JPS6284097A - Production of protected oligonucleotide - Google Patents

Abstract

PURPOSE:To obtain the titled compound rapidly deprotective under mild conditions in high yield, by condensing a raw material for synthesizing an oligonucleotide with a phosphoramidite ragent and oxidizing the formed phosphite part into phosphate. CONSTITUTION:A raw material for synthesizing an oligonucleotide expressed by formula I is condensed with a phosphoramidite reagent expressed by formula II (R1 is protecting group or residue of carrier linked through covalent bond; R2 is H or OH having protecting group; R3 is protecting group; A is protecting group eliminating by beta-cleavage; BAOC is residue of nucleoside base having no amino group or nucleoside base having amino group or imino group protected by allyloxycarbonyl type residue; NAOC is residue of nucleoside base having amino group or imino group protected by allyloxycarbonyl type residue; n is 0 or a positive integer) and the formed phosphite part is then oxidized into phosphate to afford the aimed compound expressed by formula III.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing protected oligonucleotides, and more specifically, the present invention relates to a method for producing protected oligonucleotides, and more particularly, the hydroxyl group of the phosphate moiety and the amino group of the nucleoside base moiety are removed by β-cleavage. Protecting group and allyloxycar to be removed? The present invention relates to a method for producing protected oligonucleotides using a phosphoramidide reagent protected with a nyl-type residue as a monomer component.

(Conventional technology) With the recent development of genetic engineering, its important materials such as DNA (deoxynucleotide is a nucleic acid) and RNA (lysonucleic acid)
Research is being actively conducted on methods for chemically synthesizing polynucleotides, such as the phosphodiester method, phosphotriester method, and phosphite method.

Among these, the phosphite method, which uses a phosphoramidite reagent as a monomer component, has superior reactivity compared to other methods and has recently attracted particular attention. Generally, in order to avoid side reactions in the condensation reaction, t-protected phosphoramidite reagents are used as monomer components that protect the hydroxyl group of the phosphoric acid moiety and the amino group of the nucleoside base moiety. A method has been developed in which a protecting group that is removed by β-cleavage, such as a β-cyanethyl group, a β-octahedanoethyl group, or a β-nitroethyl group, is used in place of the methyl group, which has been widely praised (e.g., WO85100816). , Tetrahedron Letters Vol. 24, No. 52, pp. 5843-5846, etc.).

According to this method, thiophenol treatment, which was a drawback of the conventional method, can be avoided because deprotection is easier compared to methyl groups, and when applied to solid phase synthesis, the support can be treated with ammonia. It has the advantage that hydroxyl groups can be deprotected at the same time when they are removed by treatment.

However, in this method, benzoyl group, imbutyryl group, etc. are used as protecting groups for amino groups, and in order to remove these protecting groups, treatment with hot ammonia water is required for a long time. There was a problem.

(Problems to be Solved by the Invention) Therefore, as a result of intensive studies to solve the drawbacks of the prior art, the present inventors found that a protective group that is eliminated by β-cleavage and an allyl group as a protective group for a hydroxyl group and an amino group, respectively, The present invention was completed based on the discovery that when a phosphoramidite reagent having an oxycarnyl-type residue is used as a monomer component, a protected oligonucleotide that can be rapidly deprotected under mild conditions can be obtained in good yield. I came to the conclusion.

(Means for Solving the Problems) Thus, according to the present invention, a raw material for oligonucleotide synthesis represented by the following general formula [I] and the following general formula (II)
Provided is a method for producing a protected oligonucleotide represented by the following general formula CI[I], which comprises condensing a phosphoramidide reagent represented by the formula, and then oxidizing the formed phosphite moiety to a phosphate. Ru.

0-A R4 , R3 represents a protecting group, and A is β-
B AOC represents a nucleoside base that does not have an amino group or a nucleoside base whose amino group or imino group is protected with an allyloxycarbinyl type residue, and NAOC represents a protecting group that is removed by cleavage. group or imino group represents a nucleoside base residue protected with an allyloxycarvinyl type residue, n is 0
or represents a positive integer. ) In the present invention, first, a condensation reaction is performed using a raw material for oligonucleotide synthesis represented by the general formula (1) and a phosphoramidite reagent represented by the general formula CID.

R1 in the above formula may be any protecting group or carrier residue commonly used in nucleoside chemistry, and specific examples of protecting groups include trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethylsilyl group. , triethylsilyl group, triphenylsilyl group, L-butyldimethylsilyl group, tetrahydropyranyl group, 4-methoxytetrahydrobilanyl group, benzoyl group, benzyl group, tetrahydrofuranyl group, methoxymethyl group, methoxyethoxymethyl group, phenoxy Examples include methyl group, methylthiomethyl group, and phenylthiomethyl group.

