JP2010248084A - Method of synthesizing oligonucleotide using novel cleaning solvent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of synthesizing an oligonucleotide using an alternative cleaning solvent. <P>SOLUTION: The method of chemically synthesizing an oligonucleotide in a solid phase according to a phosphoroamidite process is characterized by cleaning a solid state support using acetone or a liquid mixture of acetonitrile and acetone as a cleaning solvent upon completing each of its reaction processes. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an oligonucleotide synthesis method using an alternative washing solvent.

  In recent years, oligonucleotides have been widely used for a wide range of applications including PCR, antisense technology, nucleic acid structure analysis, and RNAi research. As for the chemical synthesis technology of oligonucleotides, the phosphoric acid diester method (Non-patent Document 1) of Khorana et al. Was first developed. 4) Improvement of phosphite method, phosphoramidite method using Carrthers et al. Phosphoramidite method (Non-patent Documents 5 and 6), and H-phosphonate method (Non-patent Document 7) Etc. were developed. The β-cyanoethyl phosphoramidite method (Non-patent Document 6) developed by Koster et al. As an improved method of the phosphoramidite method is widely used in DNA / RNA automatic synthesizers.

  With regard to these oligonucleotide synthesis technologies, reagents and reaction conditions suitable for each reaction have been studied and improved in detail. For example, as the yield of one oligonucleotide chain extension reaction (condensation reaction), phosphorus The acid triester method achieves approximately 90 to 95%, and the phosphite method including the β-cyanoethyl phosphoramidite method achieves approximately 98 to 99%.

  These oligonucleotide synthesis methods are considered to be almost completed as chemical reactions, but long oligonucleotides exceeding 50 mer are obtained even if the yield of one condensation reaction is 99%. In the case of synthesis, it is still a problem that the final yield is greatly reduced. In addition, although the conventional oligonucleotide synthesis method can synthesize oligonucleotides with relatively high purity, impurities such as oligonucleotides that have stopped the synthesis reaction are produced in a certain amount in the synthesis product, so high-performance liquid chromatography after synthesis. It was necessary to carry out purification by means of chromatography and the like, and this purification step was a factor that further reduced the final yield. Therefore, in order to improve the final yield, for example, a synthesis method using an unprotected nucleoside capable of omitting the deprotection step of the nucleoside base which causes a decrease in yield has been developed (Non-Patent Document 8, Patent Document 1). However, it is still desired to develop a method for synthesizing oligonucleotides with higher yield by simpler operation.

  By the way, in the oligonucleotide solid phase synthesis by the phosphoramidite method currently in practical use, in order to remove the reactant used in each step after completion of each reaction step, the solid phase carrier is washed using acetonitrile as a washing solvent. ing. Acetonitrile is a by-product produced during the manufacture of acrylonitrile, and is produced at a rate of 3% with respect to acrylonitrile. In recent years, acetonitrile has been used not only for solid-phase synthesis of oligonucleotides but also in a wide range of fields such as pharmaceutical intermediate production and chromatography. On the other hand, if acrylonitrile is reduced in production, acetonitrile is expected to be insufficiently supplied, so a solid-phase synthesis method using an alternative washing solvent capable of synthesizing oligonucleotides with at least the same efficiency as conventional methods has been developed. There was an urgent need to do.

JP 2004-99532 A

P.T.Gilham, H.G.Khorana, J. Amer. Chem. Soc., (1958) 80, 6212 R.L.Letsinger, K.K.Ogilvie, J. Am. Chem. Soc. (1967) 89, 4801 H. Ito et al., Nucleic Acids Res. (1982) 10, 1755 R.L.Letsinger, W.B.Lundsford, J. Am. Chem. Soc. (1976) 98, p.3655 M.D.Matteucci, M.H.Caruthers, J. Amer. Chem. Soc. (1981) 103, 3185 N.D.Sinha, J. Biernat and H. Koster, Nucleic Acids Res. (1984) 12, 4539 B.C.Froehler et al., Nucleic Acids Res. (1986) 14, 5399 S.M.Gryaznov, R.L.Lettsinger, Nucleic Acids Res. (1992) 20 (8): 1879-1882

  An object of the present invention is to provide an oligonucleotide synthesis method using an alternative washing solvent.

  As a result of intensive studies to solve the above problems, the present inventors can achieve an excellent cleaning effect by using acetonitrile-acetone mixed solution or acetone as a cleaning solvent instead of acetonitrile in the oligonucleotide solid-phase synthesis method. The inventors have found that synthetic oligonucleotides can be produced efficiently and have completed the present invention.

That is, the present invention includes the following.
[1] In the oligonucleotide solid phase synthesis method based on the phosphoramidite method, the solid phase carrier is washed with a mixed solution of acetonitrile and acetone or acetone as a washing solvent at the end of each reaction step. Chemical synthesis of nucleotides.

  In a preferred embodiment, a mixture of acetonitrile and acetone or acetone may be used in a mixture ratio of acetonitrile: acetone of 0%: 100% to 95%: 5%.

  In a more preferred embodiment, a mixture of acetonitrile and acetone or acetone may have a mixture ratio of 30%: 70% to 70%: 30% acetonitrile: acetone.

In the above method, the oligonucleotide solid phase synthesis method based on the phosphoramidite method is:
(a) removing the 5 ′ hydroxyl group of the nucleoside on the solid support by acid treatment;
(b) a step of activating a nucleoside-3′-O-phosphoramidite derivative with an acid catalyst and linking the nucleoside 5 ′ hydroxyl group on a solid phase carrier with a trivalent phosphate bond by a condensation reaction;
(c) capping the unreacted 5 ′ hydroxyl group, and
(d) It may be an oligonucleotide solid-phase synthesis method including repeatedly performing a reaction cycle including a step of oxidizing the trivalent phosphate bond.

  In the above method, control pore glass (CPG) or polystyrene (PS) can be suitably used as the solid phase carrier.

[2] A cleaning agent for a solid phase carrier for oligonucleotide solid phase synthesis, comprising a mixed solution of acetonitrile and acetone, or acetone as an active ingredient.
In a preferred embodiment of this cleaning agent, a mixture of acetonitrile and acetone or acetone may be used in a mixture ratio of acetonitrile: acetone of 0%: 100% to 95%: 5%.

  The present invention can provide an alternative method for synthesizing oligonucleotides that can achieve synthesis with the same efficiency and higher purity than conventional solid-phase oligonucleotide synthesis methods using acetonitrile as a washing solvent.

