JP2000344793A - Deprotection of oligonucleotides - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify an elimination process of a hydrophobic protecting group such as DMTi (dimethoxytrityl), etc., of oligonucleotide purified by a liquid chromatography method and to shorten days for the process. SOLUTION: This deprotection method comprises adding a strong acid to an eluted solution of liquid chromatography containing an oligonucleotide having a hydroxyl group protected with a hydrophobic protecting group and eliminating the hydrophobic protecting group of the oligonucleotide. The strong acid can be directly added to an eluted solution of an oligonucleotide collected from reversed-phase liquid chromatography. Hydrochloric acid, sulfuric acid, trifluoroacetic acid, trichloroacetic acid, etc., are used as the strong acid. In order to shorten the reaction time of the deprotection, preferably an aqueous solution of sodium bicarbonate is added to the eluted solution to neutralize the strong acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for purifying oligonucleotides whose hydroxyl groups are protected with a hydrophobic protecting group by liquid chromatography, and then efficiently deprotecting the oligonucleotides.

[0002]

2. Description of the Related Art DNA synthesis has become easier with the recent spread of automatic synthesizers. A synthesis method generally used in such a synthesizer is a solid-phase cyanoethyl phosphoramidite method, which is represented by a synthesis cycle as shown in FIG. That is, first, a nucleoside in which the 5′-hydroxyl group is protected with a dimethoxytrityl (DMTr) group is ester-bonded to an insoluble carrier such as CPG (control pore glass) via the 3′-hydroxyl group (FIG. 1). [I]). Then, the hydroxyl group at the 5'-position of this nucleoside unit is deprotected (FIG. 1 [II]).
The amidite form of the next nucleotide unit (Fig. 1 [II
I]). In this amidite form, the 5'-hydroxyl group of the nucleoside is protected with a DMTr group, and the 3'-hydroxyl group is provided with trivalent phosphorus, and a β-cyanoethyl group is bonded to the trivalent phosphorus as a protecting group, A dialkylamino group is bound as a leaving group. Trivalent phosphorus of the dinucleotide (FIG. 1 [IV]) thus produced is oxidized to pentavalent with iodine (FIG. 1 [V]). The unreacted hydroxyl group at the 5'-position is excluded from the subsequent extension reaction by capping by acetylation. Then, by repeating this cycle, a desired DNA is synthesized.

[0003] The synthesized DNA is cut out of the carrier with concentrated aqueous ammonia and collected in a vial.
By treating at 4 ° C. for 4 to 5 hours, the protecting group for the amino group of the base of the nucleoside and the protecting group for phosphorus (β-cyanoethyl group) are eliminated. Thereafter, the crude oligo DNA contains the above-mentioned eliminated protecting group and oligo DNA with poor condensation, and thus is purified by reverse phase liquid chromatography (reverse phase HPLC). The target DNA has a significantly different molecular weight from the removed protecting group, and since the 5 'end is protected by a hydrophobic DMTr group, it is hydrophobic with poorly condensed oligo DNA that does not have a DMTr group. Can be purified to high purity by reversed-phase HPLC.

[0004]

The target oligo DNA obtained by HPLC fractionation as described above removes the DMTr group (elimination of Tr).
The final product. Oligo DNA separated by HPLC
Is in a solution state in the HPLC eluent, so that conventionally, the eluent was removed by drying under reduced pressure, dried, then detrified for 30 minutes by adding 80% acetic acid, and dried again under reduced pressure. The acetic acid was removed and redissolved.

[0005] As described above, the conventional method for removing Tr has the following problems.
Times of vacuum drying and re-dissolving steps, and the reaction time is 3
It took about 0 minutes. In the case of phosphorothioate-type oligo DNA (oligodeoxynucleotide phosphorothioate) used for antisense nucleic acid drug research,
It may be difficult to dissolve in an 80% acetic acid aqueous solution, and the Tr removal reaction may be insufficient.

[0006] It is an object of the present invention to simplify the steps and shorten the number of days of the steps in the deTr reaction of oligonucleotides purified by liquid chromatography.

