of acetic anhydride: N-methylimidazole: THF = 1: 2: 7 for 60 seconds, and the unreacted 5 ′ hydroxyl group and the amino group of the released asymmetric auxiliary group are treated. The group was acetylated. After completion of the reaction, it was washed with acetonitrile.

 (3) Sulfidation reaction

 The ribonucleotide bound to the solid support was treated with 50 equivalents of Beaucage reagent (0.5 M) in acetonitrile solution for 60 seconds to sulfide the phosphite intermediate. After the reaction was completed, the substrate was washed with acetonitrile.

 (4) Detritylation reaction

 The ribonucleotide bound to the solid support was treated with a solution of trichloromouth acetate in dichloromethane for 60 seconds to remove the DMTr group at the 5 'end. After the completion of the reaction, the resultant was washed with dichloromethane and then with acetonitrile.

 (5) Chain extension reaction, deprotection reaction and purification

 The above-mentioned reaction operations (1) to (4) were repeated to extend the oligoribonucleotide chain on the solid support.

After the oligoribonucleotide derivative of the desired chain length has been synthesized on the solid support, the solid support is reacted with 25% aqueous ammonia: ethanol (3: 1, v / v) at 60 ° C for 15 hours. Thus, the protecting groups at the base moiety and the phosphate moiety were removed. At this time, the 3'-terminal hydroxyl-protecting group and the extraction of the oligomer from the solid support also proceeded at the same time. The solid phase carrier was removed by filtration, the filtrate was concentrated and dried under reduced pressure, Et ₃ N ′ 3HF (100 equivalents) was added, and the mixture was reacted at room temperature for 2 hours to remove the TBDMS group which is a protecting group for the 2 ′ hydroxyl group. . After completion of the reaction, Et ₃ M ′ 3HF was distilled off under reduced pressure, dried, dissolved in water (1 ml), and washed with ether (1 mix × 3 times). After concentrating and drying the aqueous layer under reduced pressure,

 (1 ml) and purified by reversed-phase HPLG to obtain the desired product in a yield of 20-70%.

 Example 3

 [Scheme 1: Synthesis of 1,2-amino amino alcohol as chiral chiral auxiliary]

 3a (R = Ph) 88% 4a (R = Ph) 60% 3b (R = Me) 98% 4b (R = Me) 88%

Synthesis of (S) -proline-N-ethyl carbamate (1)

 S-proline (5.75 g, 49.9 fractions) was added to a 10N aqueous NaOH solution (50 ml), cooled to 0 ° C, and stirred with chloroformic acid ethyl esther (5.75 ml, 60.4 g o I) for 40 minutes. ) Was added dropwise while maintaining the pH at 9-10. After stirring at room temperature for 3.5 hours, dichloromethane (30 ml) was added, and a 1N aqueous HGI solution (360 ml) was added to adjust the pH to 1, followed by extraction with dichloromethane (300 ml × 10) and anhydrous sodium sulfate. The residue was filtered and concentrated under reduced pressure to give 1 (9.19 g, 98%). Colorless transparent liquid.

¹ H NMR (CDGIg) δ 10.85-10.42 (br, 1H), 4.41-4.30 (m, 1H), 4.22-4.15 (m, 2H), 3.60-3.37 (in, 2Η), 2.32-2.20 (m, 1H ), 2.17-2.05 (m, 1H), 1.98-1.90 (m, 2H), 1.31-1.19 (m, 3H); IR (NaCI, cm- ¹ ) 3459 (one C00H), 1724 (-C00H), 1682 (N-C = 0).

 Synthesis of (S) -proline-N-ethyl carbamethyl methyl ester (2)

 Under Ar atmosphere, add methanol (150 ml) to S-proline-N-ethyl carbamate 1 (9.19 g, 49.1 Cheom I), cool to 0 ° C, and stir with thionyl chloride (5.40 ml, 74.3 raiol). ) Was added. After stirring at room temperature for 5 hours, methanol was distilled off under reduced pressure, a saturated aqueous solution of sodium hydrogen carbonate (100 ml) was added, and the mixture was extracted with chloroform (100 ml x 3), dried over anhydrous sodium sulfate, and filtered. Concentration under reduced pressure gave 2 (9.80 g, 99%). Colorless transparent liquid.