FIG. 1 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 1 synthesized on a solid support CPG using acetonitrile as a washing solvent. FIG. 2 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 1 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. . FIG. 3 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 1 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (1: 1 (v / v)) as a washing solvent. . FIG. 4 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 1 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixture (3: 7 (v / v)) as a washing solvent. . FIG. 5 is a diagram showing a MALDI Tof MS spectrum of an oligonucleotide synthesis product of sequence 1 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (3: 7 (v / v)) as a washing solvent. FIG. 6 is a diagram showing an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 1 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 7 shows a capillary electrophoresis electrogram of the oligonucleotide synthesis product of sequence 1 synthesized on solid support polystyrene (PS) using acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. FIG. FIG. 8 shows an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 1 synthesized on solid phase polystyrene (PS) using acetonitrile-acetone mixed solution (1: 1 (v / v)) as a washing solvent. FIG. FIG. 9 shows an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 1 synthesized on solid support polystyrene (PS) using acetonitrile-acetone mixed solution (3: 7 (v / v)) as a washing solvent. FIG. FIG. 10 is an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 2 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 11 is a diagram showing a MALDI Tof MS spectrum of the oligonucleotide synthesis product of sequence 2 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 12 is an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 3 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 13 is a diagram showing a MALDI Tof MS spectrum of the oligonucleotide synthesis product of sequence 3 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 14 is an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 4 synthesized on solid phase carrier CPG using acetonitrile as a washing solvent. FIG. 15 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 4 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (95: 5 (v / v)) as a washing solvent. . FIG. 16 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 4 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. . FIG. 17 is a diagram showing a MALDI Tof MS spectrum of an oligonucleotide synthesis product of sequence 4 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. FIG. 18 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 4 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (1: 1 (v / v)) as a washing solvent. . FIG. 19 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 4 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixture (3: 7 (v / v)) as a washing solvent. . FIG. 20 is a diagram showing an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 4 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 21 is a diagram showing a MALDI Tof MS spectrum of the oligonucleotide synthesis product of sequence 4 synthesized on solid phase carrier CPG using acetone as a washing solvent. FIG. 22 is a diagram showing an electrogram of capillary electrophoresis of oligonucleotide synthesis product of sequence 5 synthesized on solid phase carrier CPG using acetonitrile as a washing solvent. FIG. 23 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 5 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (95: 5 (v / v)) as a washing solvent. . FIG. 24 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 5 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. . FIG. 25 is a diagram showing a MALDI Tof MS spectrum of the oligonucleotide synthesis product of sequence 5 synthesized on a solid phase carrier CPG using acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. FIG. 26 is a diagram showing an electrogram of capillary electrophoresis of an oligonucleotide synthesis product of sequence 5 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixed solution (1: 1 (v / v)) as a washing solvent. . FIG. 27 is a diagram showing an electrogram of capillary electrophoresis of oligonucleotide synthesis product of sequence 5 synthesized on solid phase carrier CPG using acetonitrile-acetone mixed solution (3: 7 (v / v)) as a washing solvent. . FIG. 28 is a diagram showing an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 6 synthesized on solid phase carrier CPG using acetonitrile as a washing solvent. FIG. 29 is a diagram showing a MALDI Tof MS spectrum of an oligonucleotide synthesis product of sequence 6 synthesized on a solid phase carrier CPG using acetonitrile-acetone mixed solution (7: 3 (v / v)) as a washing solvent. FIG. 30 is a diagram showing an electrogram of capillary electrophoresis of the oligonucleotide synthesis product of sequence 7 synthesized on solid phase carrier CPG using acetonitrile as a washing solvent. FIG. 31 is a diagram showing a MALDI Tof MS spectrum of an oligonucleotide synthesis product of sequence 7 synthesized on a solid phase carrier CPG using an acetonitrile-acetone mixture (7: 3 (v / v)) as a washing solvent.

Hereinafter, the present invention will be described in detail.
The present invention relates to an oligonucleotide synthesis method using a phosphoramidite method. Instead of acetonitrile, which has been most commonly used as a washing solvent used at the end of each step, a mixed solution of acetonitrile and acetone, or an oligonucleotide using acetone is used. The present invention relates to a method for chemical synthesis of nucleotides. In the method of the present invention, oligonucleotides can be synthesized successfully using these alternative washing solvents as in the conventional method. In the present invention, “oligonucleotide” refers to a nucleic acid having a length of 2 bases (2 mer or 2 bp) or more, usually 2 to 1000 bases, more generally 2 to 500 bases. The “oligonucleotide” in the present invention may be DNA (oligodeoxyribonucleotide) or RNA (oligoribonucleotide).

  The oligonucleotide synthesis method of the present invention is suitable for the synthesis of long-chain oligonucleotides because the purity of the synthesized product is unlikely to decrease even when the number of synthesis cycles increases. In general, when the average condensation yield is 98% to 97%, the ratio of the target chain length (main product) to the incomplete chain length with a shorter base length becomes longer as the chain length is extended. The ratio is higher than that of a synthetic product of 99% or more, and as a result, the purity is lowered. In this method, it is possible to more effectively remove the reagent remaining on the surface of the solid phase carrier used after the condensation, which inhibits the condensation, and it is highly efficient up to the desired final chain length without reducing the condensation efficiency. The condensation reaction can be maintained. As a particularly preferred example of a long oligonucleotide, a 50mer to 100mer oligonucleotide can be efficiently produced.

  The oligonucleotide synthesis method based on the phosphoramidite method used as the basis of the method of the present invention is basically the phosphoramidite method of Caruthers et al., The β-cyanoethyl phosphoramidite method of Koster et al. It is an improved method. In these methods, the nucleoside-3′-O-phosphoramidite derivative used in the condensation reaction is activated by donating a proton to its NN-diisopropylamino group using an acid catalyst, and thereby on the solid support. To cause a condensation reaction with the 5'-hydroxyl group (5'-terminal hydroxyl group) of the 5'-terminal nucleoside of the immobilized nucleic acid (nucleoside or polynucleotide) and to bind them by a trivalent phosphate bond And oxidation of the trivalent phosphate bond to a stable pentavalent phosphate bond.

  In the method of the present invention, each step of the oligonucleotide synthesis method by a known phosphoramidite method may be carried out except that a novel washing solvent that is a mixture of acetonitrile and acetone or acetone is used. The method of the present invention can be preferably carried out, for example, by using the washing solvent according to the present invention in place of acetonitrile in the β-cyanoethyl phosphoramidite method using a β-cyanoethyl phosphoramidite nucleoside. . A typical oligonucleotide solid phase synthesis cycle by the β-cyanoethyl phosphoramidite method is shown below. “CPG” below represents control pore glass (CPG; described later) as solid phase synthesis. “B” below represents a nucleobase (usually adenine, guanine, cytosine, thymine, or uracil). DMTr represents a dimethoxytrityl group.

A mixed liquid of acetonitrile and acetone (hereinafter also referred to as an acetonitrile-acetone mixed liquid) or acetone used as a washing solvent in the present invention is acetonitrile (CH ₃ CN) and acetone ((CH ₃ ) ₂ CO) in a weight ratio. , 0%: 100% to 95%: 5% acetonitrile: acetone is preferable. Here, the cleaning solvent having a mixture ratio of acetonitrile and acetone of 0%: 100% is a solvent of acetone 100%. The mixing ratio of the cleaning solvent acetonitrile and acetone used in the present invention may also be 0%: 100% -80%: 20% by weight ratio, 0%: 100% -70%: 30% 10%: 90% to 70%: 30%, 30%: 70% to 70%: 30%, 40%: 60% to 60%: 40% It may be 50%: 50%. In order to improve the purity of the target oligonucleotide in the synthesis product obtained at the completion of the synthesis reaction compared to when acetonitrile is used as a washing solvent, the mixing ratio of acetonitrile and acetone is not limited. In order to obtain a highly pure synthetic product, it is preferable that the weight ratio is 30%: 70% to 70%: 30%, more preferably 40%: 60% to 60%: 40%. More preferably, the mixing ratio of acetonitrile and acetone is 50%: 50% by weight. The mixing ratio of the washing solvent acetonitrile and acetone can be appropriately adjusted according to the solid phase carrier used, other reagents, the length of the synthetic oligonucleotide, and the like. The amount of the washing solvent according to the present invention added to the solid phase carrier per washing will be determined by those skilled in the art depending on the amount of the solid phase carrier, the DNA / RNA automatic synthesizer used, the amount of reagent used, etc. Although it can set suitably and it is not specifically limited, Usually, 100 microliters-10mL should just be used.

