JPS6339894A - Production of oligodeoxynucleotide - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful in the field of genetic engineering, etc., by reacting a deoxynucleotide phosphorsulfide compound with a deoxynucleotide compound in the presence of a trialkylstannylazole compound and a base, followed by oxidation thereof. CONSTITUTION:A deoxynucleotide phosphorsulfide compound expressed by formula I (R<4> and R<5> represent protecting group; R<6> represents aryl; B<2> represents base residue; n<1> >= 0) is reacted with a deoxynucleotide compound expressed by formula II (R<7> represents protecting group; B<3> and B<4> represent base residue; n<2> >= 0) in the presence of both a trialkylstannylazole compound expressed by the formula R<1>3X (R<1> represents alkyl; X represents azolyl) and a base expressed by the formula R<2>NR<3>2 (R<2> represents H, alkyl or aryl; R<3> represents alkyl or two R<3>, together with the adjacent Natom, form a heterocyclic ring optionally containing two or more of N, O and S, etc., as a hetero-atom and then oxidized to afford the aimed compound expressed by formula III.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing oligodeoxynucleotides useful in fields such as genetic engineering.

Currently, methods for producing oligodeoxynucleotide F include the phosphoric acid triester method (reaction formula (1)),
The phosphorochloride method (reaction formula (2)) and the phosphoramidite method (reaction formula (3)) are widely used (for details on the phosphotriester method, see C, B, Re
eae, Tatrahedron, 34.3143
(1978).

For the phosphorochloride 0 method, R, L, Letsi
Nger et al., J. Am. Chem. Soc., 97.
3278 (1975), and for the phosphoramidite method, M. H. Caruthera et al., Tetrah
edron Lett-+ 22.185'? (198
Please refer to 1).

(In the formula, R4 and R5 represent a protecting group: R6 represents an aryl group, R' represents a protective group that may contain a polymeric carrier, B1 and B2
may be the same or different, and each may have a protecting group. Also, the dehydrating agent is 2 t.
416-1- IJ Isoglobilbenzenesulfonyl tetrazolide etc. )R"O""" P\Cl ["IXI [:■] (in the formula, R',
R5* R', B'' and B2 have the same meanings as above.) RONR2 [X] [■] [■] (In the formula, R4, R5, R7, B1 and B2 have the same meanings as above. (Also, R8 is an alkyl group, etc.) Problems to be Solved by the Invention The advantage of producing dinucleotides in oligothio using the phosphotriester method is that the production intermediate compound (Vl) Because it is stable to oxygen and water, the handling of compound [VI] and the operation of reaction (1) are easy.Therefore, the phosphotriester method is the mainstream method for producing dinucleotides in oligothio. It's here.

However, in this method, because the reaction rate of reaction (1) is not very high, 9 oligos! The disadvantage is that it takes time to build A.

- The advantage of the phosphorochloride method and phosphoramidite method is that compound (IX) and CXI, which are production intermediates,
Because the reaction with the alcoholic hydroxyl group, that is, the reaction with the compound [■], and the subsequent oxidation reaction with iodine water, etc. proceed quickly, the target compound [■]
] can be obtained in a short time. However, in the above method, the compounds [open and [X], and the separated compound [IXI
Since f is unstable to oxygen or water, care must be taken to prevent it from decomposing during storage or during reactions (2) and (3). The compound CIXI is generally difficult to isolate from the production system due to its instability, and the compound [
X] is known to be unstable to such an extent that the PN bond is easily cleaved even in the presence of a weak acid.

In view of the state of the prior art as described above, the present inventors have conducted extensive research with the aim of developing a method for quickly providing orico-0 deoxynucleotides using stable production intermediates, and as a result, the reaction ( The present invention was completed by discovering that the method shown in 4) advantageously achieves this object.

Therefore, the present invention utilizes the advantages of the method for constructing dinucleotide yJ in oligodeoxynucleotides as described above, namely, a method for producing oligodeoxynucleotides using a stable and easy-to-handle construction intermediate and through a quick reaction. Specifically, in the presence of a triple kylstannylazole compound represented by general 2B) and a base represented by general formula [■], a deoxynucleotide phosphor represented by general formula (III) is prepared. Rusulfide compounds and general formula C
The present invention provides a method for producing deogysylnucleotides represented by general formula [V], which comprises reacting with a deogysylnucleotide compound represented by IVI and then carrying out an oxidation reaction.