Further, the carrier is bonded to the 3'-hydroxyl group through a covalent bond represented by an ester bond, and specific examples thereof include modified silica dal, polyester, polyamide, polyvinyl alcohol, polysilolisane, polystyrene, and glass.

82 in the above formula is a hydrogen atom or a hydroxyl group having a protecting group, and specific examples of the protecting group include those similar to R4.

BAOC is a nucleoside base having no amino group or a nucleoside base residue in which the amino group or imino group is protected with an allyloxycarvinyl type residue.

Specific examples of nucleosides having nucleoside bases include deoxyadenosine, deoxyguanosine,
Examples include deoxycytidine, thymidine, adenosine, guanosine, cytidine, uridine, and inosine, among which thymidine, urinone, and inosine belong to those having no amino group.

The allyloxycarbinyl type residue used as a protecting group for the amine group in the base moiety may be any residue that does not essentially impair the deprotection reaction. Oxycal? Nyl group,
Crotyloxycal? nyl group, frenyloxycarbunyl M,, I'5nyloxycardenyl group, cinnamyloxycarbunyl group, chloroallyloxycar? Nyl group,
Examples include p-chlorocinnamyloxycargenyl group.

The number of carbon atoms in the allyloxycarvinyl type residue may be appropriately selected in consideration of separation of by-products generated after the reaction, ease of obtaining raw materials, etc., but those having 12 or less carbon atoms are usually used.

Further, A represents a protecting group that is eliminated by β-cleavage, that is, a protecting group represented by the following general formula [■].

Y In the formula, Y represents a hydrogen atom or a lower alkyl group such as a methyl group or an ethyl group, and 2 represents an electron-withdrawing residue, specific examples of which include a cyano group, a nitro group, a thiocyano group, fluorine, and a base. Examples include heron groups such as , bromine, phenylsulfonyl group, methylsulfonyl group, and phenyl group. At this time, the ortho-position of the phenyl group moiety and/or
Alternatively, it may have a substituent such as a halodane group, a cyan group, or a nitro group at the bara position.

Among these protecting groups, those in which 2 is a cyan group are preferred, and β-cyanoethyl group is particularly preferred.

Further, the value of n is not particularly limited as long as it is O or a positive integer, and may be 100 or more.

R2 and A in the general formula (II) are the same as above, and R3 is a protecting group commonly used in nucleoside chemistry, and specific examples thereof include the same as in the case of R1. Illustrated. Also, NAoc is BAoc except that it does not contain nucleoside bases that do not have amine groups.
is synonymous with

Further, X represents a secondary amino group, and specific examples thereof include dimethylamino group, diethylamino group, diisopropylamine group, di-t-butylamino group, methylglopylamino group, methylhexylamino group, methylcyclohexylamino group, Ethylbenzylamino group, morpholino group,
Thiomorpholino group, pyrrolidino group, piperidino group, 2.
Examples include a 6-dimethylpiveridino group, a piperazino group, an imida/dino group, a pyrolino group, and those having 10 or less carbon atoms are given the prize.

The condensation reaction between the first material for oligonucleotide synthesis and the phosphoramidide reagent is usually carried out using a condensing agent in the presence of a solvent.

Specific examples of the condensing agent used include 1-H-tetrazole, 5-nitro-1-H tetrazole, and triazole.

Further, specific examples of the solvent include pyridines such as pyridine, picoline, and lutidine, quinoline, isoquinoline, oxazole, tetrahydro7rane, dimethylformamide, and dichloromethane.

The ratio of the Orico 0 nucleotide synthesis raw material and the phosphoramidide reagent to be used is selected as appropriate, but usually the ratio is 1 equivalent of the former to 2 to 10 equivalents of the latter, and the condensing agent is 2 to 10 equivalents per mole of the synthetic raw material. It is used in a proportion of ~5 mol.

Such a condensation reaction is usually carried out at 10 to 50°C for 0.2 to 2 minutes.
takes place over time.

After the condensation reaction is complete, an oxidation reaction is carried out to oxidize the phosphite moiety formed in the product to phosphate.

This oxidation reaction may be carried out according to conventional methods, and specific examples include oxidation with iodine and water, oxidation with peroxides such as t-butyl peroxide and benzoyl 14-oxide, and oxidation with nitrogen dioxide. Examples include.

Through this oxidation reaction, the desired protected oligo9 nucleotide represented by the general formula [III] is synthesized. A series of such condensation reactions and oxidation reactions may be carried out in a liquid phase or in a solid phase. Of course, the reaction can also be carried out using a commercially available manual or automatic synthesizer.

After the oxidation reaction, the 5'-hydroxyl group remaining unreacted during the condensation reaction is inactivated with a permanent protecting group such as acetic anhydride, and then the d-group (R3) of the 5'-hydroxyl group of the product is inactivated. Remove using protonic acid or Lewis acid according to conventional methods,
It can be used as a raw material for the next condensation reaction.

