Method of solid-phase nucleic acid synthesis and solution composition for solid-phase nucleic acid synthesis

Abstract

This invention is intended to discover a novel solvent that can be used as an alternative to toluene in the step of deprotection in the method of solid-phase nucleic acid synthesis. With the use of such novel solvent, various problems caused by the use of toluene are dissolved. This invention is also intended to provide a method of solid-phase nucleic acid synthesis in which protected nucleoside phosphoramidites in which a protective group is bonded to a hydroxyl group at the 5′position or the 3′ position of a nucleoside are sequentially bound on a solid phase carrier, where a reaction of removing the protecting group from the protected nucleoside phosphoramidite is carried out in a solution comprising an acid with a pKa of 0.2 to 0.8 and acetonitrile.

Claims

1. A method of solid-phase nucleic acid synthesis in which protected nucleoside phosphoramidites in which a protective group is bonded to a hydroxyl group at the 5′ position or the 3′ position of a nucleoside are sequentially bound on a solid phase carrier, comprising deprotection of the hydroxyl group by removing the protecting group from the protected nucleoside phosphoramidite in a solution comprising an acid with a pKa of 0.2 to 0.8 and acetonitrile.

2. The method of solid-phase nucleic acid synthesis according to claim 1, wherein the protecting group is a trityl-based protecting group or a silyl-based protecting group.

3. The method of solid-phase nucleic acid synthesis according to claim 1, wherein the acid is trichloroacetic acid or trifluoroacetic acid.

4. The method of solid-phase nucleic acid synthesis according to claim 1, wherein the solid-phase support comprises porous resin beads.

5. The method of solid-phase nucleic acid synthesis according to claim 4, wherein the step of deprotection is carried out by allowing the solution to flow through a column filled with the porous resin beads.

6. The method of solid-phase nucleic acid synthesis according to claim 5, wherein a flow rate of the solution is 400 to 2,000 cm/h.

7. The method of solid-phase nucleic acid synthesis according to claim 5, wherein a length of a layer comprising the porous resin beads introduced into the column is 4 to 50 cm in a flow pass direction, provided that the length is determined when the porous resin beads are swollen with the solution.

Description

EXAMPLES

(1) Hereafter, the present invention is described in greater detail with reference to the examples, although the technical scope of the present invention is not limited to the examples below.

Example 11

(2) In Example 1, Nitto Phase HL comprising, carried thereon, T to which a dimethyltrytyl group (a protecting group) had bound was used, and a reaction of removing a protecting group was examined using various solvents in combination with acid substances. In Example 1, acetonitrile, acetone, ethyl acetate, DMF, DMSO, hexane, methanol, and ethanol were prepared as candidate solvents. In addition, dichloroacetic acid (DCA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA), p-toluenesulfonic acid (PTSA), and hydrochloric acid were prepared as acid substances.

Experimental Procedure 1

(3) Candidate solvents were mixed with acid substances in the manner described below.

(4) Preparation of Solutions of Candidate Solvents Containing 3% DCA:

(5) DCA (1.5 ml) was added to a 50-ml volumetric flask using a measuring pipette. The 50-ml volumetric flask was filled with the solutions.

(6) Preparation of 50% Mixed Solutions of Toluene and Candidate Solvents Containing 3% DCA:

(7) DCA (1.5 ml) was added to a 50-ml volumetric flask using a measuring pipette. The 50-ml volumetric flask was filled with the mixed solutions of toluene and candidate solvents at 1:1.

(8) Preparation of Solutions of Candidate Solvents Containing 9% TCA:

(9) TCA (1.5 ml) was added to a 50-ml volumetric flask using a measuring pipette. The 50-ml volumetric flask was filled with the solutions.

(10) Preparation of Solutions of Candidate Solvents Containing 3% TFA:

(11) TFA (1.5 ml) was added to a 50-ml volumetric flask using a measuring pipette. The 50-ml volumetric flask was filled with the solutions.

(12) Preparation of Solutions of Candidate Solvents Containing PTSA:

(13) PTSA (951.3 mg) was added to a 50-ml volumetric flask. The 50-ml volumetric flask was filled with the solutions.

(14) Preparation of Solutions of Candidate Solvents Containing Hydrochloric Acid:

(15) 4 N hydrochloric acid (0.1 ml) was added to a 100-ml volumetric flask using a measuring pipette, and the 100-ml volumetric flask was filled with various solvents.

Experimental Procedure 2

(16) A 10-mg fraction of Nitto Phase HL comprising dT carried on an aluminum dish obtained via weighing using a balance was added to a 50-ml volumetric flask. The 50-ml volumetric flask was filled with the solvents containing various acid substances prepared in Experimental procedure 1 and the flask was allowed to stand for 7 minutes.