R' S nX CI ] (in the formula R+
represents an alkyl group; R3 is 1 as a heteroatom together with the adjacent nitrogen atom
or a heterocyclic group which may contain two nitrogen atoms, oxygen atoms or sulfur atoms, or R2 and two R3 together with the adjacent nitrogen atoms are 1 as a heteroatom.
It refers to a heterocyclic group which may contain one to two nitrogen atoms, oxygen atoms or sulfur atoms. ) (In the formula, R4 and R5Fi represent protective groups: R6 represents an aryl group; 81 and B2 may be the same or different, and each represents a basic residue that may have a protective group; nl is 0 or a positive integer, but if nl is 2 or more, B2
are not limited to the same base residue. ) (In the formula, R has the same meaning as above: R7 represents a protecting group which may contain a polymeric carrier; B and B may be the same or different and each has a protecting group. (In the formula, B4. B5. R7, al, B2. B3
．． B4. nl and R2 have the same meanings as above. ), I-' Therefore, the reaction according to the present invention can be expressed by reaction formula (4).

[■1] QV) (V) To explain the trialkylstannylazole compound represented by the general formula CI], the organic group R1 may be an alkyl group, specifically a methyl group, an ethyl group, n
- Primary alkyl group such as globyl group, n-butyl group, i
- Secondary alkyl group such as propyl group, S-butyl group, t
Examples include tertiary alkyl groups such as -butyl group. Regarding which of these alkyl groups is used as R in the compound CI), the compound [! ] may be determined by taking into account the ease of production and its stability (although there are no particular limitations, ethyl, n-globyl, and n-ethyl groups are preferred because they are easy to produce and provide good stability). In addition, as the azolyl group X, imidazolyl group, 3-2) loimidazolychlorohenso)
Examples include an IJ group, a 5-nitrobenzotriazolyl group, and a tetrazolyl group. Which of these azolyl groups is used as X in compound CI) is not limited, but is generally determined by the ease of production of compound CI) and the degree to which reaction (4) can proceed well. Imidazolyl group, 1,2,3-triazolyl group, which provides easy processability and good processability.

1.2.4 - triazolyl group, benzotriaglyl group, tetrazolyl group, etc. are preferably used.

Compound CII can be easily produced by the method already shown by the present inventors, ie, reaction (5) (see the specification of Japanese Patent Application No. 16511/1988).

(In the formula, B and is a hydrogen atom, an argyl group (e.g., methyl group, ethyl group, n-7"ropyl M, if-ropyl group, n-butyl group, etc.) or an aryl group (e.g., phenyl group, 4-methylphenyl group, etc.) Sometimes R is an alkyl group (eg, a methyl group).

Ethyl g, n-f lopyl fi, l-7' lovyl group, n-
butyl group, etc.) or a multicyclic group which may contain 1 or 2 nitrogen atoms, oxygen atoms, or sulfur atoms when two R's together with the adjacent nitrogen atoms (for example, pi group, 2,6-zomete rubiberidino group, pyrrolyl group, 9morpholino group, thiomorpholino group, imidazolyl group, 2-methylimidazolyl group, benzimidazolyl group.

1.2.4-Triazolyl group, 3-methyl-I.

2, 4-dolyaglyl group, etc.), and furthermore, as a compound CUE, R and two R's are combined with adjacent nitrogen atoms to form one or more heteroatoms. Heterocycles which may contain two nitrogen, oxygen or sulfur atoms (e.g. pyridine, 2,6-dimethylpyridine, 4-dimethylamino-
Pyridine, oxazole, thiazole, pyrimidine, pyranone, quinoline, 8-triazine, etc.) compounds may also be mentioned. From these compounds [■], reaction (4
) There is no limitation as to which one to use, but those having a pKa in the range of 5 to 12 are preferably used.

To explain the deoxynucleotide phosphorus sulfide compound represented by the general formula [111], the protecting group R4 can be a basic V or any hydrocarbon protecting group. Is this specifically an oligodeoxynucleotide? :Hydroxy-retaining groups whose usefulness in production has been recognized, such as triphenylmethyl group and 4-nodoxytriphenylmethyl group.

4.4'-ZometoΦcitriphenylmethyl JIF triarylmethyl group, pinoyl group (i.e. 9-phenylxanthine-9-yl group), alkoxycarmenyl group,
Examples of the aryloxycarbonyl group and arylthioalkyloxycal include a nyl group and a trialkylsilyl group such as t-butyldimethylsilyl group. There is no restriction as to which one to select as the ζ4 of compound [1■] from among these hydropal protective groups, but triarylmethyl groups can be preferably used since their introduction and deprotection are easy. It is something.

