JPS59199700A - Production of oligonucleotide - Google Patents

Abstract

PURPOSE:The condensation reactions of a plurality of oligonucleotide-3'-phosphates is effected in the presence of a specific arenesulfonly chloride and an alkylimidazole at room temperature give the titled compound without side- reactions with industrial advantages. CONSTITUTION:The condensation reaction between (A) an oligonucleotide-3'- phosphate of formula I (B1, B2 are thymine, uracil, N-benzoylcytosine, N-benzoyladenine; Z is dimethoxytrityl; R1 is H, protected hydroxyl; R2 is 2-clhorophenyl, cyanoethyl; m>=0) and (B) another oligonucleotide-3'-phosphate of formula II(B3, B4 are thymine, N-benzoylcytosine; R3 is 2-chlorophenyl, cyanoethyl; n>=0) is carried out in the presence of an arenesulfonyl chloride of formula III (X1, Y1, Z1 are H, lower alkyl) or formula IV (X2 is CH, N; Y2, Z2 are H, SO2C1) and of an alkylimidazole of formula V (R is alkyl) to give a compound of formula VI.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing oligonucleotides, and as a condensing agent for the production.

This method uses allenesulfonyl chlorides and alkylimidazoles.

Oligonucleotides are important materials in genetic engineering as recombinant products.

Conventionally, as a method for producing oligonucleotides, that is, in order to form an internucleotide bond using a dehydration condensation agent, it is common to use a condensation agent as shown in formula [■]. However, their preparation is cumbersome and, although some are commercially available, they are very expensive.

There is also a method using only allenesulfonyl chloride, but 1
The disadvantages were that the reaction took a long time and there were many side reactions.

The present inventors have found that when using allenesulfonyl chlorides and alkylimidazoles as a condensing agent system, a short period of time (1
The present invention was completed based on the discovery that oligonucleotides can be produced at low cost and in high yields because the reaction is completed within a short period of time (within 10 minutes) and post-treatment is easy.

That is, in the present invention.

General formula CI) [In the formula, B1 and B2 are the same or different and represent any base of thymine, uracil, N-benzoylcytosine, N-henzoyladenine, or N-isobutyrylguanine, and Z is dimethoxy represents a trityl group, R1 represents hydrogen or a protected hydroxyl group, R2 represents any of 2-chlorophenyl, l,4-chlorophenyl, cyanoethyl, anilide, or anilide, and 2m represents an integer of ≧0. show. However, when 1m is 2 or more, B2 is not limited to the same base. ] An oligonucleotide-3'-phosphate represented by:

General formula [■] [In the formula, B3 and B4 are the same or different, thymine, N
-benzoylcytosine, N-benzoyladenine, N-
Indicates any base of isobutyrylguanine, R,,
R2 is the same as above, R3 is 2-chlorophenyl group, 4-
It represents either a chlorophenyl group, cya, noethyl group, anilide group, or aniside group, and n represents an integer of ≧0. However, when n is 2 or more, B4 is not limited to the same base.

] Oligonucleotide-3-phosphate represented by is reacted in the presence of an allenesulfonyl chloride and an alkylimidazole to form a compound of the general formula (II) [where B1
．． B2. B3. B4. R+, R2°R3, m, and n are the same as above. ] The content is to produce an oligonucleotide represented by the following.

The compound of formula (1) is an anion, the counterion of which is usually tertiary ammonium, for example.

Triethylammonium ion is selected.

The molar ratio of raw materials, reagents, etc. is usually 9 to 1 molar ratio of triester represented by general formula (I[) to 1.5 to 2.0 mol of diester represented by general formula (T), allenesulfonyl. 3 to 5 moles of chlorides, 7 moles of alkylimidazoles
It is common to use ˜10 moles.

Further, according to the present invention, the reaction is usually carried out at room temperature and is completed in 30 minutes to 1 hour, so that side reactions can be suppressed.

In the present invention, the raw material compound (1), (I[), in which m and n are 0, can be converted more easily than commercially available products.

It is preferable to apply the present invention in the production of products where m and n are 1 or more, but in addition, for example, Jour
nal of Biochemistry 81.12
37 (1979).

After the reaction, the reaction product oligonucleotide can be easily obtained by subjecting the 5 reactants to silica gel column chromatography.

Since the produced oligonucleotide has protective groups at the 5' and 3' ends, a longer chain oligoribonucleotide can be produced by using this as a raw material and then carrying out the present invention again.

The reaction of the present invention can also be carried out by a solid phase method.

For the synthesis method using the solid phase method, refer to the following literature.