[0007]

According to the present invention, the above object is achieved by adding a strong acid to an eluent for liquid chromatography containing an oligonucleotide whose hydroxyl group is protected by a hydrophobic protecting group. This is achieved by a method for deprotecting an oligonucleotide, which comprises removing a hydrophobic protecting group of nucleotides.

That is, the present invention is characterized in that oligonucleotides having a hydroxyl group protected by a hydrophobic protecting group are reacted with a strong acid while being contained in an eluent to remove the protecting group. The deprotection can be performed only by adding the strong acid directly to the eluent, so that the operation is simple. Furthermore, since the purified oligonucleotides separated by HPLC can be deprotected as they are without being subjected to the step of drying and re-dissolving the eluent under reduced pressure, the oligonucleotide purification step can be simplified and the purification can be simplified. This is convenient because the labor and time of the operation are reduced.

[0009] The deprotection method of the present invention can be applied to any kind of oligonucleotide. In the present invention, oligonucleotides are composed of nucleotides such as deoxyribonucleotides and ribonucleotides as structural units, and each nucleotide is linked by a phosphodiester bond at the 3′- and 5′-carbons of the sugar. In addition to synthetic DNAs and RNAs, the term encompasses modified products in which the phosphate moiety or nucleoside moiety is modified. Oligonucleotides in which the phosphate moiety is modified include, for example, oligodeoxynucleotide phosphorothioate (phosphorothioate-type oligo DNA). Synthetic oligonucleotides include those obtained by methods based on the known amidite method and phosphotriester method. The hydrophobic protecting group is not limited as long as it is a protecting group which can be easily removed by an acid, and typically, a dimethoxytrityl (DMTr) group which protects a hydroxyl group at the 5 'end of oligonucleotides is exemplified. Can be

In the present invention, the eluent is not particularly limited as long as it is used for liquid chromatography. Generally, reverse-phase liquid chromatography (reverse HPLC) is commonly used for the purification of synthetic oligonucleotides.
The eluent C) includes, for example, a mixture of a buffer such as triethylammonium acetate and an organic solvent such as acetonitrile.

The strong acid used in the present invention includes, for example, hydrochloric acid, sulfuric acid, trifluoroacetic acid, trichloroacetic acid and the like. Of these, hydrochloric acid is preferred from the viewpoint of easy handling.

In the present invention, the use of a strong acid is excellent in that the deprotection reaction is carried out quickly, but if the reaction time is too long, the phosphodiester bond of the oligonucleotides is hydrolyzed and recovered. The purity of the oligonucleotide may be impaired. Therefore, it is preferable to neutralize the strong acid by adding a base to the eluent in order to stop the deprotection reaction after an appropriate time has elapsed after adding the strong acid to the eluent. Such bases include aqueous sodium bicarbonate,
Ammonia water, aqueous potassium carbonate solution and the like can be mentioned. Among them, aqueous sodium bicarbonate is preferred from the viewpoint of easy handling. The appropriate reaction time for deprotection varies depending on the amount of the strong acid added. In general, it is desirable to add 100 to 400 parts by volume of a strong acid in a 1N conversion amount to 1,000 parts by volume of the eluent. The reaction time is desirably 1.0 to 3.0 minutes.

[0013]

The present invention will be described in more detail with reference to the following examples. The materials and measurement methods used in this example are as follows.

Materials and Measurement Method (1) Oligo DNA, Tr Trating Agent and Neutralizing Agent Used Oligo DNA was converted to Tr on a 1 μmol scale by a conventional method using an automatic DNA synthesizer (Expedite Model 8909, manufactured by Nippon Perceptive Co.). It was synthesized as an / ON form (a state in which the hydroxyl group at the 5 'end was protected with a DMTr group). Cutting out from the carrier was carried out using 1.0 ml of 30% concentrated aqueous ammonia in a usual manner. The deprotection of the amino group protecting group and the phosphate group protecting group of the base portion of the oligo DNA was carried out at 50 ° C. overnight in a pressure-resistant vial in the form of a concentrated aqueous ammonia solution in accordance with a conventional method. Thereafter, ammonia was removed by drying under reduced pressure, and the dried oligo DNA was 0.1
Dissolved in N-pH 7.2-triethylammonium acetate buffer. Since a 0.2 μmol synthesis scale is a normal amount, one fifth of the synthesized amount was used as a crude oligo DNA sample in one experiment. Table 1 shows the sequence of the synthesized oligo DNA. The sequence listing of the samples shown in Table 1 will be described later.