_{^{1 H NMR (CDC 1 3)}} δ 4.29 - 4.20 (m, 1H), 4.08 - 3,98 (m, 2H), 3.65 (s, 3H), 3.56 - 3.35 (m, 2H), 2.21 - 2.09 (m , 1H), 1.94-1.82 (m, 3H), 1.21-1.10 (m, 3H); IR (NaCI, cm " ¹ ) 1751 (COOMe), 1702 (NC = 0) ₀

N-ethyl carbamate- (2S)-, a-diphenyl (pyrrolidine-2-yl) meta Synthesis of knol (3a)

(S) -Proline-N-ethyl carbamate methyl esther 2 (5.03 g, 25.0 chemo 1) was repeatedly azeotroped with toluene, dissolved in THF (50 ml), and cooled to 0 ° C. PhMgBr (18.0 ml, 100.0 ml) dissolved in THF (96.2 ml) was added with stirring, and the mixture was stirred at 0 ° C for 3 hours. A saturated aqueous solution of ammonium chloride (50 ml) and a saturated aqueous solution of sodium chloride (50 ml) were added, and the mixture was extracted with chloroform (50 mix 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Then, 30 ml of hexane was added, and the mixture was vigorously stirred, filtered by suction, and dried in vacuo to obtain 3a (7.22 g, 88%). Colorless amorphous. ^ NMR (CDCI ₃ ) δ 7.40-7.12 (m, 10H), 4.94-4.87 (m, 1H), 4.19-3.98 (m, 2H), 3.45-3.35 (m, 2H), 2.17-2.02 (m, 1H ), 1.99-1.88 (m, 1H), 1.55-1.42 (m, 1H), 1.25-1.22 (t, J = 7.2 Hz, 3H); IR (NaCI, cm " ¹ ) 3375 (-OH), 1680 ( NG = 0).

 Synthesis of N-ethyl carbamate- (2S) -methyl (pyrrolidine-2-yl) ethanol (3b)

 Under an Ar atmosphere, add ether (100 ml) to magnesium (4.80 g, 197.3 mmol), cool to 0 ° C, and stir while dissolving methyl iodide (12.5 ml, 200.7 sol) in ether (50 ml). ml) was added. After stirring at room temperature for 45 minutes, the mixture was cooled to 0 ° C, and with stirring, ether (50 ml) in which (S) -Proline-N-ethyl carbamate methyl esther 2 (9.80 g, '48 .7 mmol) was dissolved was added. added. After stirring at 0 ° C for 1.5 hours, a saturated aqueous solution of ammonium chloride (75 ml) and a saturated aqueous solution of sodium chloride (75 ml) were added, extracted with dichloromethane (150 ml x 3), dried over anhydrous sodium sulfate, and filtered. After concentration under reduced pressure, 3b (9.60 g, 98%) was obtained. Yellow transparent liquid.

1H NMR (CDCI ₃ ) δ 5.72-5.66 (br, 1H), 4.09 (q, J = 6.9 Hz, 2H), 3.84 (t, J = 7.5 Hz, 1H), 3.72-3.62 (m, 1H), 3.20 -3.11 (m, 1H), 2.03-1.94 (m, 1H), 1.84-1.76 (m, 1H), 1.69-1.51 (m, 2H), 1.24-1.20 (t, J = 6.9 Hz, 3H), 1.11 (s, 3H), 1.03 (s, 3H); IR (NaCI, cm " ¹ ) 3391 (-OH), 1670 (N-G = 0).