Miyoshi + Arentzen, R
,l Huang, T., and Itakur
a, K. (1980) Nucleic Ac1
dRes, 8+5507-5517 In the present invention, those in which R1 is a hydroxyl group can be reacted in the same manner as those in which R1 is hydrogen by appropriately protecting them.

As the condensing agent system used in the present invention, the following can be mentioned.

Allensulfonyl chlorides have the following general formula (I
V) [In the formula, XI, Yl and Zl are the same or different,
Indicates hydrogen, lower alkyl, or nitro group. ] or the first-order general formula (V) [wherein, X2 represents CH or N, Y2 and z2
are the same or different and represent hydrogen or -3O2-CI. ] Compounds represented by the following can be mentioned.

In addition, as alkylimidazole, the first-order general formula (V
l) [In the formula, R represents an alkyl group. ] Compounds represented by the following can be mentioned.

The temperature of the condensing agent system used in the present invention is lower than that of MSNT (2,4,6-)limethylbenzenesulfonylnitrotriazolide), which has recently been frequently used for the same purpose.

It is stable to light, easily available, and the reaction time is about the same.
A highly purified target product can be easily obtained in high yield.

Using the method of the present invention, any long oligonucleotide can be easily produced.

According to the present invention, oligonucleotides, which are important materials in genetic engineering, can be provided accurately and at a very low cost.

Examples will be described in more detail below, and the abbreviations used in each example are as follows.

NO2HH4
Synthesis of 3-(2-chlorophenyl-2-cyanoethyl)phosphate (1) (wherein DMTr is dimethoxytrityl group, R is 2-chlorophenyl group, R+ is 2-chloropheny) Lt group, R2
represents a 2-cyanoethyl group, Bz represents a benzoyl group, T represents thymine + BzC represents N6-hendiylcytosine. The same applies to the following examples. )5'-0-dimethoxytritylthymidine-3'-(2-chlorophenyl)phosphate triethylammonium salt ■175mg (0,
15X 1.4 mmol) and N4-benzoylcytidine-3'-(2-chlorophenyl-2-cyanoethyl)
Phosphate ■86 mg (0.15 mmol) was dissolved in a small amount of pyridine, and after azeotroping twice, dry pyridine (
Dissolve n-nonylimidazole Σ
0.25ml and 198ml of mesitylenesulfonyl chloride
Add g (0,15X 3.Ommol) and leave the chamber? l
AReact for 30 minutes. After the reaction ends, +120 (1,
0 ml) and concentrated to dryness using a vacuum pump. The residue was partitioned between methylene chloride and water, and the organic layer was washed with water and then concentrated to dryness. The residue was purified by column chromatography [silica gel 60H (Merck) (3, Ogr) (developing solvent MeOH/CH
2CI2)) to give 5'-0-dimethoxytritylthymidyl-(3'-(2-chlorophenyl)-5')-N4-benzoylcytidine-3"-(2-
196 mg (101%) of chlorophenyl-2-cyanoethyl/L/)phosphate (2) was obtained.

Hexane and methylene chloride at mpHo-115°C (
Dec, ) became a colorless powder.

Examples 2 to 10 The same operations as in Example 1 were performed using the phosphodiesters and phosphotriesters listed in Table 1. Phosphodiester is 0.15X 1.4 mmol, phosphotriester is 0.15 mmol, allenesulfonyl chloride is 0.15X 3. Ommol, alkylimidazole is 0.15X 9. Ommol was used. Yields are based on phosphotriester.

Example 11 Synthesis of Tetram6r 5'-〇-dimethoxytrityl-N4-penzoylcytidylyl [3'-(2-chlorophenyl)-5')
-N6-hendiyladenosilyl-[3i2(chlorophenyl)-5']-thymidylylu (3'-(2-chlorophenyl)-5')-N4-benzoylcytidine-3
Synthesis of co(2-chlorophenyl-2-cyanoethyl) phosphate ■
)-N6-penzoyladenosine-3'-(2-chlorophenyl)phosphate triethylammonium salt ■2
1Bmg (0, I
4-benzoylcytidine-3'-(2-chlorophenyl-2-cyanoethyl)phosphate ■ (0.1 mmol
) was dissolved in a small amount of pyridine, and after azeotropic distillation twice, it was dissolved in dry pyridine C (1.2 ml) to form 1-methylimidazole a (0,075ml 1.0.1x9 mmol) and mesitylenesulfonyl chloride 3 (66mg 0.1x3+).
n+nol) and left at room temperature for 30 minutes. After the reaction is complete, add H20 (1, 1) and concentrate to dryness using a vacuum pump. The residue was partitioned between methylene chloride and water, and the organic layer was washed with water.