Isolation and purification of the product from the reaction solution is carried out by removing protective groups as necessary, and then using adsorption chromatography, ion exchange chromatography, electrophoresis, and distribution using organic solvents, which are the means of ordinary organic synthesis reactions. It is possible to appropriately select or combine known means such as crystallization and crystallization.

In the case of a solid-phase reaction, after removing the protective group if necessary, the carrier can be removed according to a conventional method, and the product can be isolated and purified by the same method as described above.

(Effects of the Invention) In the thus obtained protected oligonucleotide of the present invention, the protecting groups of both the hydroxyl group of the phosphoric acid moiety and the amino group of the nucleoside base can be rapidly removed under mild conditions. For example, 2'-hydroxyl group, 3'-hydroxyl group,
- After removing the protective group or carrier for the hydroxyl group and/or 5'-hydroxyl group, treatment under neutral conditions with a zero-valent palladium compound and a nucleophilic reagent such as an amine or formate can be used for a short period of time at room temperature. The protecting group on the nucleoside base moiety can be removed in time.

Furthermore, the hydroxyl group moiety can be easily deprotected by treatment with aqueous ammonia at room temperature for 0.1 to 5 hours. In the case of solid phase synthesis using a carrier, cleavage from the carrier can be carried out simultaneously during this treatment.

In addition, protecting groups and allyloxycargenyl-type residues that are removed by β-cleavage can be used under general conditions used in deprotection reactions of sugar hydroxyl groups (e.g., deprotection of 5'-hydroxyl groups with trichloroacetic acid, tetra-n - deprotection of the 3'-hydroxyl group with butylammonium chloride, etc.).

(Example) The present invention will be described in more detail with reference to Examples below.

Example 1 3.12 mmol of β-cyanoethoxymonochloro(N,N-diisopropylamino)phosphine (compound 2) obtained by reacting β-cyanoethoxydichlorophosphine with diisopropylamine was dissolved in 4 ml of acetonitrile, and then dissolved in tetrahydrofuran. : 5' dissolved in 15 ml of mixed solvent of acetonitrile-1=2
-o-dimethoxytrityl-N-allyloxycar-nyldeoxycytidine (compound 1) 3 mSol and 1-H
3.6 mmol of -tetrazole was added dropwise at 20° C. over 20 minutes and stirred for 1 hour to obtain phosphoramidide (compound 3).

Next, 2.86 mmol of 3'-o-t-butyldimethylsilylthymidine (compound 4) and 1-H-
3.42 mmol of tetrazole was added and stirred at 20°C for 2 hours.

Then, it was cooled to -78°C, a dichloromethane solution containing 4.86 mmol of nitrogen dioxide was added, and after oxidation for 30 minutes,
Thirty milliliters of a 0.5 molar aqueous sodium sulfite solution was added. After returning to room temperature, chloroform and saturated brine were added, and the separated aqueous layer was extracted with chloroform. After drying, it was separated by chromatography (Silica Dal 120?, methanol:chloroform-1:30-1:20), and β-cyaethyl-5'-〇-dimethoxytrityl-N-allyloxycar was separated from erdeoxycytidylyl. Roux(
3'→5')-3'-ot-butyldimethylsilylthymidine (compound 5) was obtained in a yield of 79 elb.

The physical properties of this substance are as follows.

0 elemental analysis (C53H650,5N6SiP) Calc
d, C58,67H6,00N 7.75 Foun
d, C58,57H6,06N 7.98H Compound 3. 2 Compound 5 Example 2 An experiment was carried out according to Example 1 except that phosphoramidide (compound 6) described below was used instead of compound 30 used in Example 1, and β-cyanoethyl-5'-〇- Dimethoxytrityl-N-allyloxycal? Nyldeoxyadenosilyl-(3'→5')-3'-ot-butyldimethylsilylthymidine (Compound 7) was obtained with a yield of 88%.

The physical properties of this substance are as follows.

・Elemental analysis value (C54H6,014N8PSi) Cal
cd, C58,48H5,87N 10.11Fo
und, 05B, 29 H5, 80N 10.
09NCCH2Cl2-0-P-N(CH(CH,)2
) 2 (Compound 6) (Compound 7) (AOC=allyloxycarbonyl group) Reference Example 1 50.3 mmol of the compound obtained in Example 1 and 0.95 mmol of triphenylphosphine were added to 50 milliliters of torr. After placing in a Kolben and creating an argon atmosphere, it was dissolved in 3 ml of tetrahydrofuran, and then 0.6 mmol of n-butylamine and 0.6 mmol of formic acid were sequentially added dropwise. 015 mmol dissolved in 2 ml of tetrahydrofuran and toluene was added thereto, and the mixture was stirred at room temperature for 10 minutes.