[0015]

[Table 1]

A 1N aqueous hydrochloric acid solution used as a detrending agent was prepared by adding 25 ml of sterilized water to 25 ml of 2N hydrochloric acid solution and stirring.
A sodium bicarbonate solution used as a neutralizing agent to stop the deTr reaction is
It was prepared by dissolving 4.20 g of sodium bicarbonate in 50 ml of sterile water. The sterilized water was ultrapure water (resistivity: 10.0 MΩ · cm or more, filtered with 0.2 μm) in an autoclave at 120 ° C, 1
It was prepared by sterilizing for 5 minutes.

(2) Oligo DNA concentration measurement method The oligo DNA solution is put into a quartz cell (optical path length = 10.0 mm),
The absorbance at 260 nm was measured with a spectrophotometer (V-550: manufactured by JASCO Corporation), and the absorbance (unit: ABS) was used as the DNA concentration (unit: ODu / ml). That is, absorbance 1.0 (abs) is DN
Corresponds to an A concentration of 1.0 (ODu / ml). Since the measurement limit of the above spectrophotometer was 1.8 ABS, when the concentration of the oligo DNA solution exceeded this measurement limit, the DNA solution was diluted with pure water, and the absorbance was measured. The DNA concentration was determined by multiplication. The amount of DNA was determined by multiplying the DNA concentration by the volume of the solution.

(3) Method for Measuring DeTr-Treatment Rate A sample of the oligoDNA detrified according to the method of the present invention was treated with a high-performance liquid chromatography apparatus (manufactured by Nippon Waters Co., Ltd.) to remove the oligoDNA (Tr). / ON body)
And the oligo DNA that was not removed from Tr (Tr / OFF form), the area of both peaks was determined, and the removal rate of Tr was determined according to the following formula.

[0019]

(Equation 1)

The column is a WS-DNA (manufactured by Wako Pure Chemical Industries) column (4.6 mmφ), which is a C18 silica-based reverse phase column (ODS column).
× 150 mm). The detector wavelength used for analysis is 2
The analysis temperature was 40 ° C. The gradient table was as shown in Table 2.

The sample was prepared by diluting it in a 1.0 ml buffer after the Tr removal reaction (neutralized), and then diluting the solution 15-fold.

[0022]

[Table 2]

(4) Purity analysis method of oligo-DNA detrified In order to examine the effect of oligo-DNA degradation (hydrolysis of phosphodiester bond) by the deTr-treatment, deTr-treatment was performed according to the method of the present invention. Oligo DNA samples were separated by capillary gel electrophoresis analysis to determine purity. In this capillary gel electrophoresis, oligos with a chain length
Even DNA can be separated as completely different peaks. The analysis was performed using a capillary gel electrophoresis analyzer CAPI-3100 (manufactured by Otsuka Electronics Co., Ltd.). As a capillary gel column, μPAGE-5 (manufactured by J & W Scientific) was used. The specifications of this column are as follows: Gel: polyacrylamide gel (5% T, 5% C) Buffer: 100 mmol / 1 tris-borate, 7 mol / 1 urea (pH 8.3) Size: 75 μmφ × 50 cm (Effective length).

The analysis conditions were as follows: Electrophoresis voltage: -13 kV Injection: Voltage equation: -10 kV, 2.0 to 2.5 seconds Analysis temperature: 25 ° C. Detector: 260 nm.

[0025] The sample was prepared by diluting it in a 1.0 ml buffer after the deTr conversion reaction (neutralized), dialysis for 2.0 hours, and diluting the solution 15-fold.