Furthermore, as the organic group R in the compound On], basically all phosphate protecting groups are possible. Specifically, protecting groups developed for the production of nucleotides, such as lower alkyl groups having up to 5 carbon atoms, cyano alkyl groups, and haloalkyl groups.

Examples include an arylsulfonylalkyl group, an allyl group, an aryl group, and a haloaryl group. - Among these, there is no restriction as to which one to select as R in compound [III], but the phosphate protecting group for producing oridenucleotides [methyl group whose usefulness has been confirmed], β -Cyanethyl group, allyl group and 2-
A chlorophenyl group is preferably used.

Next, to explain the organic group R of compound CI[I], it can be any aryl group, specifically phenyl group, 2-methylphenyl group, 4-methylphenyl group, 2-methylphenyl group,
．． 4-tmethylphenyl group.

2.6-dimethylphenyl group, 2.4.6-)rimeterphenyl group, 2-10lophenyl group, 4-nitrophenyl! , 3.4-zochlorophenyl group.

Examples include α-naphthyl group. The choice of which aryl group to use as 86 from among these aryl groups depends on the type of protecting group R in compound (IV), so it cannot be limited; Considering the 2-methylphenyl group, which generally gives ease and good stability,
An aryl group having a substituent at the 2-position such as 2.6-dimethylphenyl group is preferably used.

Next, the compound (base residues in [3, B and B2
To explain, representative examples include a thymine residue, which may have a protecting group at the 3-position, as shown by the general formula CXII, and a thymine residue, which may have a protecting group at the 4-position, as shown in the general formula [XI[ ]. There are also cytosine residues, adenine residues that may have a protecting group on the amino group at the 6-position represented by the general formula CXIVI, amide groups at the 2.6-position, amino groups, and Examples include guanine residues that may have a protecting group on the keto group.

[M] [■] As protective groups for these bases, doarylmethyl groups and trialkylsilylalkyl groups are recognized as useful in the production of oligonucleotides.

Arylthioalkyl group, phthaloyl group, aryloxycarbonyl group, alkoxycarvinyl group.

Diarylcarbamoyl group, arylcar? Nyl group, alkylcarbonyl group, allyl group, etc. can be mentioned (general formulas C, 'G) ~ [XIV] The question of which group to use for the base residue has already been discussed. (For example, see Akira Hatatsuji et al., Journal of the Society of Organic Synthetic Chemistry, 42.429 (+984)).

Next, explaining n in compound (III), it can be 0 or a positive integer. Although it is not possible to limit the actual number of n in compound [■] as an intermediate for the production of oligodeoxynucleotides, judging from the ease of production of compound [■], The case of 0 or 1ltl is common) and is preferably used.

The phosphorus sulfide compound represented by the general formula [■] is
As shown in reaction formula (6), it is produced by the reaction of a thiodinucleotide represented by the general formula (XVI) and a 1,2,4-triazolylphosphine compound represented by the general formula [■]. (For details on manufacturability, see Special Title No. 1988-245326, filed by the applicant himself, and Japanese Patent Application No. 1983-245, filed on July 23, 1988.
Please refer to the specification. ).

11+-"nl [W] [Tame] 5/P\6 10 SR HI (In the formula, R'+ R5, R6, B", B2 and nl have the same meanings as above.0) Next, general To explain the deoxynucleotide f compound represented by formula (IVI), its organic group R5 is a phosphate protecting group as described above, and the base residue B3
and B4 are basic residues having the structure shown in the general formulas c to c and which may have a protecting group as described above. In addition, the organic group R of the compound [■] is basically used for producing oligodeoxynucleotides ((the developed 3'-hydroxy protecting group is possible. Specifically, an acedel group, a benzoyl group, a regulinyl group, Examples include a t-butyltmethylsilyl group or an organic group having a structure represented by the general formula.

[F]-yl-NHCO-Yl-CO-[XIU (wherein [F] represents a polymer carrier: Y1 and Y
1 is an organic group t that can form a covalent bond with the adjacent polymer 7 carrier, amide group, and carbonyl group.