Synthesis of (2S) -of, H-diphenyl (pyrrolidine-2-yl) methanol (4a) N-ethyl carbamate- (2S)-a, a-diphenyl (pyrrol idi η-2-y I) methano I 3a (6.51 g, 20.0 raiol) Potassium (11.2 g, 200.0 ol) was added. After heating and refluxing for 4 hours while stirring, methanol was distilled off under reduced pressure, 50 ml of water was added, extracted with dichloromethane (50 ml 2), and washed with saturated saline (100 ml x 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Hexane (50 ml) was added to the obtained crystals, and the mixture was vigorously stirred, followed by suction filtration to obtain a white powder. When the obtained white powder was measured with ¹ HNR (GDGI ₃ ), the chemical shift was different from that described in the literature.When measured with ¹³ C rigid R (CDGI ₃ ), the carbon number was 14, Further, the obtained white powder (5 mg) was allowed to form a salt with mandelic acid (3 mg), and measured by ¹ H NMR (CDGI ₃ ). Powder! In addition, it was determined that an oxazolidinone ring was formed instead of the target product 4a. There, they were all recovered and reacted under the same conditions for 6 hours. Purification was performed in the same manner to obtain 4a (2.99 g, 60%). Colorless amorphous.

¹ H NMR (CDCI ₃ ) δ 7.57-7.11 (m, 10H), 4.26 — 4.21 (m, 1H), 3.06-2,89 (m, 2H), 1.78-1.52 (m, 4H); IR (KBr, cm- ¹ ) 3350 (-OH, NH).

 Synthesis of (2S) -Hymethyl (pyrrolidine-2-yl) ethanol (4b)

N-ethy I carbamate- (2S)-a? -Methyl (pyrrol idi η-2-y I) ethano I 3b (9.60 g, 48.7 mmol), add methanol (50 ml), cool to 0 ° C, With stirring, potassium hydroxide (27.0 g, 481.1 mmol) was added. After heating and refluxing for 4 hours while stirring, methanol was distilled off under reduced pressure, 50 ml of water was added, concentrated hydrochloric acid was added until the pH became 1 and the mixture was washed with ether (100 ml x 2) to produce The aqueous phase was recovered from the precipitate. Potassium hydroxide was added until pH 12, the precipitate was removed by suction filtration, extracted with dichloromethane (200 ml x 6), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. (5.54 g, 88%). Yellow needle crystals. ¹ H Go R (CDCI ₃ ) δ 3.03-2.87 (m, 3H), 2.66-2.48 (br, 2H), 1.80-1.58 (in, 4Η), 1.16 (s, 3H), 1.13 (s, 3H); IR (KBr, cm- ¹ ) 3376 (-0H, NH) ₀ [Scheme 2: Synthesis of phosphitylating agent]

5a (

 5b (

 Synthesis of (4S) -2-chlorotetrahydro-1H, 3H-pyrro [1, 2-G] -5,5-diphenyl-1,3,2-oxazaphospholidine (5a)

 (2S)-,-diphenyl (pyrrol id in-2-yl) methanol 4a (1.27 g, 5 mmol) ¾: The residue was azeotropically dried with toluene and dissolved in 2.5 ml of toluene. N-methylmorpholine (1.1 ml, 10.0 fractions) was added to the solution, and this mixed solution was added dropwise to a toluene solution of phosphorus trichloride (0.44 ml, 5.0 mmol) at 0 ° C. with stirring under an Ar atmosphere. After stirring the reaction mixture at room temperature for 30 minutes, the resulting salt was filtered off at -78 ° C under an Ar atmosphere, and the filtrate was concentrated under reduced pressure under an Ar atmosphere to obtain 5a (1.79 g, crude).