  In the present invention, as a solid carrier to be washed with the washing solvent, any solid carrier used for oligonucleotide solid phase synthesis, for example, polystyrene (PS), silica gel, polyethylene glycol, porous having pores controlled by the production method Control pore glass (CPG) which is a glass bead can be used. In practice, it is convenient to use polystyrene (PS) or control pore glass (CPG). The solid phase carrier can be used in any form, but in a DNA / RNA automatic synthesizer or the like, it is preferably used in a form packed in a container such as a column. In the present invention, although not limited thereto, a solid support on which a nucleoside containing a 3′-terminal base of an oligonucleotide to be synthesized, preferably a derivative having succinic acid introduced at the 3′-hydroxyl group, is fixed, It is preferable to use for oligonucleotide synthesis reaction as a starting material. Immobilization of the nucleoside on the solid support can be performed by a conventional method, for example, preferably via a linker. Specifically, the solid support on which the nucleoside is immobilized may be a solid support in which an aminoalkyl group such as an aminopropyl group is introduced into a silanol hydroxyl group on the solid support and a nucleoside-3′-O-succinyl form is bound. . Alternatively, a protected nucleoside may be introduced into a solid support using a Q linker, or a nucleoside amidite may be introduced into a solid support universal support as a known technique in the present invention.

  In the method of the present invention, a nucleoside-3′-O-phosphoramidite derivative is used for the condensation reaction as in the oligonucleotide synthesis method using the phosphoramidite method used as the basis of the method of the present invention. In the present invention, the nucleoside-3'-O-phosphoramidite derivative refers to a so-called amidite reagent in the field of oligonucleotide synthesis, and specifically, a nucleotide in which the 3'-position of the sugar moiety is phosphitylated. Point to.

  In the present invention, the nucleoside-3′-O-phosphoramidite derivative may be a deoxyribonucleoside-3′-O-phosphoramidite derivative or a ribonucleoside-3′-O-phosphoramidite derivative, the functional group of which is It may be protected or unprotected. The nucleoside-3′-O-phosphoramidite derivative used in the present invention is typically a compound of the following formula (I).

（式中、Bは核酸塩基を表し、R₁は水素原子、水酸基、又は水酸基の保護基を表し、R₂は水酸基の保護基を表し、R₃はリン酸の保護基を表し、R₄は置換又は非置換のアミノ基を表す）

(In the formula, B represents a nucleobase, R ₁ represents a hydrogen atom, a hydroxyl group, or a hydroxyl protecting group, R ₂ represents a hydroxyl protecting group, R ₃ represents a phosphate protecting group, R ₄ Represents a substituted or unsubstituted amino group)

  The amino moiety (amino group) of the base part (B in formula (I)) of this nucleoside-3′-O-phosphoramidite derivative may be protected. In the nucleoside-3′-O-phosphoramidite derivative in the present invention, the amino group of the base moiety is not limited, but in terms of ease of operation, a protecting group that is eliminated under basic conditions, such as an acyl group. It is preferably protected with an acyl protecting group such as a group or an amidine protecting group, specifically, a benzoyl group, an isobutyryl group, an acetyl group or the like. Although not limited, when B is adenine, cytosine, or guanine, it is more preferable that the amino moiety of the base is protected with the above protecting group, and when B is thymine or uracil, Since the nature is originally low, the base part may be unprotected.

The 2′-position of the nucleoside-3′-O-phosphoramidite derivative (R ₁ in formula (I)) is a hydrogen atom in the case of the deoxyribonucleoside-3′-O-phosphoramidite derivative, and the ribonucleoside In the case of a -3′-O-phosphoramidite derivative, it is a hydroxyl group or a protecting group for protecting the hydroxyl group. In the ribonucleoside-3′-O-phosphoramidite derivative used in the present invention, this 2′-hydroxyl group is preferably protected. Protecting groups for 2'-hydroxyl groups include t-butyldimethylsilyl group (TBDMS), triisopropylsilyloxymethyl group (TOM), bis (2-acetoxyethyloxy) methyl group (ACE), 2-cyanoethoxymethyl group (CEM) and the like.

In the nucleoside-3′-O-phosphoramidite derivative in the present invention, it is more preferable that the 5′-hydroxy group of the sugar moiety corresponding to the 5′-terminus is protected. The 5′-hydroxyl-protecting group (R ₂ in formula (I)) can be easily and selectively removed (specifically under acidic conditions), and the absorbance of the deprotected protecting group can be measured. It is preferable that it is a thing. Representative examples of the 5′-hydroxyl protecting group include 4,4′-dimethoxytrityl group (DMTr) or a derivative thereof. The 5′-hydroxyl protecting group of the nucleoside-3′-O-phosphoramidite derivative according to the present invention may be further modified with a fluorescent dye, biotin, phosphate group, amino group or the like.

In the nucleoside-3′-O-phosphoramidite derivative according to the present invention, the phosphate group is preferably protected by a protecting group. Examples of the protecting group for the phosphate group (R ₃ in formula (I)) include, but are not limited to, a β-cyanoethyl group, a nitrophenylethyl group, and a methyl group. In terms of the ease of removal of the protecting group, the protecting group for the phosphate group of this nucleoside-3′-O-phosphoramidite derivative in the present invention is not eliminated under acidic conditions but is easily removed under basic conditions. More preferred is a protecting group which is eliminated by, for example, a β-cyanoethyl group.

The nucleoside-3′-O-phosphoramidite derivative according to the present invention has a substituted or unsubstituted amino group (R ₄ ). Specific examples of the substituted or unsubstituted amino group include amine derivatives containing amines such as a diisopropylamino group, a diethylamino group, an ethylmethylamino group, or a morpholino group.

  As the nucleoside-3′-O-phosphoramidite derivative used in the present invention, in the case of DNA synthesis, deoxyribonucleoside-3′-O-phosphoramidite derivative, ie, protected or unprotected deoxyadenosine, protected or unprotected Each 3′-O-phosphoramidite of deoxyguanosine, protected or unprotected deoxycytidine, protected or unprotected thymidine can be used. In the case of RNA synthesis, ribonucleoside-3′-O-phosphoramidite derivatives, ie protected or unprotected adenosine, protected or unprotected guanosine, protected or unprotected cytidine, protected or unprotected uridine 3′- each. A 0-phosphoramidite body may be used.

  The nucleoside-3′-O-phosphoramidite derivative used for the condensation reaction in the method of the present invention can be obtained by, for example, converting a 3′-hydroxyl free partially protected or base unprotected nucleoside to (2-cyanoethyl) (N , N-diisopropylamino) chlorophosphine, or amidating a protected nucleoside with bis- (N, N-diisopropylamino)-(2-cyanoethyl) -phosphine. In the present invention, the nucleoside-3′-O-phosphoramidite derivative can also be used by purchasing a commercially available product. Nucleoside-3′-O-phosphoramidite derivatives used in the present invention include, for example, Wako Pure Chemical Industries, GE Healthcare, Applied Biosystems, Thremo Fisher, Glen Research Co., Ltd., Proligo Co., Ltd., etc. Available from suppliers. The protected nucleoside-3′-O-phosphoramidite derivative can also be obtained as a raw material for oligonucleotide synthesis from Thermo Fisher, Applied Biosystems, Proligo and the like.

  Specifically, the method of the present invention is based on an oligonucleotide synthesis method based on a phosphoramidite method, and (a) a step of removing a 5′-hydroxyl protecting group of a nucleoside on a solid phase carrier by acid treatment (5 ′ -Position protecting group removal step), (b) The nucleoside-3'-0-phosphoramidite derivative is activated with an acid catalyst, and the condensation reaction causes the 5 'hydroxyl group of the nucleoside on the solid support and the trivalent phosphate bond (C) a step of capping the unreacted 5′-hydroxyl group (capping step), and (d) a step of oxidizing the trivalent phosphate bond (oxidation step). An oligonucleotide solid-phase synthesis method comprising repeatedly performing a reaction cycle (two or more times depending on the chain length to be synthesized), at the end of at least one of the steps (ie, one step and During the next step), the reaction system To remove the remaining excess reagent is a chemical synthesis method of oligonucleotides and performing solid-phase carrier cleaning process using the cleaning solvent of the present invention. In the method of the present invention, the steps up to the completion of the synthesis of the target chain length can be performed at room temperature (about 20 to 25 ° C.). In the method of the present invention, both DNA and RNA can be synthesized. The method of the present invention is suitable for carrying out using an automatic DNA / RNA synthesizer.