) Silicadel is used as a single molecular carrier. Examples include polystyrene. Although Yl and Yl are not limited, alkylene groups having a p'-i number of 1 to 10 are often used. Among the above-mentioned protecting groups, compounds (R of IVI
Which one to choose as the reaction is determined by the reaction form of reaction (4). That is, when producing oligodeoxynucleotides by dissolving compounds [■] and [■] in a reaction solvent and performing a so-called liquid phase homogeneous reaction, acetyl groups, benzoyl groups, t-butyltmethylsilyl groups, etc. used. In addition, when producing an oligo compound by reacting compound [1■] and ([V) in a solid phase heterogeneous system, organic group [A1]
is used.

The n Fi of compound [IV:l] is determined by the length of the oligodeoxynucleotide to be produced, but generally when producing an oligo by the liquid phase method described above, B2
It is generally in the range of 0 to 30, and in the case of producing it by a solid phase method, the range is generally in the range of 0 to 200.

Reaction (4) according to the present invention is usually carried out by carrying out Kag IJ Nog reaction with compound [■] and (IVI) in the presence of compound [CI] and compound 1 [], and then phosphorusing the coupling product. First, to explain the coupling reaction between the compound [113 and [■], which consists of a reaction that oxidizes an atom, the reaction proceeds smoothly in an organic solvent. Such solvents include methylene chloride, chloroform, 1.1 −
Examples include zochloroethane, 1,2-dichloroethane, tetrahydrofuran, p-zoogisane, benzene, and toluene. It is preferable to use these solvents after drying them with a suitable desiccant and then purifying them by distillation or the like. The reaction is usually carried out at room temperature, but between 0° and 35°C.
It may be carried out within the range of °C. The molar ratio of Compound CI) to Compound (IVI) is 1 to 100 times equivalent of Compound CII and 1 equivalent of Compound [■] to 1 equivalent of Compound CIVI.
It is common to use 1 to 40 times equivalent of compound CLIII. The reaction is generally completed within one hour, but when the protecting group R of the compound [IVI is a thyl group, benzoyl group, regulinyl group, or t-butyltmethylsilyl group at 7-, that is, as described above, the reaction is completed in a liquid phase. If it is a reaction, the final reaction can be confirmed by thin layer chromatography (TI, C'), II NMR, etc., and it is best to carry out the oxidation reaction after confirming this. In addition, R in compound CIV) has the general formula [Tao]
In the case of an organic group shown by perform an oxidation reaction,
The generated orico-9 deoxynucleotide compound (VIE
The protective group R of is deprotected, and the reaction termination time is determined from the amount produced. In this case, if R4 is a triarylmethyl derivative such as a dimethoxytrityl group or a monomethoxytrityl group, the absorbance of the trityl cation generated during deprotection with an acid (this is the so-called trityl cation test, and the dimethoxytrityl cation is 4'? g nm,
It can be easily determined by measuring the monomethoxytrityl cation (at 475 nm).

Next, the oxidation reaction in reaction (4) according to the present invention will be explained. The reaction is an iodine-water system.

This can be carried out using m-chloroperbenzoic acid, iodobenzene no tate, nitrogen oxide, or the like. Among these reagents, there is no limitation as to which one to use for the oxidation reaction of reaction (4), but iodine-water is preferably used because it is easily available and inexpensive. In this case, the oxidation reaction is carried out in the range of -78°C to 35°C, using 1 to 200 times the amount of compound [Iv], and
It is recommended to react for 5 minutes.

If it is desired to produce an oligo having a longer chain than the oligodeoxynucleotide compound CVI produced by reaction (4), the retaining group R of compound [V] can be selectively removed by a known method. (If R4 is a triarylmethyl transparent conductor such as a dimethoxytrityl group or a monomethoxytrityl group, benzenesulfone, zochloro16fi, l) A protonic acid such as chloroacetic acid or a Lewis acid such as zinc bromide may be used. often used), compound 0
GI) and then use this instead of compound CIVI to carry out reaction (4).

In this case, in the liquid phase method described above, after the compound [ice 1] is isolated and purified by silica gluchromatography,
This is generally reacted with compound (111).

However, in the case of the solid phase method, it is not possible to isolate and purify the compound, so before removing the retaining group R of the compound CV) to form the compound, reaction(
The 5'-011 group of compound CIV) which became unreacted in 4) was capped using an appropriate method.
) and then, after removing the protecting group R4, the compound (m]
A method is used to react with

(In the formula % R5, R'* B'+ B2+ B3+ B
'*nl and R2 have the same meanings as above. ) The above-mentioned life-yampinog reaction is basically a compound (fV)
Any reaction that forms a stable chemical bond with the 5-〇H group of ) system is often used.