¹ H NMR (CDGI ₃ ) δ 7.57-7.01 (m, 10H), 4.68 — 4.51 (m, 1H), 3.44-

3.35 (m, 1H), 3.17-3,07 (m, 1H), 2.06-1.89 (m, 2H), 1.67-1.24 (m,

2H); ³¹ P NMR (121 MHz, CDCI ₃ ) δ 158.2 · (71%), 173.6 (29%) ₀

 Synthesis of (4S) -2-chlorotetrahydro-1H, 3H-pyro [1,2-c] -5,5-dimethyl-1,3,2-2-oxazaphospholidine (5b)

(2S) -oi-methyl (pyrrol idin-2-yl) ethanol 4b (1.95 g, 15.1 mmol) was azeotropically dried using toluene and dissolved in 5.0 ml of toluene. N-methylmorpholine (3.3 ml, 29.8 mmol) was added to the solution, and this mixed solution was added dropwise to a toluene solution of phosphorus trichloride (1.4 ml, 16.0 mmol) at 0 ° C. with stirring under an Ar atmosphere. After stirring the reaction mixture at room temperature for 30 minutes, the resulting salt was filtered off at -78 ° C under an Ar atmosphere, and the filtrate was concentrated under reduced pressure under an Ar atmosphere. Purification was attempted by vacuum distillation (bp. 55; C / 0.2 mmHg). crude). Colorless transparent liquid. ·

¹ H NMR (CDCIg) δ 3.70-3.61 (m, 1H), 3.53-3.40 (m, 1H), 3.19-3.05 (m, 1H), 2.21-2.04 (m, 2H), 1.84-1.71 (m, 2H ), 1.53 (s, 3H), 1.37 (s, 3H); ³¹ P NMR (121 MHz, CDGI ₃ ) δ 171.0 (35%), 164.5 (26%), 161.6 (39%).

 [Scheme 3: Synthesis of oxazaphospholidine derivative]

Table 2 dr

 5 temp., Time yield

 (Sp) -7: (flP) -7 *

 7a (R = Ph) 3.3 equiv reflux 15 h> 99: 1 70%

 7b (R = Wle) 2.4 equiv Π2 h 98: 2 36%

* Measured by ³¹ P MR

 5'-0- (tert-butyldiphenylsilyl) -3'-0-[(2S, 5R) -5,5-diphenyl-tetrahydro-1H, 3H-pyrro [1,2-c] Synthesis of [1,3,2-2-Year-Oxazaphospholidine-2-yl] thymidine (7a)

5′-0- (tert-ButyIdiphenyIsiIyI) thymidine 6 (722.3 mg, 1.5 mmol) was repeatedly azeotroped with pyridine and toluene to obtain a THF solution. After adding Et ₃ N (1, 1 ml, 7.9 mmol) and cooling to -78 ° C, under Ar atmosphere (4S)-2-ch I orotetrahydro-1 H, 3H-Pyr ro [1, 2 -c] -5,5-Dipheny 1-1,3,2-oxazaphosphol idine 5a in 0.22 THF (22.5 ml, 5.0 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours. Since the reaction was not completed, the mixture was heated and refluxed overnight. A saturated aqueous sodium hydrogen carbonate solution (75 ml) and black mouth form (75 ml) were added. After separating the organic phase, it was washed with a saturated aqueous sodium hydrogencarbonate solution (75 ml × 2), and the collected washings were extracted with a black hole form (75 ml × 2). The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate and added dropwise to hexane to reprecipitate the target compound. The solid was collected by suction filtration and washed with hexane to give 7a (801.6 mg, crude). Colorless amorphous.

¹ HR (CDGI ₃ ) δ 7.65-7.12 (m, 11H), 6.13 (dd, ³ J _HH = 7.8, 7.8 Hz, 1H), 4.65-4.57 (m, 1H), 4.55-4.49 (m, 1H), 3.79 (dd, ² J _HH = 11.3 Hz, ³ J _HH = 2.4 Hz, 1H), 3.84 (dd, ² J _HH = 11.7 Hz, ³ J _HH = 2.4 Hz, 1H), 3.57-3.48 (m, 1H) , 3.45-3.44 (m, 1H), 3.17-3.07 (m, 1H), 2.33-2.25 (m 1H), 1.92-1.82 (m, 1H), 1.81-1.50 (m, 4H), 1.49 (s, 3H) ), 1.06 (s, 9H); IR (KBr, cm " ¹ ) 3423, 2930, 1688, 1466, 1448, 1278, 1113, 1066, 958,