  In a more preferred embodiment, the method of the present invention comprises a step of completing an oligonucleotide chain extension reaction for one base comprising the following steps (a) to (d) and washing steps (i) to (iv): This can be done by cycling and repeating it until the desired chain length is reached. However, a step that becomes unnecessary depending on the reagent to be used and a cleaning step at the end of the step may be omitted.

(a) 5'-position protecting group removal step In this step, the nucleic acid (oligonucleotide or nucleoside) immobilized on the solid phase carrier is treated with an acid, thereby 5'-hydroxyl group of the 5'-terminal nucleoside of the nucleic acid. The 5'-hydroxyl group is removed by removing (deprotecting) the protective group (hereinafter also simply referred to as 5'-hydroxyl group) under acidic conditions and leaving the 5'-hydroxyl group free. The 5'-hydroxyl protecting group is not limited, but is most commonly a dimethoxytrityl group (DMTr). The elimination of the dimethoxytrityl group from the 5′-hydroxyl group is not limited, but a 3% trichloroacetic acid-dichloromethane solution or a 3% dichloroacetic acid-dichloromethane solution can be preferably used. These acid treatment reagents are available from many suppliers such as Wako Pure Chemical Industries, GE Healthcare, Applied Biosystems, Thremo Fisher, Glen Research Co., Ltd., Proligo Co., Ltd., and the like.

  In the present invention, the “nucleoside on the solid phase carrier” refers to the 5′-terminal nucleoside of the oligonucleotide immobilized on the solid phase carrier, or the nucleoside immobilized on the solid phase carrier. Furthermore, “the 5′-hydroxyl group of the nucleoside on the solid phase carrier” means the 5′-terminal nucleoside of the oligonucleotide immobilized on the solid phase carrier or the 5′-position of the nucleoside immobilized on the solid phase carrier. Refers to a hydroxyl group.

(i) Cleaning process-1
After completion of the 5′-position protecting group removal step, the washing solvent according to the present invention (acetonitrile-acetone mixed solution or acetone) is added to the solid support, and the acid treatment reagent and the like are washed away from the solid support. This solid phase carrier washing operation may be performed once, but may preferably be performed a plurality of times, for example, twice. After the washing solvent is added to the solid phase carrier, the washing solvent can be efficiently removed by a flash operation. In the present invention, the flush operation refers to an operation of removing the column content liquid by blowing an inert gas such as argon gas or nitrogen gas to the column. By this washing step, the solid phase carrier is sufficiently washed and dehydrated. This dehydration treatment is necessary to suppress the inactivation of the nucleoside-3′-O-phosphoramidite derivative in the next step (b).

(b) Condensation process
In this step subsequent to the 5′-position protecting group removal step (a) and the washing step (i), the nucleic acid on the solid phase carrier having the 5 ′ hydroxyl group deprotected in the above step (a) is then added to the nucleic acid. A nucleoside-3′-O-phosphoramidite derivative to be linked and an acid catalyst as an activator are added. As a result, the nucleoside-3′-O-phosphoramidite derivative is activated by the acid catalyst, and the free 5 ′ hydroxyl group of the nucleic acid on the solid phase carrier reacts therewith, and both are linked by a condensation reaction. The bond produced by this condensation reaction is a trivalent phosphate bond.

  In this step, the nucleoside-3′-O-phosphoramidite derivative is not limited, but is preferably added to the solid phase carrier as a solution in a solvent such as acetonitrile. The nucleoside-3′-O-phosphoramidite derivatives that can be used in the method of the present invention will be described later.

  The acid catalyst is not limited, and a known acid catalyst (activator) such as 1H-tetrazole, 5′-ethylthio-1H-tetrazole, benzylthio-1H-tetrazole, dicyanoimidazole, etc. can be used. One example is 1H-tetrazole. The acid catalyst is preferably added as a solution dissolved in a solvent such as acetonitrile, and the acid catalyst solution can be 0.1M to 0.45M, preferably 0.25M to 0.45M. The concentration is not limited and can be adjusted as appropriate by those skilled in the art.

  For the deprotected 5'-terminal nucleoside of nucleic acid immobilized on a solid support, usually 6-12 equivalents of a solution containing a nucleoside-3'-O-phosphoramidite derivative, acid catalyst The solution is preferably added in an amount of 12 to 24 equivalents, but is not limited thereto. Although reaction time is not limited, Usually, it is preferable to set it as 30 to 120 second.

(ii) Cleaning step-2
After completion of the condensation step (b), the washing solvent according to the present invention (acetonitrile-acetone mixed solution or acetone) is added to the solid phase carrier, and the acid catalyst and nucleoside-3′-O-phosphoramidite derivative, etc. from the solid phase carrier Wash away excess reagent. After the washing solvent is added to the solid phase carrier, the washing solvent can be efficiently removed by a flash operation. This solid phase carrier washing operation may be performed once, but may preferably be performed a plurality of times, for example, twice.

(c) Capping step Following the condensation step (b) and the washing step (ii), another protection other than the deprotection of the unreacted 5′-hydroxy group of the nucleoside on the solid support in step (a) By protecting with a group, it can be inactivated (ie capped).

  The phosphoramidite method is the most active phosphoric acid ester condensation reaction developed so far, and the reaction is 100% complete in the liquid phase, but the phase tends to be heterogeneous in the solid phase and the reaction efficiency is slightly lower. descend. For this reason, a nucleoside having an unreacted 5′-hydroxyl group remains on the solid phase carrier, but this becomes an impurity that is difficult to separate when an extension reaction occurs in the next chain length extension reaction cycle. It is preferable to stop the extension reaction by inactivating the unreacted 5′-hydroxyl group by capping so that the unreacted nucleoside or nucleotide having the 5′-hydroxyl group in the free state of the reaction is not carried over to the next cycle. .

  This capping can be performed by a conventionally known method, but is preferably performed by adding a protecting group that can be eliminated only under basic conditions. Generally, the unreacted 5 ′ hydroxyl group is acetylated. It is preferable to carry out by adjusting. The acetylation of the unreacted 5′-hydroxyl group is not limited, but acetylation with acetic anhydride or phenoxyacetic anhydride can be used. For capping the unreacted 5′-hydroxy group, it is preferable to add a solution containing acetic anhydride to a solid support together with a basic catalyst for forming a salt with acetic acid generated during the reaction. The solution containing acetic anhydride and the solution containing 1-methylimidazole as a basic catalyst are preferably prepared separately and prepared at the time of use in the capping step.

The basic catalyst to be added together with acetic anhydride ((CH ₃ CO) ₂ O or Ac ₂ O) is not limited, but includes 1-methylimidazole (1-MeIm), pyridine (Pyr) which is an aromatic amine 2,6-lutidine (Lut) and the like. Acetic anhydride and a basic catalyst may be added as a solution dissolved in an appropriate solvent (for example, tetrahydrofuran (THF), acetonitrile, etc.) These reagents (capping reagents) used in this step are, for example, Wako Pure Chemical Industries, Ltd. , GE Healthcare, Applied Biosystems, Thermo Fisher, Glen Research Co., Ltd., Proligo Co., Ltd., etc. Added to solid support usable in the method of the present invention. Examples of combinations of acetic anhydride solution and basic catalyst solution to be listed are listed in Table 1, but are not limited thereto.

  After adding the capping reagent and reacting, it is preferable to remove the reaction solution from the solid phase carrier by a known operation such as a flash operation.

(iii) Cleaning step-3
After completion of the capping step (c), the washing solvent according to the present invention (acetonitrile-acetone mixed solution or acetone) is added to the solid phase carrier, and excess acetic anhydride, basic catalyst, and a solvent in which they are dissolved from the solid phase carrier. Wash away the capping reagent. After the washing solvent is added to the solid phase carrier, the washing solvent can be efficiently removed by a flash operation. This solid phase carrier washing operation may be performed once, but may be performed a plurality of times, for example, twice or more.

