JPH0368594A - Production of polynucleotide - Google Patents

Abstract

PURPOSE:To obtain a polynucleotide in high yield and short time without causing side reaction by reacting a nucleoside phosphorous acid ester derivative with a nucleoside in the presence of an imidazolinium compound. CONSTITUTION:The objective polynucleoside is produced by condensing a nucleoside phosphorous acid ester derivative of formula I (A is pyrimidine base or purine base: R<1> is hydroxyl-protecting group or mono or polynucleoside phosphorous acid ester residue; R<2> is H or cationic group) with a nucleoside of formula II (B is pyrimidine base or purine base; R<3> is same as R<1>) in the presence of an imidazolinium compound of formula III (R<4> and R<5> are lower alkyl; X and Y are halogen) in a solvent (e.g. acetonitrile) at room temperature and oxidizing the resultant polynucleoside phosphorous acid ester of formula IV. The amounts of the compound of formula I and the compound of formula III are 1-50mol and 1-250mol per 1 mol of the compound of formula II, respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing polynucleotides, and more particularly to a novel chemical synthesis method for polynucleotides useful as genetically recombinant materials.

[Conventional technology and its problems]

In recent years, advances in molecular biology, particularly in gene modification technology, have been remarkable, and along with this, the importance of chemical synthesis of genes is also increasing. Conventionally, methods for synthesizing genes, that is, polynucleotides, include the phosphodiester method C)I;
G, Khorana; rsome RecentD
developments in the Chemises
try of Phosphateasters of
8io1logical rnterastJ, Jo
hn Wileyand 5ons, Inc.
New York (1961)) is widely known, but it has the disadvantage that the condensation reaction takes a long time and separation is difficult.

Improvements to the phosphoric acid diester method include the phosphoric acid triester method and the phosphite method [R,L.

Letsinger, W.B., Lun5f.
ord; J, A+n, Cham, Sac,
.

98, 3655 (1976)), phosphoramidite method [5°, Beaucage, M.
H, Caruthers; Tetrahed
ron Lett, 22.1859 (1981)
) etc. have been reported.

By the way, the so-called H-phosphonate method [8, C, Froe
hler and M, D, Mattaucci;
etrahedron Lett,, 27.469
(1986) and P, J.

Garegg, T., Regberg, J., Sta.
winski, and R.

Str6mbarg, Tetrahedron La
tt., 27.4055 (1986)) has a short reaction time, the nucleoside phosphite unit is chemically stable, and it does not require a phosphoric acid protecting group.
It has advantages such as easy purification.

However, this H-phosphonate method has drawbacks such as the condensing agent pivaloyl chloride used is unstable in a solvent and side reactions occur.

Therefore, it has been desired to develop a method for producing polynucleotides that is a further improvement over the H-phosphonate method, which has various advantages.

[Means to solve the problem]

In view of these circumstances, the present inventors have conducted various studies to solve the above problems, and have found that when a specific imidazolium compound is used as a condensing agent for nucleoside phosphite in the H-phosphonate method, side reactions occur. Without,
The present invention was completed by discovering that polynucleoside phosphites can be obtained in extremely high yields and that polynucleotides can also be efficiently produced by oxidizing this.

That is, the present invention relates to the following general formula (1,) below the margin (III) X [wherein A represents a pyrimidine base or purine base,
' represents a protecting group for a hydroxy group or a mono- or polynucleoside phosphite residue, and R2 represents a hydrogen atom or a cationic group] (hereinafter abbreviated as H-phosphonate unit) and the following general formula (II) [wherein R4 and Rs represent a lower alkyl group, and X and Y represent a halogen atom]. Nucleosides represented by the formula (rV) [wherein B represents a pyrimidine base or a purine base, and R3 represents a hydroxy group protecting group or a mono- or polyscleoside phosphite residue],
Provides a method for producing a polynucleoside phosphite represented by the following general formula [wherein ASB SR' and R1 have the same meanings as above], and a method for producing a polynucleotide using the condensation reaction. It is something to do.

The present invention is shown by the following reaction formula.

[In the formula, R1-R5, ^, 8, x and Y have the same meanings as above] H-phosphonate unit (1
), A is a pyrimidine base or a purine base, specifically adenine, guanine, cytosine or thymine. As the hydroxy group-protecting group represented by R', any hydroxy group-protecting group generally used in the synthesis of polynucleotides can be used, including dimethoxytrityl group, 9-fluorenylmethyloxycarbonyl group, etc. , levunyl group, etc. are preferred. Further, examples of the cationic group represented by R2 include tertiary bases such as triethylamine and 1,5-diazabicyclo[5,4,0]undec-5-ene (OBtl).