5 '-0- (tert-butyldiphenylsilyl)-3'-0-[(2S, 5R) -5, 5-dimethyl-tetrahydro-1H, 3H-pyrro [1,2-G]- Synthesis of 1,3,2-2-oxazaphospholidine-2-yl] thymidine (7b)

5 '-0- (tert-Buty I dipheny I si Iy I) thymidine 6 (1.31, 2.72 mmol) was dried by repeated azeotropes with pyridine and toluene, and the THF solution (7.50 ml) was added. did. After adding Et ₃ N (1.9 ml, 13.6 1 ol) to the mixture and cooling to -78 ° C, under Ar atmosphere (4S) -2- 2-chlorotetrahydro-1H, 3H-Pyrro [1,2-c]- A 0.38 M THF solution of 5, 5-dimethyl-1,3,2-oxazaphospholidine 5b (10.0 ml, 3.81 mmol) was added dropwise. After the reaction mixture was stirred at room temperature for 30 minutes, a saturated aqueous solution of sodium hydrogencarbonate (100 ml) and chloroform (100 ml) were added. After separating the organic phase, the organic phase was washed with a saturated aqueous solution of sodium hydrogencarbonate (100 ml × 2), and the collected washings were extracted with chloroform (200 mlxl). The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [4x16 cm, 100 g of silica gel, hexan-ethy I acetate-triethyl amine (50: 50: 5,

v / v / v) → Hexan-ethy I acetate-triethylamine (50: 50: 2, v / v / v)]. Fractions containing 7b are collected and saturated aqueous sodium bicarbonate solution (100 After washing with 1 ml × 1), it was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 7b (614.8 mg, 36%). Light brown amorphous.

¹ H MR (CDCI ₃ ) δ 9.83-- 9.63 (br, 1Η), f. 68-- 7, 34 (m, 11H), 6.42 (dd, ³ J _HH = 8.1, 8.1 Hz, 1H), 4.90-4.85 ( m, 1H), 4.09-4.08 (m, 1H), 3.99 (dd, ² J _HH = 11.7 Hz, ³ J _HH = 2.1 Hz, 1H), 3.84 (dd, ² J _HH = 11.1 Hz, ³ J _HH = 2.1 Hz, 1H), 3.53-3.47 (m, 2H), 3.09-2.09 (m, 1H), 2.53-2,46 (m, 1H), 2.24-2.15 (m, 1H), 1.85-1.65 (m , 4H), 1.58 (s, 3H), 1.47 (s, 3H), 1.20 (s, 3H), 1.11 (s, 9H); ³¹ P NMR (121 MHz, CDCI ₃ ) δ 152.2 (98%), 142.9 (2D; IR (KBr, cm— ¹ ) 3423, 2963, 1689, 1467, 1428, 1273, 1113, 1074, 956. '

 [Scheme 4: Condensation of 7 and 9]

Table 3 dr dr

 e

 (Sp) -7: tim

(Rp) -7 * (Rp) -10: (Sp) -10 ^:

7a (R = Ph)> 99: 1 20 min 87: 13

 7b (R = Me) 98: 2 <5 min 97: 3

 99: 1 <5 min 98: 2

* ³¹ Measured by PR.

Monitoring condensation of 7 and 9 in the presence of 8 by ³¹ P NMR spectroscopy. Representative monitoring of the condensation of 7a-b and 9 in the presence of 8

刚 7a (41.9 mg, 55 mol) and 9 (17.8 mg, 50 03812

The mol) was vacuum-dried for 12 hours over P ₂ 0 _5, 8 and dried for 8 hours (ΑΟΟμ I, 100 mo I) of 0.25 Micromax Asetonitoriru solution and GD ₃ GN (100 i I) was added under an Ar atmosphere MS 3A Was. Three minutes later, the integration by R was started, and the diastereomer ratio of the reaction was determined by the integration ratio of the NMR signal.