The meanings of the abbreviations used in this specification are as follows.

Bt: z-thyl group, n-Pr: n-globyl group.

n-Bu: n-butyl group, Bz: benzoyl group.

TBDMS: t-butylzomethylsilyl group, D
M'rr: 4.4'-dimethoxytrityl group 1MMT
r: 4-monomethoxytrityl group, T: thymine residue.

1) Z, 4 C,N-benzoylcytosine fi group, A”: N6-
pen! Yladenine residue, Glb: N2-isobutyrylguanine residue.

EXAMPLES The present invention will be specifically explained with reference to Examples, but the present invention is not limited to the Examples.

Example 1 [111][IVI 3'-〇-penzoylthymizone (IVI (1,731
5 mmol), 5'-0-nomethoxytritylthymisin-3'-0-(2-chlorophenyloxy-2-methylphenylthio)phosphine [:lII] (s, 7 g
17 mmol), iJ ethylstannonyl-1°2.4
-1-Riazole CII (4,1+g, 15 mmol) and 1,2-nochloroethane (50 ml) were added in this order. Immediately afterwards, pyricin (4,0d,
50 mi! 7 mol) was added. After stirring for 5 minutes in chamber B, iodine (6,359,25 mmol) was added to tetrahydrofuran (lOOd), pyridine (5,5 mg)
and a solution in water (5,5d) was added. After stirring at room temperature for 1 minute, the reaction solution was washed with a saturated aqueous sodium bicarbonate solution (50-X3), and the organic layer was dried over anhydrous magnesium sulfate. After f1 filtration, the r liquid was concentrated under reduced pressure, and the residue was passed through silica gel column chromatography and eluted with chloroform-methanol (10:I). The eluate was concentrated to dryness, and the foamy solid produced was dried under reduced pressure to obtain 4.84 g (yield: 91 pieces) of P-2-chlorophenyl-5'-〇-zomethoxytritylthymicyl (3'→
5')-3'-0-pendiylthymidine [V) was obtained.

Rf (CHC/3:MeOH=10:I)
= 0-57I'HNMR (CDC/3.TMS)
: δ1.40 (a+3n+s-c!!,)*1, g
2 (S, 3H, 5-CR2), 2.10 −
2.90 (m, 4H, 2X2.

3.10-3.65 (m, 2H, 5'), 3.66
(!l-6H, 2XC sio-).

4.05-4-70 (m, 4H, 2X4' and 5'
), 5.20-5.60 (m, 2H, 2X3'
), 6.38 (t, 2H, J=7.OHz,
2X one.

6.60-6.90 (m, 4H, ph), 6.90
-8. OO(m, 20H.

2×6 and ph) and 9J8 (s12H, NFI
)ppm.

Table 1 - Same as Example 1 using deoxynucleotide phosphorus sulfide compound ([I[:1.) realkylstannylazole (I), m group [■] and deoxynucleotide compound CrV] described in Table 1 The dideoxynucleotides or trideoxynucleotides listed in Table 1-BK were obtained.

Example 6 ([][IV] (V) 5'-〇-dimethoxytritylthymizone supported on polystyrene ([F]) (supported amount 120 micromol/g
Thymidine polystyrene resin (IV
Tohi ethylsmennylimidazole (fl (l)) was added to I (l OBi, 1.2 micromol).

"i'IP, 36 micromol) and the 1,2-
Add dichloroethane (0,2-) solution and immediately add 5'-
〇-dimethoxytritylthymizone-3'-0-(2-chlorophenyloxicu-2-methylphenylthio)phosphine Cl111 (20F, 24 micromol) 1,2
-dichloroethane (0.15 tnt) solution was added.