(d) Oxidation step Following the capping step (c) and washing step (iii), the trivalent phosphate bond of the nucleotide chain-extended in the condensation step (b) is added to the solid phase carrier by adding an oxidation reagent. Oxidizes and converts to a stable pentavalent regular phosphate bond. This is because the trivalent phosphate bond (trivalent phosphate triester bond) is easily hydrolyzed and unstable. As the oxidizing reagent, a known oxidizing reagent can be used. For example, an aqueous solution or peroxide containing iodine can be preferably used. As a specific example, a solution obtained by dissolving a 0.1 M iodine-pyridine solution in an aqueous solvent or an organic solvent, for example, a peroxide such as iodine-pyridine-water-tetrahydrofuran solution or t-butyl hydroperoxide-methylene chloride is used. However, it is not limited to this.

(iv) Cleaning step-4
After completion of the oxidation step (d), the cleaning solvent according to the present invention (acetonitrile-acetone mixed solution or acetone) is added to the solid phase carrier, and excess capping reagent such as an oxidation reagent is washed away from the solid phase carrier. After the washing solvent is added to the solid phase carrier, the washing solvent can be efficiently removed by a flash operation. This solid phase carrier washing operation may be performed once, but is preferably performed a plurality of times, for example, twice or more.

  After the washing step (iv) is completed, if the synthetic oligonucleotide has not yet reached the target chain length, the process returns to the 5′-position protecting group removal step (a) and the above cycle is repeated.

  In the present invention, an oligonucleotide is synthesized at a level equivalent to or higher than conventional by synthesizing an oligonucleotide by repeating the cycle including the steps (a) to (d) and the washing steps (i) to (iv). Further, the purity of the target oligonucleotide in the synthesis product can be improved. When a basic reagent is used in the capping step and / or the oxidation step, the purity of the target oligonucleotide in the synthesized product can be particularly remarkably improved.

(e) Desorption step from solid phase carrier and deprotection step After the synthesis of the target chain length oligonucleotide is completed, the desorption step of the synthetic oligonucleotide from the solid phase carrier, the phosphate group of the oligonucleotide, A step of deprotecting the protecting group of the amino group can be performed.

  The desorption step and deprotection step of the oligonucleotide synthesized by the above method from the solid phase carrier can be performed by a conventional method. The elimination of the synthesized oligodeoxyribonucleotide from the solid phase carrier can be usually carried out by placing the solid phase carrier carrying the synthetic oligodeoxyribonucleotide under basic conditions. Suitably, for example, by adding concentrated aqueous ammonia to a solid phase carrier supporting a synthetic oligodeoxyribonucleotide and allowing it to react for 15 minutes or longer, preferably 30 minutes or longer, more preferably 1 hour or longer, Synthetic oligonucleotides can be desorbed from the solid support. Alternatively, the elimination of the synthetic oligodeoxyribonucleotide from the solid support can be promoted by heating after the addition of concentrated aqueous ammonia. Deprotection of the protecting group of the phosphate group or amino group of the oligodeoxyribonucleotide can be performed by a conventional method. Typically, in the desorption step from the solid phase carrier, a basic reagent (for example, concentrated aqueous ammonia) is added to the solid phase carrier and reacted for a certain period of time, followed by heat treatment (for example, 1 to 12 at 55 to 60 ° C). By the treatment of time, the protecting group of amino group and phosphate group added to the nucleotide can be deprotected. For example, a β-cyanoethyl group at the 3′-position used as a phosphate group protecting group derived from a nucleoside-3′-O-phosphoramidite derivative, a benzoyl group or an isobutyryl group commonly used as an amino acid protecting group at the base moiety Acyl groups such as phenoxyacetyl group and acetyl group can be easily removed by heat treatment in a basic solvent such as aqueous ammonia (particularly preferably concentrated aqueous ammonia) or concentrated aqueous ammonia-40% methylamine solution. Can be protected. In the case of having a methyl group as a phosphate protecting group derived from a nucleoside-3′-O-phosphoramidite derivative, deprotection is preferably performed using thiophenol before the desorption step from the solid phase carrier. be able to.

  As the desorption step and deprotection step of the synthetic oligoribonucleotide from the solid phase carrier, a basic reagent such as 40% methylamine aqueous solution or ammonia-ethanol 3: 1 mixture is added to the solid phase carrier, for example, ammonia-ethanol. By heat treatment at 55 ° C to 60 ° C for a certain time (for example, 1 to 12 hours, preferably 2 to 3 hours) with a 3: 1 mixture, or at room temperature for 2 hours (preferably 3 hours with a 40% methylamine aqueous solution) ) A method may be used in which the elimination from the solid phase carrier and the deprotection of the base moiety and the β-cyanoethyl group which is a protecting group for phosphoric acid are carried out in the same step. Removal of the protecting group added to the synthesized oligoribonucleotide can also be carried out by a conventional method. For example, when the 2′-hydroxyl group of the synthesized oligoribonucleotide is protected with a silyl protecting group such as t-butyldimethylsilyl group, or protected with a 2-cyanoethoxyethyl group, tetra The protecting group can be removed using a fluorine ion compound such as -n-butylammonium fluoride-tetrahydrofuran solution.

  The oligonucleotide synthesis method of the present invention may further include steps other than the steps (a) to (e) and the washing steps (i) to (iv). For example, the synthetic oligonucleotide desorbed from the solid phase may be acid-treated to desorb the 5'-hydroxyl DMTr group. Further, the synthesis product obtained by the above-described oligonucleotide synthesis procedure may be further purified by gel filtration, desalting such as ethanol precipitation, high performance liquid chromatography (HPLC) purification, PAGE purification or the like.

  The present invention further provides a detergent for a solid phase carrier for oligonucleotide solid phase synthesis, which contains a mixed solution of acetonitrile and acetone, or acetone as an active ingredient. The active ingredient of this cleaning agent may be, but is not limited to, a mixture of acetonitrile and acetone or a mixture of acetone and acetone in which the mixing ratio of acetonitrile: acetone is 0%: 100% to 95%: 5%. . Furthermore, the mixing ratio of acetonitrile: acetone in the active ingredient of this cleaning agent may be 0%: 100% -80%: 20% by weight ratio, 0%: 100% -70%: 30%. 10%: 90% to 70%: 30%, 30%: 70% to 70%: 30%, 40%: 60% to 60%: 40% It may be 50%: 50%. The detergent for the solid phase carrier for oligonucleotide solid phase synthesis according to the present invention preferably contains no moisture.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[Example 1]
Based on the conventional phosphoramidite method except that an automatic DNA / RNA synthesizer (Applied Biosystems Model 392 or 3900) is used and a mixture of acetonitrile and acetone or acetone is used as a washing solvent as described below, Table 2 DNA or RNA synthesis of the indicated oligonucleotides (sequences 1-7) was performed. As a control experiment, a conventional phosphoramidite method using acetonitrile as a washing solution was also performed.

  The oligonucleotide sequence synthesized by the method of the present invention (Table 2) is a sequence that does not take structural polymorphism such as hairpin loops and duplexes, and whose purity can be clearly determined by electrogram in capillary electrophoresis. It is predicted.

  In the oligonucleotide synthesis of this example, nucleoside-3′-CPG or nucleoside-3 using control pore glass (CPG) (manufactured by Proligo) or polystyrene resin (PS), which is a porous glass, as a solid support. '-PS was used. All reactions were performed at room temperature (about 20-25 ° C.).

  The nucleoside derivative used as a raw material for synthesis in this example is the following protected nucleoside-3′-O-phosphoramidite derivative (hereinafter sometimes simply referred to as an amidite form) produced by a conventional method shown below. It is a commercial product.