The H-phosphonate unit of formula (I) is, for example, a tris(1,l, 1°3,
It can be produced by reacting phosphines such as 3.3-hexafluoro-2-propoxy>phosphine, bis(N,N-diisopropylamino)chlorophosphine, and bis(N,N-diethylamino>chlorophosphine).

Nucleosides (II), the other raw material for the method of the present invention
Examples of the pyrimidine base and purine base represented by B include those mentioned above. Furthermore, examples of protecting groups for hydroxy groups include protecting groups and solid supports that are generally used in the base of polynucleotides. Examples of the solid support include polystyrene, polydimethylacrylamide, polyacrylmorpholide, silica gel, porous glass beads, and the like. Note that the bonding with such a solid phase carrier is usually performed via a crosslinking agent such as a succinate ester.

Mononucleosides (I
In I), for example, a 5'-protected nucleoside is reacted with succinic anhydride, and the obtained 5'-protected 1ii-3'-〇-succinyl nucleoside and porous glass beads are treated with dicyclocarbodiimide, triethylamine, and dimethylaminopyridine. It can be obtained by reacting in the presence of

Further, the imidazolinium compound (III) which is a condensing agent used in the present invention is, for example, the following general formula (V) which is known as an easily available solvent (wherein R4 and R5 have the above-mentioned meanings) It is produced by reacting 1,3-dialkyl-2-imidazolidinone represented by the formula with a halogenating agent.

Examples of the halogenating agent used here include oxalyl halide, phosphorus trihalide, phosphorus pentahalide, phosphorus oxyhalide, phosgene, trichloromethyl chloroformate, and the like.

The reaction is carried out by dissolving either the 1,3-dialkyl-2-imidazolidinone or the halogenating agent in a suitable solvent such as carbon tetrachloride, adding the other one little by little to this, and then
The reaction is carried out by reacting at room temperature to 70°C for several hours to over ten hours.

1,3-dialkyl-2-halogeno- thus obtained
Although the imidazolinium halide can be isolated, its reaction solution can also be used in the condensation reaction of the present invention without isolation.

To carry out the present invention, H-phosphonate unit (I) is used in an amount of 1 to 50 times the nucleoside (If) in the presence of 1 to 250 times the mole of imidazolinium compound (III) for several minutes. The condensation reaction may be carried out for several tens of minutes. The reaction temperature is preferably room temperature, and the reaction solvent is preferably acetonitrile, pyridine, a mixed solvent thereof, or the like.

A polynucleotide can be produced by oxidizing the polynucleoside phosphite obtained by sequentially repeating such a condensation reaction according to a conventional method.

In the production of polynucleotides, the synthesis of the polynucleoside phosphites described above is preferably carried out in a solid phase. Oxidation of the polynucleoside phosphite on a solid support can be carried out, for example, by reacting iodine in tetrahydrofuran-pyridine-water. Furthermore, in order to remove the polynucleotide on the solid support, treatment with an alkali such as aqueous ammonia or the like may be performed. It is preferable to use a commercially available DNA kit for such polynucleotide synthesis.

In order to purify the obtained polynucleotide, it is preferable to use, for example, ion exchange or reversed phase silica gel high performance liquid chromatography.

〔Example〕

EXAMPLES Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 (1) Production of 5'-〇-dimethoxytrityl deoxyribonucleoside: A small amount of anhydrous pyridine was added to thymidine (10 mM, 2.4 g) and the mixture was dried under reduced pressure. After repeating this operation three times to dehydrate, dissolve in anhydrous pyridine (50-), add dimethoxytrityl chloride (12mM, 4.1g), and search by TLC at room temperature every 30 to 60 minutes to find the spot of the raw material thymidine. The reaction was allowed to occur until it disappeared. Ice water was poured into this reaction solution, extracted with methylene chloride, the organic layer was washed several times with water, dehydrated with sodium sulfate, filtered off, and the organic layer was condensed under reduced pressure. When dissolved in methylene and reprecipitated with a 5-10% ether-hexane solution, 5'-〇-dimethoxytritylthymidine (
d-DMTrT) white crystals (8.8 g, 1 ad,
A yield of 80% was obtained.