Experimental Procedure 3

(17) After the reaction, polymer beads in the solution were separated via filtration through a filter. The absorbance of the filtrate (412 nm) was determined using a spectrophotometer.

(18) Results:

(19) Deprotection reactivity of DMT groups caused by various solvents in combination with acids was evaluated based on color development. The results of evaluation are summarized in Table 1 below. In Table 1, (*1) shows the results of evaluation using a mixed solution comprising toluene and a solvent at 1:1. In Table 1, the symbol “∘” indicates the absorbance within 20%, the symbol “Δ” indicates the absorbance of 50% or lower, and the symbol “x” indicates the absorbance of 5% or lower, in comparison with 3% DCA/toluene.

(20) TABLE-US-00001 TABLE 1 Acidic substance 3% Hydro- 3% DCA chloric DCA (*1) TCA TFA PTSA acid Candidate Toluene ○ ○ x solvent Acetonitrile x Δ ○ ○ ○ ○ Acetone x x x x Ethyl acetate x x x x DMF x x x x DMSO x x x Hexane x Δ x x Methanol x x x Ethanol x x x

(21) As shown in Table 1, provided that an acetonitrile solvent was used, acid substances other than DCA were found to have the effects of deprotection equivalent to those achieved with the use of 3% DCA/toluene. In Example 1, trichloroacetic acid (TCA), trifluoroacetic acid (TFA), p-toluenesulfonic acid (PTSA), and hydrochloric acid were subjected to primary screening as alternatives to DCA used for the deprotection reaction.

Example 2

(22) In Example 2, solid-phase nucleic acid synthesis was carried out using trichloroacetic acid (TCA), trifluoroacetic acid (TFA), p-toluenesulfonic acid (PTSA), and hydrochloric acid subjected to primary screening in Example 1 in the step of deprotection, and the purity of the synthesized nucleic acids was evaluated.

Experimental Procedure

(23) In Example 2, nucleic acid synthesis was carried out using an AKTA 10 synthesizer (Amersham Biosciences). A 1.2-ml stainless steel column (diameter: 1 cm; height: 1.5 cm) was used. A nucleic acid sequence to be synthesized was designated as 5′-ATA CCG ATT AAG CGA AGT TT-3′ (SEQ ID NO: 1). As a solid-phase support, 350 to 370 μmol/g Nitto Phase HL (Nitto Denko Corporation) was used. In addition, nucleic acid synthesis was carried out by removing a protecting group (a DMT group) from a base at the terminus, and amine wash was carried out using 0.1 N diazabicycloundecene for 30 minutes. As an activator, 5-ethylthio-1-tetrazole (ETT) was used. In Example 2, the step of deprotection was carried out in a reaction column at a linear flow rate of 400 cm/h.

(24) In Example 2, the cleaving reaction was carried out by, at the outset, transferring 50 mg of Nitto Phase HL after the nucleic acid synthesis to a plastic tube with a screw cap. Subsequently, 1 ml of an aqueous solution of 28% ammonia was added to the plastic tube with a screw cap, and the plastic tube was allowed to stand at 55° C. for 16 hours. Thereafter, beads were separated via filtration using 50% ethanol, the reaction product was introduced into a volumetric flask, and the total amount of the content in the volumetric flask was adjusted to 5 mg.

(25) In Example 2, the synthesized nucleic acids were analyzed using an anion exchange column. Specifically, Chromeleon, Thermo Fisher Scientific DNApac PA200 columns (Lot. 014-27-136) were used. The synthesized nucleic acids were identified at the column temperature of 30° C. and at 260 nm. The sample concentration was designated at 1.5 OD/ml and the amount of the sample was designated at 2 μl. As Eluate E1, 20 mM Tris buffer (pH 8.0) was used, and a mixture prepared by adding 1.25 M NaCl (pH 8.0) to Eluate E1 was used as Eluate E2. Eluate E1 and Eluate E2 were supplied to the columns in the manner as shown in Table 2.

(26) TABLE-US-00002 TABLE 2 Time (min) E1 E2 0 80 20 10 55 45 10.1 25 75 11.2 80 20 14.0 80 20

(27) The results of calculation of the purity of the nucleic acids to be synthesized are shown in Table 3. The purity (%) was calculated in the manner described below. Specifically, a peak area of the target of synthesis was designated as the numerator, the total peak area including the peak arising from impurities was designated as the denominator, and the purity (%) was then calculated.