After shaking at room temperature for 4 minutes, 1,2-dichloroethane/
and excess fH compounds [I], CII) and [III
] was separated through a G-4 glass filter, and the remaining polystyrene resin was mixed with methanol (3-) and pyridine/(3-).
d). Then tetrahydrofuran/pyricine/water (o, a s −/0.025-70.025
*) Iodine (30i1F, +20 micromol) was added to the mixture, and the mixture was shaken and reacted at room temperature for 2 minutes. The tetrahydrofuran/pyricine/water mixture containing excess iodine was separated through a G-4 glass filter, and the remaining polystyrene resin was filtered with pyricine (5dX2) and 1.
Washed with 2-dichloroethane (5d). Absorbance measurements at 4'? g nm, details of this test method can be found for example in H
, G., Gagsen et al., “Chemical a
nd Enzymatic Synthesis of
Gene Fragmenta”, Verlag
See Chemie (Weinheim) + 1982 and references therein. ) showed that the above reaction was proceeding with a yield of 98%. This yield was confirmed by deprotecting the protecting group of the product by the method shown below and then analyzing the obtained deprotected product by high performance liquid chromatography (HPLC). That is, the above resin is
yn-4-nitrobenzaldosim (50■, 30
0 micromol) and 1,1,3,3-tetramethylguanidine (34,511?, 300 micromol) in p-
A solution of dioxane (0,3d) and water (0,3-) was added, and the mixture was left to react at room temperature for 10 hours. Thereafter, this mixture was filtered, the p liquid was concentrated, and the residue was directly
Vinegar rR(+, 5fRt)V was added, and the mixture was allowed to react at room temperature for 30 minutes. Acetic acid and water were distilled off under reduced pressure, and water (Iy
) and ether (5-) were added, and the deprotected product was extracted with water. After further washing the aqueous layer with ether (5-x4), HPLC analysis of the aqueous layer (see Figure 1) revealed that it contained about 98% of thymidine dimer (d(TpT)) and thymidine (dT). It was shown that it was generated at a ratio of 20 to 20%. The results shown in Figure 1 and Figures 2 to 4 below are based on UNISIL PACK (SC) as a column.
I8-250 Base A, Gascro Industry 5! ! p
H7.0 in 0.1M triethylammonium acetate 5~
This was obtained by gradient elution with 25 liters of acetonitrile for 30 minutes.

Example 7 [■] 00 [V] Nomethoxytritylthymidine supported on polystyrene ([F]) IC (supported amount 120 micromol/g).

Thyminone boristyrene resin CIVI obtained by reacting commercially available product) with 5-tidolychloroacetic acid in 1,2-dichloroethane
(l Ovq, 1.2 micromol)
-butylstannyl-1,2,4-triazole CD
(34,4v, 96 micromol) and 2-methylimidazole [■] (9,8511F, 120 micromol) in 1,2-dichloroethane (0,20d) were added immediately. -Zomethoxytrityldeogicithinone-3'-0-(2-chlorophenyloxy-2-methyl7z-nylthio)phosphine CI
II) 1 of (22111P, 24 micromol)
,2-dichloroethane (o,+s-) solution was added.

After a shaking reaction at room temperature for 4 min, 1,2-dichloroethane and excess compounds CI), Cn] and ([II]
Is it the G-4 glass filter? The remaining polystyrene resin was mixed with methanol (3-) and pyridine/(3y
d). Then tetrahydrofurano/pyricin/water (0,45 W1t10,025 d/0.
025 d) Iodine in the mixture (30 ■.

120 micromol) was added and the reaction was incubated with shaking for 2 minutes at room temperature. The tetrahydroflough/pyrison/water mixture containing excess iodine is separated through a G-4 glass filter, and the remaining polystyrene resin is treated with pyricine (5ix2).
and 1,2-dichloroethane (5 gLt). A trityl cation test on a portion of the resin showed that the reaction was proceeding in 98-yield. This yield was confirmed by HPLC analysis of the deprotected product produced by deprotection of the product. That is, ayn
-4-nitrope/dualdoxime (50119,300
A solution of 1.1.3.3-tetramethylguanisine (34.5η, 300 micromol) in p-dioxane (0.3 m/) and water (0.3 d) was added, and the solution was heated to room temperature. The mixture was allowed to react for 10 hours. Thereafter, 28 thiammonia water (1-) was added, and the mixture was reacted at 600C for 5 hours. This mixture'? IF', the P solution was concentrated, 80 thiacetic acid (1.5 m/) was added to the residue, and the mixture was reacted at room temperature for 30 minutes. Acetic acid and water were distilled off under true pressure, and water (1-) was added to the residue.
and ether (5-) were added, and the deprotected product was extracted with water.

After washing the aqueous layer with ether (5iX4), the aqueous layer was subjected to HPLC analysis (see Figure 2).
It is a dimer of deoΦcycytidine and thymidine (d(C
pT).

[V')) and thymidine (dT) were shown to be produced at a ratio of approximately 98:2.

H [V'] Example 8 CIII) [:[V:] [V] Thymidine [, IV
](10, 1,2 micromol), tri-n-butylstannylbenzo) U7:,'-[11(3q
η.