(2) Production of deoxyribonucleotide-3'-H-phosphonate: 5'-dimethoxytritylthymidine (DMTrT) (3
, 5mM, 1.92g) was added with a small amount of anhydrous pyridine and dried under reduced pressure three times to dehydrate. This was added to anhydrous pyridine (17
, 5mM, anhydrous triethylamine (31.5mM
M, 4.4mt') and further tris(1,1,
Add 1,3゜3.3-hexafluoro-2-propoxy)phosphine (10.5mM, 3.12m1!) and search by TLC every 30 minutes at room temperature under a nitrogen atmosphere, and after the reaction is completed, 0.1M1.5 -diazabicyclo[5,4,
0] undec-5-ene (080) aqueous solution, extracted three times with methylene chloride, and extracted the organic layer with 0.1 M triethylammonium bicarbonate (TBAB): ) ethylamine (Bt, N) = 50: 1 ( V/v
), 0. IM TBAB'? 'Washing was performed sequentially 5 to 10 times. At this time, the aqueous layer is occasionally searched by TLC to confirm whether any phosphonate units remain. After washing, the organic layer was concentrated under reduced pressure, and finally this was
Isolation and purification by silica gel column chromatography using a methylene chloride eluate containing % BtaN yielded the desired DBtl salt of nucleoside-H-phosphonate (3.03 mM, 87%, 2.306 g).

(3) Production of deoxyribonucleoside-H-phosphonate diester: 5'-dimethoxytritylthymidine obtained in (2)
080 salt of 3'-H-phosphonate 1 mmof, 3'-
〇-Benzoylthymidine 1.1mmo! , and 2.0 mmol of 1°3-dimethyl-2-chloro-imidazolinium chloride in acetonitrile-pyridine (1
:1) Stirred for IO minutes in a solvent at room temperature. 3IP reaction
- When monitored by NMR, the signal of the raw material H-phosphonate completely disappeared, and a new signal appeared at 8
．． 21 and 7.20 ppm, confirming the production of deoxyribonucleoside-H-phosphonate diester.

Example 2 (1) Introduction of nucleosides into control bore grass (CPG) l) Synthesis of 5'-〇-dimethoxytrityl-3'-0-succinylthymidine 5'-〇-dimethoxytritylthymidine (DMTrT)
(10mM, 5.44g) was dehydrated with a small amount of anhydrous pyridine, and then anhydrous pyridine (30Tnl) was added.
, dimethylaminopyridine (15mM, 1.8g)
.

Succinic anhydride (15 mM, 1.5 g) was added, stirred at room temperature for 5 hours, and reacted until the OMTrT spot disappeared by TLC. After the reaction was completed, this was concentrated under reduced pressure, extracted with methylene chloride (100 mg), and 5% carbonated)
Wash 3 times with IJum aqueous solution, 3 times with water, and rinse with sulfuric acid)
Dehydrate with um. This was concentrated under reduced pressure to completely remove the pyridine odor and reprecipitated (5% ether-hexane solution), resulting in the desired 5'-〇-dimethoxytrityl-3'-
〇-Succinylthymidine (DMTrTCO(CI(-)
*C00H) was obtained in 50% yield.

1f) Alkylamino CPG (raminopropyl CPG AMPO[l 500BJ, BLB(
'TR0-NtJCLBONICS, 1.5g)
and 5'-〇-dimethoxytrityl-3'-〇-succinylthymidine (1.4mM).

1, 204g) and dichlorocarbodiimide (7mM).

1.442g), anhydrous triethylamine (1.4tn
M, 0.196-), anhydrous dimethylformamide (7
mM, add dimethylaminopyridine (several pieces),
The reaction was carried out for 12 hours at room temperature using an agitator. After the reaction was completed, the CPG carrying the nucleoside was washed successively with anhydrous dimethylformamide, pyridine, methanol, and anhydrous ether, and dried under reduced pressure. A small amount of this was measured, perchloric acid was added to remove the dimethoxytrityl group, and the trityl cation was colorimetrically determined. As a result, it was confirmed that the target nucleoside was introduced in a supported amount of 39 μIIIo1/g.

This was converted into acetic anhydride:pyridine=1:9 (V/V)
The unreacted portions of the alkylamino groups were acylated by capping with several pieces of dimethylaminopyridine for 1 hour.