(28) TABLE-US-00003 TABLE 3 Acid/solvent Purity (%) 3% DCA/toluene 87.9 9% TCA/acetonitrile 88.3 5% TFA/acetonitrile 82.7 p-Toluenesulfonic acid/acetonitrile 0 (up to 4-mer) HCl/acetonitrile 0 (up to 3-mer)

(29) When an acetonitrile solution containing TCA as an acid substance was used, as shown in Table 3, the results of nucleic acid synthesis were equivalent to those attained with the use of a toluene solution containing DCA as an acid substance. When an acetonitrile solution containing TFA as an acid substance was used, also, the results of nucleic acid synthesis were satisfactory.

(30) When the TCA concentration in an acetonitrile solution was 7% or 5%, the results were equivalent to those attained at the TCA concentration of 9%.

(31) When p-toluenesulfonic acid and hydrochloric acid were used as acid substances, however, a nucleic acid having a chain length of interest could not be synthesized.

(32) As is apparent from the results attained in Examples 1 and 2, use of an acid with a pKa of 0.2 to 0.8 (TCA is with a pKa of 0.66 and TFA is with a pKa of 0.2) is sufficient when performing the step of deprotection with the use of an acetonitrile solution. DCA has a pKa of 1.25, p-toluenesulfonic acid has a pKa of −2.8, and hydrochloric acid has a pKa of −8.0.

Example 3

(33) In Example 3, solid-phase nucleic acid synthesis was carried out in the same manner as in Example 2, except that a length of the column used in the reaction of nucleic acid synthesis was increased from 4 cm to 8 cm, and the purity of the synthesized nucleic acids was evaluated. In Example 3, the step of deprotection was carried out using a 3% DCA/toluene solution (linear velocity: 400 cm/h) or a 9% TCA/acetonitrile solution (linear velocity: 800 cm/h).

(34) When a solid-phase support comprising porous resin beads (e.g., Nitto Phase HL) is used, the upper limit of the liquid flow rate is 400 cm/h with the use of a DCA/toluene solution as a deprotection solution for the following reasons. That is, porous resin beads are swollen and softened in a toluene solution, a spherical form cannot be retained due to the pressure applied at a high flow rate, and the flow pass is blocked. When a TCA/acetonitrile solution is used, in contrast, a degree of swelling of porous resin beads in acetonitrile is small, and a spherical form thereof can be maintained at a high flow rate. As a deprotection solution, accordingly, a TCA/acetonitrile solution can be used at the flow rate of 800 cm/h or higher. Example 3 was intended to examine whether or not the duration of acid contact can be shortened and whether or not the purity of nucleic acid synthesis can be improved with the use of a TCA/acetonitrile solution as a deprotection solution at a high flow rate.

(35) The results are shown in Table 4. The purity shown in Table 4 was determined in the same manner as in Example 2.

(36) TABLE-US-00004 TABLE 4 Average deprotection Acid/solvent Purity (%) duration (min) 3% DCA/toluene 79.3 9.5 ± 2.0 9% TCA/acetonitrile 83.5 4.7 ± 0.5

(37) As shown in Table 4, the purity attained when deprotection was carried out with the use of 3% DCA/toluene according to a conventional technique was 79.3%. That is, such purity was apparently lower than the purity attained when synthesis was carried out with the use of a column with a length of 1.5 cm. Because of an increased column length, specifically, a contact time between a nucleic acid and an acid substance is increased, and the purity is decreased due to the depurination reaction or other reasons. As shown in Table 4, in contrast, a 9% TCA/acetonitrile solution can be used at a high flow rate. Thus, an acid contact time can be shortened, and an increase of 4% or more was observed in purity, compared with the use of a DCA/toluene solution.

(38) In Example 3, the solvents used in Example 1 were quantitatively examined in terms of the effects of swelling in Nitto Phase HL, which is a porous resin bead. Specifically, 1 g of Nitto Phase HL was fractionated, introduced into a 10-ml measuring cylinder, and the cylinder was soaked in the various solvents. The cylinder was allowed to stand overnight, and the volume after swelling was assayed. The results are shown in Table 5.

(39) TABLE-US-00005 TABLE 5 Bead Read value (ml) Degree of Solvent weight (g) Initial Swollen swelling (%) Hexane 1.0002 2.68 2.90 1.08 Methanol 1.0007 2.70 3.00 1.11 Ethanol 1.0001 2.72 3.30 1.21 Acetonitrile 1.0047 2.75 4.05 1.47 Acetone 1.0002 2.70 5.61 2.08 Ethyl acetate 1.0016 2.72 6.00 2.21 Toluene 1.0011 2.71 6.00 2.21 DMF 1.0000 2.70 6.96 2.58 DMSO 1.0005 2.70 9.60 3.56

(40) As shown in Table 5, porous resin beads were quantitatively found to be less likely to swell in acetonitrile, compared with toluene.

(41) All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.