96 micromol), 1-methylimidazole CITE
(9,8519,120 micromol) and furnace-penzoyl-5'-0-nomethogycitrityldeoxyadenosine-3'-0-(2-chlorophenyloxy-2-
Methylphenylthio)phosphine [:[I] (231
! ? .
As a result, it was confirmed that the above reaction was proceeding with a yield of 99-yield, and HPLC revealed that 99-yield of the product was a dimer of deoxyadenosine and thymidine (d(ApT
)) It was shown that.

Example 9 [[■] [V] Thymidine [■] ([F]) supported on polystyrene ([F])
When n2=0, I O"7.1.2ff (0 mole).

) IJ -It-globylstanylpenzoi77zole CI) C3sη, 96 micromol), 1-methylimidazole [■] CC1,F55f! , 120 micromol) and 5'-0-nomethoxytritylthyminone-3'-0-(2-chlorophenyloxy-2-methylphenylthio)phosphine C11l] (20 m9°2
4 micrometers) in the same manner as in Example 6,
Tetrahydrofuran/bilinone/water (0,45 td/0.
025-/0.025 d) An oxidation reaction was carried out in the same manner as in Example 6 using the mixed solution. G the mixture containing excess iodine
4-dimethylaminopyridine (12,2Trq).

A mixture of 41 acid anhydride (0.18 J and pyridino (+, 2-) containing 100 micromoles) was added and shaken for 2 minutes at room temperature to perform a capping reaction. Resin 71,2-sochloroethane (5r=
E), add polytrichloroacetic acid α (2 ml) in 1,2-nochloroethane, and stir for 1 minute.
The nomethoxytrityl group was removed by a reaction. Separate excess of the above reagent through a G-4 glass filter,
The residual resin was washed with pyricin (5TI@t) and 1,2-dichloroethane (5-). This resin (formula [1■] above)
When n2=1), the same amount of compounds CI3~[■] as above was added, and the coupling reaction was carried out in the same manner. Furthermore, oxidation reaction and capping reaction were performed in the same manner as above, and the residual resin was washed with 1,2-dichloroethane (s-). The cyclotrityl group was then deprotected with 5-titrichloroacetic acid in 1,2-dichloroethane and n2
=2, ie tri-i- of thymidine supported on polystyrene was obtained. After repeating this operation five more times (that is, up to n=7 in the above formula CIVI) to obtain an octamer of thymizone, using the same amounts of compounds [1] to [1■] and iodine as above, coupling reaction and oxidation reaction were carried out. was carried out in the same manner, and the resin was treated with pyricin (5dX
After washing with 2) and 1,2-dichloroethane (5-), a trityl cation test was performed in the same manner as in Example 6, and compound CVI (when n2 = 7) was produced in a total yield of 85 yen. It was confirmed that there is. Then 5yn-4-nitropenzaltoxime (150”F).

900 micromol) and 1,1,3,3-tetramethylguanisine (103,5 cape, 900 micromol) and p-zoogysan (0,9 m) and water (0,9 tTlt).
The mixture was further deprotected in the same manner as in Example 6 using 80 polyacetic acid (2-). HPLC analysis of the obtained crude product (4th
(see figure), the presence of thymidine nonamer was confirmed.

The physical properties of the deo-Φ-cynucleotide phosphorus A phyto compound (III) used as a production intermediate in the method of the present invention are as follows.

[Example 1-4.6 and 9] Rf value (CHCl5:MeOH=go:I)
=O-30'HNMR (CDCl3.T station) δ:
1.46 (8,3H,5-CH5).

2.20-2.85 (m, 5H, 2,2g and 2.38
2' and CH3C6H45-),
3.35-3.65 (m, 2H, da), 3.69 (
a, 3u, c■, QC6H40-), 3.70 (+
s, 3u, c! ! 3oc6u40-).

4.25-4.50 (m, IH, 4'), 5.
45-5.80 (rn, IH, 3').

6.49 (t, lH, J=7.0Hz, l'), 6
．． 65-6.90 (m, 4H.

ph), 6-90-7.75 (m, l8H-6 and ph), 10.3 (s.

H(, N1() ppm.

[Example 5] Rf value (CHCl:MeOH=20:I)=
0.36'HNMR (CDC73, TMS) δ: 1
135 (also 813H15-C).

1.84 (g, 3H, 5-C tans), 1-90-2.
85 (m, including singlet in 7H12-34, CdiC6
H,S- and 2X2'), 2.85-3.
65 (m, 2H, 5'), 3.73 (8,6H, 2
XCH,QC6H,-), 3.95-4.70 (m
, 4H, 2X4' and 5') 5.05-5.65
(rn, 2H, 2X3'), 6-05-6.55 C
m, 2H, 2Xl'), 6.60-645 (m.