Concentrate to dryness. The residue was purified by silica gel 60H (Merck
)(3,Ogr) C)+2-C12-MeOQ system separates and purifies tetramer ■212 mg (91%)
I got it.

Triturate with hexane and methylene chloride.

mp 129-134' (dec) Uv:λ
mM (Eton) 236. 260 Example 12 Synthesis of 01igomar (deoxynonamer) by solid phase method 5'CTCTCTCTG3' 0operation of 5solid-Phase
5ynthesis aminomethylated 1%Poly
For the 3' side of styrene (commercially available), mononucleoside is bound to the resin as described above, and the following oper
Diester was condensed four times according to ation.

1 CH2-CI2 MeOH(7-3) 2
ml x3 0.7 m1n2
2%BSA in CH2-CI2-MeOH (7
-3) 3ml 1.03 CH2-
CI2-MeOH (7-3) 2ml X 1
0.24 2%BSA in CH2-
C12-MeOH (7-3> 2ml xiO
,55CH2-012-MeOH(7CH2-0l2-
0.26 pyridins
2ml×30.
27 pyridine 0.2 ml
(azeotropic) 8 diester-p
Tscl/1-methylimidazole-
Py 409 pyridine 2 m
lX 2 (washing) 11 0
, IM DMAP/pyridine 1
．． 8ml 10Ac
2 0 0.2 ml 12 pyridine 2 ml, X 3
0.2 In the formula, BSA is benzenesulfonic acid diester, pTscl is para-toluenesulfonyl chloride DMAP
indicates N,N-dimethylaminopyridine・5tep
8 is diester (20mg) + pTsc
l (20mg) + 1-Methylimi
dazole in pyridine (0,08
m1/ml) solution to 300μβ-330μ! Using.

After all reactions are completed, cut out from the resin as usual.

and deprotection.

That is, 1M tetramethylguanidine-pyridine aldoxime in dioxane (0.75 ml), dioxane (0.60 ml), shaken with H20 (0.15 ml) at room temperature for 46 hours, and then washed the resin with 50% pyridine water (10 ml) and the filtrate. After concentrating, pyridine (0.5 ml),
Add concentrated ammonia water (10ml) and heat at 55℃ for 5 hours.
Warm up. After the reaction, concentrate to dryness and apply a reversed phase open column (
C+s).

When separated and purified using an ion exchange oven column, pure nonamer dCp 'rp cp 'rp cp 'rp cp
TpGo)I was obtained. The correctness of the base sequence was confirmed by two-dimensional homochromatography.

Applicant: Nippon Shinyaku Co., Ltd. 1゜Representative
Patent attorney Hiroshi Katama-84'/

Claims (1)

[Scope of Claims] General formula (I) [In the formula, B1 and B2 are the same or different bases of any one of thymine, uracil, N-benzoylcytosine, N-benzoyladenine, and N-isobutyrylguanine; , Z represents a dimethoxytrityl group, R1 represents hydrogen or a protected hydroxyl group, R2 represents any of a 2-chlorophenyl group, 4-chlorophenyl group, cyanoethyl group, anilide group, or aniside group, and 2m represents a Indicates an integer of ≧0. However, when 9m is 2 or more, B2 is not limited to the same base. ] An oligonucleotide-3'-phosphate represented by: General formula [■] ] [In the formula, B3 and B4 are the same or different, thymine, N
-henzoylcytosine, N-benzoyladenine, N-
Indicates any base of isobutyrylguanine, R,,
R2 is the same as above, R3 is 2-chlorophenyl group, 4-
It represents either a chlorophenyl group, a cyanoethyl group, an anilide group, or an aniside group, and n represents an integer of ≧0. However, when n is 2 or more, B4 is not limited to the same base. ] Oligonucleotide-37-phosphate represented by the formula is condensed to form the general formula (I[[) [wherein, B, , B2. B3. B4. R,, R2゜R3,
m and n are the same as above. ] In producing the oligonucleotide represented by the general formula (IV) 1 [wherein] I, Yl and Zl are the same or different,
Indicates hydrogen, lower alkyl, or nitro group. ] or General Formula (V) 9 [In the formula, x2 represents CH or N, and Y2 and Z2 are the same or different and represent hydrogen or -302-C1. Allensulfonyl chloride represented by ]. and general formula (VI) [wherein R represents an alkyl group]. ] is characterized in that the reaction is carried out in the presence of an alkylimidazole represented by A method for producing an oligonucleotide represented by the general formula (I[[).