(a) Protected adenosine- or protected deoxyadenosine-3'-O-phosphoramidite derivative
For DNA synthesis, a protected deoxyadenosine-3′-O-phosphoramidite derivative was used. In this amidite, an acyl protecting group such as a benzoyl group or an amidine protecting group is introduced into 2'-deoxyadenosine as a protecting group for the amino group at the base, and then a dimethoxytrityl group is introduced as a protecting group for the 5'-hydroxyl group. The 3′-hydroxyl free nucleoside was prepared and then phosphilated with (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine to prepare a 3′-phosphoramidite. It is what was done.

  For RNA synthesis, protected adenosine-3′-O-phosphoramidite derivatives were used. In this amidite body, the base part of adenosine is previously protected with an acyl protecting group such as a benzoyl group or an amidine protecting group, the 5′-hydroxy group is protected with a dimethoxytrityl group, and the 2′-hydroxy group is protected with t. 3'-Phosphoramidite obtained by phosphilation with (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine for 3'-hydroxyl free 5'-protected nucleoside protected with -butyldimethylsilyl group, etc. It was obtained by preparing the body.

(b) Protected guanosine- or protected deoxyguanosine-3'-O-phosphoramidite derivative
For DNA synthesis, a protected deoxyguanosine-3′-O-phosphoramidite derivative was used. In this amidite, an acyl protecting group such as an isobutyryl group or an amidine protecting group is introduced into 2'-deoxyguanosine as an amino protecting group at the base portion, and then a dimethoxytrityl group is introduced as a protecting group for the 5'-hydroxyl group. The 3′-hydroxyl free nucleoside was prepared and then phosphilated with (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine to prepare a 3′-phosphoramidite. It is what was done.

  For RNA synthesis, protected guanosine-3′-O-phosphoramidite derivatives were used. In this amidite body, the base part of guanosine is previously protected with an acyl protecting group such as an isobutyryl group or an amidine protecting group, the 5′-hydroxy group is protected with a dimethoxytrityl group, and the 2′-hydroxy group is t protected. 3'-Phosphoramidite obtained by phosphilation with (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine for 3'-hydroxyl free 5'-protected nucleoside protected with -butyldimethylsilyl group, etc. It was obtained by preparing the body.

(c) Protected cytidine- or protected deoxycytidine-3′-O-phosphoramidite derivatives
For DNA synthesis, a protected deoxycytidine-3′-O-phosphoramidite derivative was used. In this amidite, 2′-deoxycytidine was introduced with an acyl-based protecting group such as a benzoyl group as a protecting group for the amino group at the base portion, and then a dimethoxytrityl group was introduced as a protecting group for the 5′-hydroxyl group. It was obtained by preparing a 3'-phosphoramidite body by preparing a hydroxyl group-free nucleoside and then phosphating it with (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine. .

  A protected cytidine-3′-O-phosphoramidite derivative was used for RNA synthesis. In this amidite body, the base part of cytidine is previously protected with an acyl protecting group such as a benzoyl group, the 5′-hydroxy group is protected with a dimethoxytrityl group, and the 2′-hydroxy group is protected with a t-butyldimethylsilyl group. 3'-hydroxyl free 5'-protected nucleoside protected with phosphite is prepared by phosphilation with (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine to prepare 3'-phosphoramidite Is obtained.

(d) Protected thymidine-3'-O-phosphoramidite derivative
A protected thymidine-3′-O-phosphoramidite derivative was used for DNA synthesis. This amidite is prepared by introducing a dimethoxytrityl group as a 5'-hydroxyl protecting group into thymidine to prepare a 3'-hydroxyl free nucleoside, and then (2-cyanoethyl) (N, N-diisopropylamino) chlorophosphine. To obtain a 3′-phosphoramidite body by phosphilation.

(e) Protected uridine-3'-O-phosphoramidite derivatives
A protected uridine-3′-O-phosphoramidite derivative was used for RNA synthesis. This amidite is a 3'-hydroxyl free 5'-protected nucleoside in which the 5'-hydroxyl group of uridine is protected with a dimethoxytrityl group and the 2'-hydroxyl group is protected with a t-butyldimethylsilyl group or the like. This was obtained by preparing a 3′-phosphoramidite body by phosphilation with (cyanoethyl) (N, N-diisopropylamino) chlorophosphine.

  Oligonucleotide synthesis was performed by sequentially joining nucleoside derivatives one by one from the 3 ′ end according to the base sequence of each oligonucleotide to be synthesized.

  Specifically, when synthesizing oligodeoxyribonucleotides, a bound solid phase carrier in which a 2′-deoxyribonucleoside-3′-O-succinate body was bound to the 3′-terminal side was prepared. Using this solid phase carrier and the protected deoxyribonucleoside-3′-O-phosphoramidite derivative (amidite body), the following steps A to D are repeated to obtain a nucleoside derivative immobilized on the solid phase carrier. Protected deoxyribonucleoside-3′-O-phosphoramidite derivatives were coupled stepwise step by step.

  On the other hand, when synthesizing oligoribonucleotides, a bound solid phase carrier having a 2′-position protected ribonucleotide-3′-O-succinate bound to the 3′-terminal side was prepared and used. By using this solid phase carrier and the protected ribonucleoside-3′-O-phosphoramidite derivative (amidite body), the following steps A to D are repeated to obtain a nucleoside derivative immobilized on the solid phase carrier. A step of attaching the protected ribonucleoside-3′-O-phosphoramidite derivative step by step step by step was performed.

Step A: 5'-position protecting group removal step
For removal (deprotection) of 5'-hydroxyl group (5'-terminal hydroxyl group), acid treatment by adding 140 μL of 3% trichloroacetic acid-dichloromethane solution to a column packed with nucleoside-3'-O-succinate-bonded solid support did. As a result, the protecting group DMTr at the 5 ′ end was eliminated from the nucleoside derivative on the solid support, and the 5′-hydroxyl group of the nucleoside derivative was released. The same acid treatment operation was further performed twice. Next, the 3% trichloroacetic acid-dichloromethane solution used was removed by flash operation.

  Subsequently, as a washing operation, 280 μL of a washing solvent was fed to a column packed with a solid phase carrier, and further a flush operation was performed to remove trichloroacetic acid adhering to the solid phase carrier. As the washing solvent, acetonitrile-acetone mixed liquid or acetone prepared at various mixing ratios was used (Table 3, described later). The same washing operation was repeated again. By this washing operation, trichloroacetic acid was completely removed from the solid phase carrier, and at the same time, dehydration was performed.

Step B: Condensation Step 0.05M to 0.1M amidite solution (dissolving the amidite to be bound) is dissolved in a solid phase carrier carrying a nucleoside derivative in which the 5′-hydroxyl group (5′-terminal hydroxyl group) is released in the condensation process step A. Acetonitrile solution) 45 μL and activator (acid catalyst) solution (0.45M 1H-tetrazole, 0.25M 2,5-dicyanoimidazole, 0.25M 5-ethylthio-1H-tetrazole, or 0.25M 5-benzylthio-1H- Acetonitrile solution containing any one of tetrazole as an activator (65 μL) was added and allowed to stand for 60 seconds. As a result, the added amidite is activated and causes a condensation reaction with the free 5′-hydrogen group of the nucleoside derivative on the solid phase carrier, resulting in the elongation of the oligonucleotide chain on the solid phase carrier. Subsequently, the reaction solution was removed from the solid support by a flash operation.

  Subsequently, as a washing operation, 280 μL of the same acetonitrile-acetone mixed solution or acetone used in Step A as a washing solvent is fed to the column packed with the solid phase carrier, and further a flush operation is performed. The solution was removed. By this washing operation, the unreacted amidite and the activator were removed from the solid support, and at the same time, dehydration was performed.