4H, ph), 6.85-7.60 (m, 23H
, ph and 2X6).

9.28 (s, IH, Nu), 9.36 (s, I
I, Nu) ppm.

[Example 7] Rf value (CHCts: Meat (= go:l)
= o, 4g'HNMR (CDCl3, TMS
) δ: 2-20-2.70 (m, 4H.

including singlets at 2.29 and 2.36, 0.5X2' and CH5C6H4S -) *2, gO-3,10 (
m, lH, 0,5X2'), 3.40-3. '? O(m
el, 5' and 2XCH3QC6H,-), including singlets at 3.65 and 3.69.

4-30-4.60 (m, lIl, 4'), 5.40
-5.70 (m, I) I, 3').

6.36 (t, IH, J=7.0Hz, I'),
6.65-7.70 (m, 26H.

ph), 7. IE -8,00(m,+H,s),
8.10-8.30 (rn.

IH, 6), 10.6 (8, IH, NH) pp
m.

[Example 8] Rf value (C'dC15: !, 1eO11=go: l
) =O-34'HNMR(cDCt5.TMS
) δ: 2.3+ (s, 1.5H+0-5XCHCH
5-)-2,37(s, 1.5H, 0,5xC is also C6H
45-).

2.55-3.25 (m, 2H, 2'), 3.25
-3.60 (rn, 2H, 5').

3-70 (', 6H-2XCHsOC6H4"), 4
-35-4.65 (m, IH14'), 5.45-
5.75 (m, lH, 3'), 6.30-6J5
(m, 5H.

1' and ph), 6-85-7.60 (m, 20
H, ph), 7.75-8.05 (m, 2IL, p
h), g, os -8,20 (m, lH, 2),
C55-C75 (m, IFI, g), F5. gg(
a, lH, NH) pprn.

[Brief explanation of the drawing]

! Figures X1 to 4 are Examples 6, 7.8 and 9, respectively.
The elution curve of the high performance liquid chromatography of the uncoupling product of the objective compound oligodeoxynucleotide (v) of the present invention prepared in
pT) is a dimer of thymidine, d(CpT)
is a dimer of deoxycytinone and thymidine, dApT
represents a dimer of deoxyadenosine and thymidine, and d(Tp)8T represents a nonamer of thymidine, respectively. Figure 1 Figure 2 Figure 3

Claims (1)

[Claims] A deoxynucleotide phosphorus sulfide compound represented by the general formula [III] in the presence of a trialkylstannylazole compound represented by the general formula [I] and a base represented by the general formula [II] A method for producing an oligodeoxynucleotide represented by the general formula [V], which comprises reacting the deoxynucleotide compound represented by the general formula [IV] with the deoxynucleotide compound represented by the general formula [IV], and then carrying out an oxidation reaction. R^1_3SnX[I] (In the formula, R^1 represents an alkyl group; X represents an azolyl group.) R^2NR^3_2[II] (In the formula, R^2 represents a hydrogen atom, an alkyl group, or an aryl group. Represents a group; R^3 represents an alkyl group, or a hetero atom in which two R^3s may contain 1 or 2 nitrogen atoms, oxygen atoms, or sulfur atoms as heteroatoms together with adjacent nitrogen atoms. Represents a cyclic group, or R^2 and two R^3 together with adjacent nitrogen atoms represent a heterocyclic group which may contain 1 or 2 nitrogen atoms, oxygen atoms or sulfur atoms as heteroatoms. ) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [III] (In the formula, R^4 and R^5 represent protecting groups; R^
6 represents an aryl group; B^1 and B^2 may be the same or different and each represents a base residue that may have a protecting group; n^1 represents 0 or a positive integer, but When n^1 is 2 or more, B^2 is not limited to the same base residue. ) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [IV] (In the formula, R^5 represents the same meaning as above; R^7 represents a protective group that may contain a polymeric carrier; B^3
and B^4 represent base residues that may be the same or different and each may have a protecting group; n^2 represents 0 or a positive integer, provided that when n^2 is 2 or more, B^4 is not limited to the same base residue. ) ▲There are mathematical formulas, chemical formulas, tables, etc.▼[V] (In the formula, R^4, R^5, R^7, B^1, B^2, B
^3, B^4, n^1 and n^2 have the same meanings as above. )