NMR sample tube, 7b (35.1 mg, 55 jumol ) and 9 (17.8 mg, 50 〃Mol) was vacuum-dried for 12 hours over P ₂ 0 _5, 8 and dried for 8 hours at MS 3A (400 il, 1O0〃 mol) of 0.25 M acetonitrile and CD ₃ CN (100 jw I) were added under an Ar atmosphere.

 Scheme 5

 (flp): (Sp) = 97: 3

5'-0- (tert-butyldiphenylsilyl) thymidine -3'-yl 3'-0- (tert-butyldimethylsilyl) thymidine-5'-yl M-acetyl- (2S) -bi-methyl Synthesis of (pyrrolidin-2-yl) ethanoyl phosphite (12b)

During NMR sample tube, 7b (35.1 mg, 55〃Mol) and 9 (17.8 mg, 50 / mol ) was vacuum-dried for 12 hours over P ₂ 0 _5, 8 and dried for 8 hours at MS 3A (400〃 I,

(100 imol) in 0.25 M acetate nitrile solution and GD ₃ GM (100 I) under Ar atmosphere It was. Five minutes later, pyridine (40.11, 500 mol) and acetic anhydride (9.5 I, 100 ol) were added. Three minutes later, black-mouthed form (30 ml) was added, washed with a saturated aqueous solution of sodium hydrogencarbonate (15 ml × 2), and the collected washings were extracted with black-mouthed form (30 ml × 1). The collected organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and azeotroped with toluene to obtain 12b (86.9 mg, crude).

_{^{] W NMR (CDC 1 3)}} δ 9.73 (br, 2H), 7.63 - 7.61 (m, 4H), 7.46 - 7.36 (m, 7H), 6,39 (dd, 3 J HH = 6.6, 3 Hz, 1H ), 6.23 (t, 6.0 Hz), 4.89 (m, 1H), 4.30 (m, 2H), 4.08--1.80 (m, 6H), 3.58--1.38 (m, 2H), 2.45 (m, 1H), 2.34 -2.23 (m, 3H), 2.23-1.98 (m, 4H), 1.89 (s, 3H), 1.60 (s, 3H), 1.46 (s, 3H), 1.41 (s, 3H), 1.10 (s, 9H ), 0.87 (s, 9H), 0.05 (6H); ³¹ P NMR (121 MHz, CDCI ₃ ) δ 137.2 (79%), 137.0 (7%), 136.7 (8%), 135.7 (1%), 135.2 (5%); IR (KBr, cm- ¹ ) 3430, 2930, 1742, 1694, 1471, 1274, 1112 ,. 1034, 966, 835.

 (Rp) -5'-0- (tert-butyldiphenylsilyl) thymidine-3'-yl 3'-0- (tert-butyldimethylsilyl) thymidine-5'-yl H-phosphonate [(Rp)- 11]

CH ₂ GI ₂ (20 ml) in which distilled TFA (2 ml) was dissolved was added to 12b (86.9 mg, crude) which had been made into a foam and dried under vacuum for 5 hours under an atmosphere of At ′. After stirring at 0 ° C for 2 minutes, dichloromethane (100 ml) was added, and the mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate (50 ml x 2), and the collected washings were extracted with dichloromethane (100 ml 1). . The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography [4x16 cm, 100 g of si I ica gel, hexan-ethyl acetate (1: 1, v / v) → hexan-ethy I acetate (1: 2, v / v)-→ hexan-ethy I acetate (1: 3, v / v) → hexan-ethy I acetate (1: 4, v / v)]. The fractions containing (Rp) -11 were collected, concentrated under reduced pressure, added with chloroform (50 ml), washed with saturated aqueous sodium hydrogen carbonate (50 mlxl), and washed with chloroform (50 ml XI) The collected organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 11b (43.4 mg, 84% (purity 93%)). It was. Colorless amorphous.