Process C: Capping process (acetylation)
After the above flash operation, in order to acetylate and protect (cap) the unreacted 5 ′ hydroxyl group of the nucleotide derivative on the solid phase carrier, acetic anhydride--as a capping reagent is added to the column packed with the solid phase carrier. Pyridine-tetrahydrofuran 1: 1: 8 mixed solution (Applied Biosystems) 30 μL and 10% 1-methylimidazole-tetrafuran mixed solution (Applied Biosystems) 30 μL were fed. Thereafter, a flash operation was performed to remove the reaction solution.

  Next, before the next step, as the washing operation, the same acetonitrile-acetone mixed solution or acetone (280 μL) as used in step A is sent as a washing solvent to the column packed with the solid phase carrier, and further, A flash operation was performed to remove the reaction solution. By this washing operation, the acetylating reagent was removed from the solid support.

Step D: Oxidation Step In order to stabilize the oligonucleotide chain extended on the solid phase carrier as described above by oxidation, 0.02M iodine solution (tetrahydrofuran-pyridine-water solution; tetrahydrofuran: pyridine: water = 90 μL of 70: 28: 2 (v / v) mixed solution prepared so that the iodine concentration was 0.02 M (manufactured by Wako Pure Chemical Industries, Ltd.) was fed. Thereafter, a flash operation was performed to remove the reaction solution.

  Next, before the next step, as a washing operation, the same acetonitrile-acetone mixed solution or acetone (280 μL) used in Step A is sent as a washing solvent to the column packed with the above solid phase carrier, The reagent adhering to the solid support was washed away. The acetonitrile-acetone mixed solution or acetone (280 μL) was fed again, and the flushing operation was performed to remove the oxidizing reagent from the solid phase carrier.

  A dinucleotide chain was synthesized on the solid support by performing the above steps A to D once. Therefore, the same synthesis reaction with steps A to D as one cycle was repeated to synthesize the target oligonucleotide chain.

Oligodeoxyribonucleotide deprotection step After completion of the synthesis reaction of each oligodeoxyribonucleotide, concentrated aqueous ammonia (saturated aqueous ammonia) was added to the solid support carrying the synthetic oligodeoxyribonucleotide, and the mixture was allowed to stand at room temperature for 1 hour. Synthetic oligodeoxyribonucleotides were desorbed from the phase support. Then, the reaction solution was heated at 55 ° C. for 8 hours to deprotect the nucleotide base part and the phosphate protecting group. After this deprotection operation, ammonia was distilled off under reduced pressure. Subsequently, the obtained reaction product was dissolved again in distilled water, and extracted by adding diethyl ether to remove carboxylic acid amides and acrylonitrile generated by deprotection. In this way, a synthetic product containing each oligodeoxyribonucleotide (DNA) shown in Table 2 was obtained.

Oligoribonucleotide deprotection step After completion of the synthesis reaction of each oligoribonucleotide, methylamine aqueous solution (0.7 mL) was injected into a solid phase carrier (CPG column) carrying the synthetic oligoribonucleotide and allowed to stand for 2 hours. . Thereafter, an aqueous methylamine solution was allowed to flow out from the column. Thereafter, an ethanol-acetonitrile-water mixture (3: 1: 1 (v / v)) was injected into the column to wash the solid phase carrier in the column. The washing solution and the eluate (effluent) were concentrated under reduced pressure.

  After concentration under reduced pressure, 100 μL of triethylamine trihydrofluoride was added and dissolved. After dissolution, the mixture was allowed to stand at room temperature for 12 hours. After 12 hours, 400 μL of butanol was added and stirred with a vortex mixer. Thereafter, centrifugation (3000 rotations) was performed for 5 minutes in a centrifuge. The supernatant was discarded, the pellet was dissolved in sterilized water, and desalting and simple purification were performed by simple reverse phase chromatography. In this way, a synthetic product containing each oligoribonucleotide (RNA) shown in Table 2 was obtained.

[Example 2]
Each oligonucleotide synthesis product obtained in Example 1 was subjected to capillary electrophoresis using a capillary electrophoresis apparatus (Beckman Coulter P / ACE). Based on the electrophoresis result (electrogram), the purity was compared between the synthesized products using different washing solvent compositions and / or solid phase carriers.

  Furthermore, using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI Tof MS, Voyager of Applied Biosystems), mass analysis of the synthesized product was performed, and the molecular weight of the oligonucleotide contained in the resulting synthesized product Confirmation was also performed.

Table 3 shows the washing solvent composition, solid phase carrier, and absorbance (OD value) used for the synthesis of each oligonucleotide synthesis product. Table 3 also shows the correspondence between the electrophorograms of capillary electrophoresis of each synthesized product and the figure showing the MALDI Tof MS spectrum. In Table 3, “CH ₃ CN: (CH ₃ ) ₂ CO (v / v)” is the weight ratio of acetonitrile (CH ₃ CN) and acetone ((CH ₃ ) ₂ CO) mixed with the composition of the washing solvent. It is shown by. Absorbance indicates the nucleic acid yield in each synthetic product.

  1 to 9 show the analysis results of the synthesized product of the oligonucleotide of sequence 1. In the conventional method (control experiment) using acetonitrile (100%; not containing acetone) as a washing solvent, although a mixture of oligonucleotides having different chain lengths was confirmed in an electrogram of capillary electrophoresis (FIG. 1). A), it was shown that the oligonucleotide of sequence 1 was successfully synthesized at a sufficient level (FIG. 1b) (FIG. 1). On the other hand, acetonitrile-acetone mixed solution (7: 3 (V / V)) (Fig. 2), acetonitrile-acetone mixed solution (1: 1 (V / V)) (Fig. 3) or acetonitrile-acetone mixed solution (3 : 7 (V / V)) (Fig. 4) or acetone (100%) (Fig. 6) (control pore glass (CPG) is used as the solid support). It was shown that the oligonucleotide of sequence 1 could be synthesized at a level comparable to the conventional method using acetonitrile (100%). Furthermore, when an acetonitrile-acetone mixture (7: 3 (V / V)) (Fig. 2) and an acetonitrile-acetone mixture (1: 1 (V / V)) (Fig. 3) are used, the conventional method is used. The mixture of other oligonucleotides of other chain lengths in the synthesized product (a in FIG. 1) was significantly reduced, indicating that a synthesized product containing the oligonucleotide of sequence 1 with very high purity was obtained. . The purity obtained here was so high that a subsequent purification step became unnecessary. Even when an acetonitrile-acetone mixture (3: 7 (V / V)) (FIG. 4) and acetone (100%) (FIG. 6) were used, the purity of the oligonucleotide of sequence 1 in the synthesized product was the same as that of the conventional method. Equivalent or better. In addition, most of the other oligonucleotides of chain length mixed in the synthesized product are incompletely synthesized oligonucleotides whose synthesis was stopped during the chain extension. Further, from FIG. 5 showing the MALDI Tof MS spectrum of the synthesized product, it was confirmed that the oligonucleotide having the molecular weight as predicted from Sequence 1 was indeed synthesized, that is, the synthesis reaction proceeded without side reactions. Indicated.

  Furthermore, when synthesis was performed using polystyrene (PS) as a solid phase carrier and acetonitrile / acetone mixtures having different ratios, acetonitrile (100%) was used as a washing solvent as shown in FIGS. The oligonucleotide of sequence 1 could be synthesized at a level comparable to the conventional method used. Furthermore, when an acetonitrile-acetone mixture (1: 1 (V / V)) was used, the mixture of oligonucleotides with other chain lengths in the synthesized product found by the conventional method (a in FIG. 1) was marked. Reduced, indicating that a synthetic product was obtained that contained the oligonucleotide of sequence 1 in very high purity (FIG. 8). Even when an acetonitrile-acetone mixture (3: 7 (V / V)) was used, the purity of the oligonucleotide of sequence 1 in the synthesized product was significantly improved compared to the conventional method (FIG. 9). Even when an acetonitrile-acetone mixture (7: 3 (V / V)) (FIG. 7) was used, the purity of the oligonucleotide of sequence 1 in the synthesized product was equal to or higher than that of the conventional method.