Example The above crude oligo DNA sample was purified by a high performance liquid chromatography apparatus (manufactured by Nippon Waters), and a Tr / ON form was collected. Purification conditions were as follows: Column: WS-DNA (manufactured by Wako Pure Chemical Industries, Ltd.), 10 mmφ × 15
0 mm Developing solvent: Gradient mode of buffer / acetonitrile Buffer = 0.1 N-pH 7.2-Triethylammonium acetate (TEAA) buffer Injection amount: 0.2 μmol Synthetic scale equivalent Gradient parameters: As shown in Table 3.

[0027]

[Table 3]

Fraction collector parameters: as shown in Table 4.

[0029]

[Table 4]

The buffered oligo DNA (Tr / ON form) in a buffer / acetonitrile solution (1000 μl) was added to 50, 100, 20
Add 0, 400 or 1000 μl of 1N aqueous hydrochloric acid and add 0.5, 1.0, 2.
After 0, 3.0, 6.0, 10.0 or 30.0 minutes, 1N aqueous sodium bicarbonate (neutralizing agent) corresponding to twice the amount of hydrochloric acid was added to stop the reaction. The stirring was performed within 10 seconds after the addition of each of the 1N hydrochloric acid solution and the 1N sodium bicarbonate solution. The reaction time corresponds to the time from the addition of 1N aqueous hydrochloric acid to the addition of 1N aqueous sodium bicarbonate.

With respect to the oligo DNA subjected to the DeTr treatment, the deTr ratio and the purity were measured in accordance with the above-mentioned methods. The results are shown in Tables 5 and 6.

[0032]

[Table 5]

[0033]

[Table 6]

Table 5 shows that Tr removal can be achieved by directly adding hydrochloric acid to the HPLC fraction of the Tr / ON form. In particular, 1N aqueous hydrochloric acid was added to 1000 µl of the HPLC aliquot for 10 µl.
It was found that adding 0 μl or more and making the reaction time 1.0 minute or more is preferable. In addition, from Table 6, 1000 µL
The amount of 1N hydrochloric acid solution is set to 400 μl or less with respect to
In the case of 3.0 minutes or less, detrenching is performed while maintaining high purity without affecting the phosphodiester bond of the DNA strand.
It was shown that it can be converted.

From the above, in the method of the present invention, the amount of hydrochloric acid added was 100 to 400 parts by volume (in terms of 1N hydrochloric acid) with respect to 1,000 parts by volume of the HPLC aliquot, and the reaction time was:
It was shown that it is preferable to set the time to 1.0 to 3.0 minutes.

[0036]

As described above in detail, according to the present invention, a method for deprotecting a hydrophobic protecting group such as a DMTr group by directly adding a strong acid to oligonucleotides in a chromatography eluent. Provided, eliminating the conventional step of removing and re-dissolving the eluent prior to deprotection,
Work time can be greatly reduced. In addition, since the reaction time can be easily controlled by adding a neutralizing agent, the degradation of DNA can be easily suppressed, and high-purity DNA can be recovered.

[0037]

[Sequence List] SEQUENCE LISTING <110> TOAGOSEI CO., LTD. <120> METHOD FOR DEPROTECTION OF OLIGONUCLEOTIDES <130> IDP-265 <160> 1 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Experimental <400> 1 gattgcctac catccgttag caactggtga t 31

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing an oligonucleotide synthesis reaction cycle.

Claims (4)

[Claims] 1. The method according to claim 1, wherein a strong acid is added to an eluent of liquid chromatography containing an oligonucleotide whose hydroxyl group is protected by a hydrophobic protecting group, and the hydrophobic protecting group of the oligonucleotide is eliminated. A method for deprotecting oligonucleotides. 2. The method according to claim 1, wherein the eluent containing the oligonucleotides has been eluted from reverse phase liquid chromatography. 3. The method according to claim 1, wherein the strong acid is selected from the group consisting of hydrochloric acid, sulfuric acid, trifluoroacetic acid and trichloroacetic acid. 4. The method according to claim 1, wherein a base is added to the eluent to neutralize the strong acid in order to stop the reaction for removing the hydrophobic protecting group. Item 2. The method according to item 1.