(2) Thymidine-introduced CPG (28 cm) was placed in a glass reaction tube with an inner diameter of 15 mm and equipped with a d-Tpl 4T bottom glass filter, and the CPG was first capped. Acetic anhydride: Lutidine: T
A solution of HF=10:10:80 and 7% dimethylaminopyridine in tetrahydrofuran was added to each IWL and reacted for 30 minutes. After the reaction, the mixture was washed with anhydrous acetonitrile. Next, the dimethoxytrityl group was completely removed with a 2.5% dichloroacetic acid solution in methylene chloride, washed successively with methylene chloride and anhydrous acetonitrile, and dried under reduced pressure for 10 minutes. After drying under reduced pressure, H-phosphonate unit (55μmol1゜50
eq), 1. 3-dimethyl-2-chloro-imidazolinium chloride (137,5μm01゜125
eq) to 1 ml of anhydrous acetonitrile: anhydrous pyridine =
It was dissolved at a ratio of 1:1 (V/V) and reacted for 10 minutes. After the reaction, washing was performed 3 to 5 times with anhydrous dimethylformamide, anhydrous acetonitrile:anhydrous pyridine=1:1 (V/V), anhydrous acetonitrile, and methylene chloride in this order. Repeat this series of operations 14 times.d-DMTr
Tpr<To/XAJCPG was synthesized. Each condensation yield is perchloric acid:ethanol = 3:2 (V/V)
The color development (Ls't) of the trityl cation was determined by colorimetry using the visible spectrum. The results are shown in Table 1.

Blank space below (3) Oxidation, elimination, purification, and structure confirmation of d-TP, TO~~CPG The oxidation of d-Tp + aToNXJcPG is O, 1M
Iodine in tetrahydrofuran: pyridine: water = 44:
3:3 solution at room temperature for 1 hour, and then washed with anhydrous acetonitrile. This oxidized the phosphonate to phosphate. The product was further removed from CPG by reacting with 28% ammonia water at room temperature for 30 minutes. This was mixed with an ammonium formate aqueous solution (pH 6,8) containing 20% acetonitrile (OJ8-0, 94M
) IPLC (
TSK get DRAB-23W) was isolated and purified.

As a result, the target product d-'rp14T (100 mouths) was obtained around 20 minutes. When this target product was confirmed by 20% polyacrylamide gel electrophoresis, it matched with the comparative oligothymidine, so the target d-TP, T
It was confirmed that

Example 3 Using 30 eq of H-phosphonate unit, 1°3
c+-'rp l 4T was synthesized in the same manner as in Example 2, except that 150 eq of -dimethyl-2-chloro-imidazolinium chloride was used. The yield in this case is shown in Table 2.

Furthermore, oxidation was performed in the same manner as in Example 2 (3), and d-T
p taT (2°50D) was obtained.

Below is the margin Example 4 Using 40 eq of H-phosphonate unit, 1°3
d-TI'+*T was synthesized in the same manner as in Example 2, except that 100 eq of -dimethyl-2-chloro-imidazolinium chloride was used. The yield in this case is shown in Table 3.

Further, d-Tp was oxidized in the same manner as in Example 2 (3).
+mT (1800) was obtained.

The following blanks [Effects of the Invention] As described above, according to the present invention, the condensation reaction of H-phosphonate (1) and nucleosides (II) can be carried out using a stable imidazolinium compound ( III)
By using , the reaction can be carried out in a short time and in high yield without causing side reactions. Therefore, the method of the present invention is extremely useful for gene synthesis, especially ON8 on a solid phase. This method can also be applied to automatic synthesizers.

that's all

Claims (1)

[Claims] 1. The following general formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, A represents a pyrimidine base or a purine base, and R
^1 represents a protecting group for a hydroxy group or a mono- or polynucleoside phosphite residue, R^2 represents a hydrogen atom or a cationic group] and a nucleoside phosphite derivative represented by the following general formula ( II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) [In the formula, B represents a pyrimidine base or a purine base, and R
The nucleosides represented by the following general formula (III) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) [In the formula, R^4 and R^5 represent a lower alkyl group, and
and Y represents a halogen atom] General formula (IV) characterized by reaction in the presence of an imidazolinium compound represented by ▲There are mathematical formulas, chemical formulas, tables, etc.▼(IV) B, R^1 and R^3 have the same meanings as above.] A method for producing a polynucleoside phosphite represented by: 2. In a method for producing a polynucleotide in which nucleoside phosphites are sequentially condensed and the obtained polynucleoside phosphites are oxidized, the condensation reaction of nucleoside phosphites is carried out by the following general formula (III) ▲Math. There are chemical formulas, tables, etc.▼(III) [In the formula, R^4 and R^5 represent lower alkyl groups, and
and Y represents a halogen atom] A method for producing a polynucleotide, which is carried out in the presence of an imidazolinium compound represented by the following. 3. Condensing nucleoside phosphites sequentially on a solid support, oxidizing the obtained polynucleoside phosphite to form a polynucleotide, and then
In a polynucleotide production method in which the polynucleotide is detached from a solid phase, the condensation reaction of nucleoside phosphites is carried out using the following general formula (III) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) [In the formula, R ^4 and R^5 represent lower alkyl groups, X
and Y represents a halogen atom] A method for producing a polynucleotide, which is carried out in the presence of an imidazolinium compound represented by the following.