  From the above results, the acetonitrile-acetone mixture or acetone newly used as a washing solvent in place of conventional acetonitrile (100%) is equivalent to conventional acetonitrile, whether the solid support is CPG or polystyrene (PS) or It was shown that the oligonucleotide can be synthesized with high efficiency by having an excellent washing property more than that, and as a result, using an acetonitrile-acetone mixed solution or acetone as a washing solvent in the solid phase nucleic acid synthesis method. Furthermore, when an acetonitrile-acetone mixture having a mixture ratio of acetonitrile and acetone of 3: 7 (30%: 70%) to 7: 3 (70%: 30%) is used, the oligo of sequence 1 in the synthesized product is used. The tendency to improve the purity of the nucleotide was shown, but this was due to the activation of the amidite with an activator (acid catalyst) and the basic reagents used in the subsequent steps of the condensation reaction (1-methylimidazole, pyridine, etc.) ) Was effectively removed by the above-mentioned acetonitrile-acetone mixture used as a washing solvent, so that the inhibition of the activity of the acid catalyst by the basic reagent was suppressed in the next chain elongation cycle, and as a result, the chain elongation was more efficient. Had suggested that happened. Furthermore, it was considered that the acetonitrile-acetone mixture was mixed with the water present on the solid support, and the dehydration effect was obtained more effectively.

  Further, in FIGS. 10 and 11, unlike sequence 1, synthesis was performed by synthesizing an oligonucleotide of sequence 2 containing 4 bases of adenine, cytosine, guanine and thymine using acetone (100%) as an alternative washing solvent. The capillary electrophoresis electrogram and MALDI Tof MS spectrum of the product are shown respectively. FIGS. 10 and 11 show that the oligonucleotide of sequence 2 was certainly synthesized at a level comparable to that of the conventional method, and that the purity of the synthesized oligonucleotide was improved as compared with the conventional method.

  Similarly, FIGS. 12 and 13 are synthetic products obtained by synthesizing an oligonucleotide of sequence 3 containing 4 bases of adenine, cytosine, guanine and thymine, unlike acetone, using acetone (100%) as an alternative washing solvent. 2 shows an electrogram and MALDI Tof MS spectrum of capillary electrophoresis. FIGS. 12 and 13 also show that the oligonucleotide of sequence 2 was indeed synthesized at a level comparable to that of the conventional method.

  FIGS. 14 and 13 show capillaries of synthetic products obtained by synthesizing oligonucleotides of sequence 3 containing 4 bases of adenine, cytosine, guanine and thymine, unlike acetone, using acetone (100%) as an alternative washing solvent. The electrogram and MALDI Tof MS spectrum of electrophoresis are shown. FIGS. 12 and 13 also show that the oligonucleotide of sequence 3 was indeed synthesized at a level comparable to the conventional method.

  Similarly, for the sequence 4 which is a 60-mer long-chain oligonucleotide, electrophorograms of capillary electrophoresis of synthetic products obtained by synthesis using acetonitrile-acetone mixtures in different ratios as washing solvents (FIGS. 15 to 16, 18) -20) and the MALDI Tof MS spectrum (FIGS. 17 and 21) also show that the oligonucleotide of sequence 4 was indeed synthesized at a level comparable to that of the conventional method (FIG. 14). From this, it was shown that a 60 mer long oligonucleotide can be synthesized by the method of the present invention.

  Furthermore, for the sequence 5 which is an 80-mer long-chain oligonucleotide, capillary electrophoresis electrograms of synthetic products obtained by synthesis using different ratios of acetonitrile-acetone mixtures as washing solvents (FIGS. 23 to 24, 26) 27) and the MALDI Tof MS spectrum (FIG. 25) also show that the oligonucleotide of sequence 5 was indeed synthesized at a level comparable to that of the conventional method (FIG. 22). From this, it was shown that a longer oligonucleotide of 80 mer can be synthesized by the method of the present invention.

  On the other hand, FIG. 29 shows a MALDI Tof MS spectrum of a synthetic product obtained by synthesizing a mixture of acetonitrile and acetone as a washing solvent for the sequence 6 which is RNA unlike the sequences 1 to 5 which is DNA. It was shown that the oligonucleotide (RNA) of sequence 6 was also able to synthesize the oligonucleotide of sequence 6 at a level comparable to that of the conventional method (FIG. 28), and the purity was improved. From this, it was shown that RNA can also be synthesized by the method of the present invention.

  Similarly, FIG. 31 shows a MALDI Tof MS spectrum of the synthesized product obtained by synthesis using the acetonitrile-acetone mixed solution as the washing solvent for the sequence 7, which is RNA. It was also shown that the oligonucleotide of sequence 7 (RNA) could certainly synthesize the oligonucleotide of sequence 7 at a level comparable to that of the conventional method (FIG. 30), and its purity was improved. From this, it was shown that RNA can be synthesized with various sequences by the method of the present invention.

  The above results showed that an acetonitrile-acetone mixture and acetone were effective as an alternative washing solvent for acetonitrile used in the solid phase nucleic acid synthesis method. In particular, the acetonitrile-acetone mixed solution also exhibited an unexpected effect of improving the purity of the synthesized oligonucleotide.

  The oligonucleotide solid-phase synthesis method of the present invention using acetone or an acetonitrile-acetone mixed solvent as an alternative washing solvent for acetonitrile is equivalent to or higher in purity than the conventional method while reducing the amount of acetonitrile used with a small supply amount. It can be used to synthesize the target oligonucleotide with the above efficiency. In particular, the method of the present invention, which is performed under conditions capable of synthesizing oligonucleotides with high purity, can produce products having the same quality as conventional purified products by simplifying or omitting the post-synthesis purification steps that were conventionally required. Can be used to provide.

SEQ ID NOs: 1-5 are synthetic DNAs.
SEQ ID NOs: 6 and 7 are synthetic RNAs.

Claims (7)

  In the oligonucleotide solid-phase synthesis method based on the phosphoramidite method, the chemistry of the oligonucleotide is characterized in that the solid phase carrier is washed with a mixture of acetonitrile and acetone or acetone as a washing solvent at the end of each reaction step. Synthetic method.   The method according to claim 1, wherein the mixture of acetonitrile and acetone or acetone is of a mixture ratio of acetonitrile: acetone of 0%: 100% to 95%: 5%.   The method according to claim 1, wherein the mixture of acetonitrile and acetone or acetone has a mixture ratio of acetonitrile: acetone of 30%: 70% to 70%: 30%. An oligonucleotide solid phase synthesis method based on the phosphoramidite method is
(a) removing the 5 ′ hydroxyl group of the nucleoside on the solid support by acid treatment;
(b) a step of activating a nucleoside-3′-O-phosphoramidite derivative with an acid catalyst and linking the nucleoside 5 ′ hydroxyl group on a solid phase carrier with a trivalent phosphate bond by a condensation reaction;
(c) capping the unreacted 5 ′ hydroxyl group, and
(d) The method according to any one of claims 1 to 3, which is an oligonucleotide solid-phase synthesis method comprising repeatedly performing a reaction cycle including a step of oxidizing the trivalent phosphate bond.   The method according to any one of claims 1 to 4, wherein the solid support is control pore glass (CPG) or polystyrene (PS).   A cleaning agent for a solid phase carrier for oligonucleotide solid phase synthesis, comprising a mixed solution of acetonitrile and acetone, or acetone as an active ingredient.   The cleaning agent according to claim 6, wherein the mixed solution of acetone and acetone or acetone has a mixing ratio of acetonitrile: acetone of 0%: 100% to 95%: 5%.

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