H NMR (CDGI3) «59.45-9.33 (br, 2H), 7.64-7.60 (m, 4H), 7.47-7.37 (m, 8H), 6.90 (d, J _PH = 715.0 Hz, 1H) 6.39 (dd, ³ J _HH = 6.0, 6.0 Hz, 1H), 6.14 (t, ³ J _HH = 7.2 Hz, 1H), 5.23— 5,22 (m, 1H), 4.46— 4.40 (m, 1H), 4.35-4.19 ( m, 2H), 4.18 (s, 1H), 3.99-3.78 (m, 3H), 2.65-2.55 (m 1H), 2.33-2.28 (m, 3H), 1.91 (s, 3H), 1.57 (s, 3H ), 1.06 (s, 9H), 0.89 (s, 9H), 0.10 (s, 6H); ³¹ P NMR (121 MHz, GDCI ₃ ) δ 9.3 (3% for (Sp) -11), 7.8 (97% for (Rp) -11); IR (KBr, cm- ¹ ) 3448, 2930, 1695, 1471, 1276, 1114, 1035, 971, 837.

 (Scheme 6)

Table 4 (Sp) -13: (flp) -13 (Sp) -18: (fip) -18 Yield of 18

98.1: 1.9 98.6: 1.4

 1.9: 98.1 1.9: 98.1 Typical procedure for manual solid-phase synthesis

(1) 3% DCA in CH ₂ CI ₂ ; 15-20 sx4

(2) Washing (GH ₂ CI ₂ fol lowed by CH ₃ CN)

(3) Coupling (0.2 M monomer 13 and 1.0 M 8 in CH ₃ CN; 3 min)

(4) Protection 0 ₂ 0-1 ^ -11161 1! 1 ^ 0 | 3∑0 | 6-chome (1: 2: 7, v / v / v); 30 s]

(5) 1% TFA in CH ₂ CI ₂ ; 15-20 sx4

(6) Sulfide [10% S ₈ in CS ₂ -Py-Et ₃ N (35: 35: 1, v / v / v); 3 h]

(7) Cleaning [GS ₂ -Py-Et ₃ N (35: 35: 1, v / v / v) fol lowed by Py]

(8) 25% NH ₃ aq. (5.0 ml; 1h)

(9) suction filtration, washed _{(H 2 0; 1.0 ml x} 5)

 (10) Evaporation of solvent under reduced pressure

(11) Dilution (H ₂ 0; 5.0 ml)

(12) Washing (Et ₂₀ ; 5.0 ml x 3)

 (13) Evaporation of solvent under reduced pressure

 (14) Freeze drying

The collected residue was dissolved in water (0.2 ml) and analyzed by reverse phase HPLC.

 (Scheme 7)

Table 5

(Sp) -13: (flp) -13 (Sp) -19: Yield of (fip) -19 19

98.1: 1.9 96.5: 3.5 97,5 1.9: 98.1 4.2: 95.8 97.6 Typical procedure for manual solid-phase synthesis

(1) 1% TFA in CH ₂ CI ₂ ; 15-20 sx4

(2) cleaning _{(CH 2 CI 2 fol lowed by} CH 3 CN)

(3) Coupling (0.2 monomer 13 and 1.0 M 8 in CH ₃ CN; 3 min)

(4) protected _{[Ac 2 0-N- methyl imidazole} - THF (1: 2: 7, v / v / v); 30 s]

(5) 1% TFA in CH ₂ GI ₂ ; 15-20 sx4

(6) oxidative Amino reduction (saturated _{NH 3 in CG 1 4 - dioxane} (4: 1, v / v); 0 ° C, 30 min)

 (7) Suction filtration and washing (dioxane; 1.0 mix 2)

 (8) Evaporation of solvent under reduced pressure

(9) Dilution (H ₂ 0; 5.0 ml)

 (10) Evaporation of solvent under reduced pressure

 (11) Freeze drying

 The collected residue was dissolved in water (0.2 ml) and analyzed by reverse phase HPLG.