After the reaction, the solvent was distilled off, and then diluted with ammonia solution at room temperature for 2
The aqueous ammonia was distilled off, the residue was dissolved in ethyl acetate, washed with water and saturated brine, dried, and separated by chromatography, and the allyloxycarbyl group and β-cyanethyl group were separated. removed 5'-o-dimethoxytritylcytidylyl-(3'→5')-3'-o
-t-Butyldimethylthymidine was obtained. The yield was 91 mol.

Reference Example 2 A reaction was carried out in the same manner as in Reference Example 1 except that O,t 5 m+) moles of Compound 7 was used in place of Compound 5 and 9. As a result, 5'-〇-dimethoxytrityldeoxyadenosilyl-(3'→5')-3'-ot-
Butyldimethylsilylthymidine was obtained in a yield of over 90%.

Example 3 CPG (Controlled Pare Gras)
s) 30 milligrams of 5'-〇-dimethoxytritylthymidine (compound 8) bound to the resin via an ester bond is placed in a glass exhibition reactor, and a dichloromethane solution of trichloroacetic acid is added to deprotect the 5'-hydroxyl group. After that, it was washed with acetonitrile. Next, 0.03 mmol and 1 phosphoramidite (compound 3) in which the amino group of the cytidine base was protected with an allyloxycarbunyl group were added.
9. 0.04 mmol of -H-tetrazole was dissolved in an acetonitrile-tetrahydrofuran mixed solvent and reacted for 2 minutes at room temperature.

Next, after washing with acetonitrile, iodine solution 1.
2 milliliters (11.6 grams of iodine, 18 milliliters of water, 180 milliliters of luthidine,
The C-T dimer (compound 9
) was obtained. The yield was 80% (measured by color development due to elimination of the 5'-dimethoxytrityl group, which will be described later).

Confirmation of this compound was performed according to the following procedure.

First, the carrier containing Compound 9 was washed with acetonitrile, acetic anhydride was added to cap the unreacted 57-hydroxyl group, and acetonitrile was added to wash the carrier.

Then triphenylphosphine 0.095 mmol, n
-butylamine 0.6 mmol, formic acid 0.6 mmol and tetrakis(triphenylphosphine)palladium (
0) 0.015 mmol dissolved in tetrahydrofuran was added and reacted at room temperature for 10 minutes to deprotect the allyloxycarbunyl group in the base moiety.

After washing with tetrahydrofuran and dichloromethane, the dimethoxytrityl group of the 5'-hydroxyl group was removed with trichloroacetic acid to obtain Compound 10. Then /λJ 30% ammonia aqueous solution was added and allowed to stand at room temperature for 30 minutes to remove the β-cyanoethyl group of the phosphoric acid moiety and the carrier, resulting in C-T
A dimer (compound 11) was obtained.

After distilling off the ammonia, dissolve it in 200 microliters of water, collect 5 microliters of the aqueous solution,
This was followed by [γ-32P)ATP (PB-170.

1 microliter of flax jam) was added and dried. Add 2μ of caination powder and fur (X 2.5) to this.
t, T4 nucleotide kinase (Zenshuzo.

2.5 u/μt) and 2 μt of water were added, and Kainesi q7 was performed at 37°C. TLC (Polygram,
CELL300 DEAE/HR-2/15,
(Maakerey-Nags 1) and developed in an RNA homomix chair to obtain a -dimensional autoradiograph and confirm that it was a single spot. From TLC,
One spot was extracted, digested with penomphosphodiesterase and nuclease P1, and the digested products were developed by electrophoresis using DEAR-Cellulose Eper, transferred to TLC, and two-dimensionally analyzed using a homomix chair. It was expanded to After 2D expansion, TLC auto°
By taking radiographs, it was confirmed that a CT dimer from which the protecting group had been deprotected was generated from the spot position.

(AOc Compound 8 0-CH2C)12CN Compound 9

Claims (1)

[Claims] 1. A raw material for oligonucleotide synthesis represented by the following general formula [I] is condensed with a phosphoramidite reagent represented by the following general formula [II], and then the formed phosphite moiety is converted into a phosphate. A method for producing a protected oligonucleotide represented by the following general formula [III], which is characterized by being oxidized. ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[II] ▲There are mathematical formulas, chemical formulas, tables, etc.▼...[III] (Formula where R_1 represents a protective group or a residue of a carrier bonded via a covalent bond, R_2 represents a hydrogen atom or a hydroxyl group having a protective group, R_3 represents a protective group, and A
represents a protecting group that is eliminated by β-cleavage, and B^A^
O^C represents a nucleoside base that does not have an amino group or a nucleoside base residue whose amino group or imino group is protected with an allyloxycarbonyl type residue, and N^
A^O^C represents a nucleoside base residue whose amino group or imino group is protected with an allyloxycarbonyl type residue, and n represents 0 or a positive integer. )