Claims

 The scope of the claims

Again ^

 Expression

 (/ \

[Wherein, R ¹ and R ′ may be the same or different and represent a hydrogen atom, an alkyl group having 1 to 3 carbon atoms or an aryl group having 6 to 14 carbon atoms,

R ² and R ″ may be the same or M, and represent a hydrogen atom, an alkyl group having 1 to 3 carbon atoms, or an aryl group having 6 to 14 carbon atoms,

R ³ represents an alkyl group having 1 to ³ carbon atoms,

R ⁴ represents a hydroxyl-protecting group, D, represents one OR ⁵ (where R ⁵ is a hydroxyl-protecting group), a hydroxyl group or a hydrogen atom;

 B s is

Represents a group derived from peracyl, adenine, cytosine, guanine, thymine or a derivative thereof represented by However, R ² and R ³ may form a monocyclo structure or a bicyclo structure together with the nitrogen atom. ]

An optically active nucleoside 3'-phosphoramidite represented by

(II)

[Wherein, R ⁶ is a hydroxyl-protecting group and E, is one OR ⁷ (where R ⁵ is a hydroxyl-protecting group), a hydroxyl group or a hydrogen atom, and Bs has the same meaning as described above. ]

And a nucleoside represented by

 —General formula (III)

[Wherein, X- is BF ₄ —, PF ₆ —, T f CT (T f is CF ₃ S 0 ₂ —; the same applies hereinafter), T f ₂ Ν−, As F ₆ — or S b F ₆ — is indicated. The cyclic structure A represents a monocyclo or bicyclo structure having 3 to 16 carbon atoms formed together with a nitrogen atom. ]

Characterized by reacting with an electrophile and deprotecting after condensing using an activator represented by the formula: A method for producing a nucleotide analog and a deoxyliponucleide analog.

[In each formula, Y is a linear or branched alkyl group having 1 to 10 carbon atoms, a linear or branched alkoxy group having 1 to 10 carbon atoms, a linear or branched chain having 1 to 10 carbon atoms. Hydroxyalkyl group of chain, aryl group having 6 to 14 carbon atoms, alkyl group having 1 to 10 carbon atoms, acyl group having 1 to 10 carbon atoms, amino group, alkyl group having 1 to 10 carbon atoms Mino-go, dialkylamino group having 1 to 10 carbon atoms, or Ζ = Υ 'Ζ ⁺ (Υ' is S-, Se-, BH ₃ —, Z ⁺ is ammonium ion, primary to Represents a quaternary lower alkyl ammonium ion or a monovalent metal ion). B s has the same meaning as described above, and two B s in each formula may be the same or different. D ₂ and E ₂ represent a hydroxyl group or a hydrogen atom. ]

 2. —The optically active nucleoside 3'-phosphoramidite represented by the general formula (I) is reacted with the optically active 1,2-amino amino alcohol represented by the general formula (VI) and phosphorus trichloride. 2. The process according to claim 1, which is obtained by reacting the obtained optically active phosphitylating agent represented by the general formula (VII) with a nucleoside represented by the general formula (VIII).

[Wherein, R ¹ , R ² , R ³ , R ⁴ , D, and B s have the same meaning as described above. ]

3. The general formula (I), wherein R ¹ and R ′ are the same or different and are an alkyl group having 1 to 3 carbon atoms or an aryl group having 6 to 14 carbon atoms. Manufacturing method.

 4. A highly stereoregular oligoribonucleotide analog represented by the general formula (XII I) and an oligodexoxy lipoate represented by the general formula (XII I), characterized by repeating the reaction in the production method according to claim 1. A method for producing a nucleotide analog.

[In the formula,丫, B, 0 ₂ and已₂ Ha general formula (IV), the integer n is 1-1 50 shows the same meaning as (V). ]