JPH03181334A - Production of carrier for synthesis of nucleic acid - Google Patents

Abstract

PURPOSE:To obtain a carrier for synthesis of nucleic acid not causing scattering or sticking during reaction by dehydrating granular high purity silica gel having a specified diameter and a specified pore diameter and allowing the dehydrated silica gel to react with a silane coupling agent having alkylamino groups. CONSTITUTION:Spherical silica gel or porous glass having such high purity as >=95% silicon dioxide content and contg. <=5% boron oxide, <=5% aluminum oxide and <=5% other elements is used. The spherical silica gel having 30-200mum diameter and 900-2,000Angstrom pore diameter or the porous glass subjected to phase separation treatment and leaching by the principle of spinodal decomposition is satisfactorily dehydrated in an org. solvent or in a flow of gas and then allowed to react with a silane coupling agent having alkylamino groups. These alkylamino groups are bonded to isolated silanol groups on the surface of the silica gel or porous glass through alkoxysilane groups by 0.3-2.5mumol/m<2>.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for producing a carrier for nucleic acid synthesis, and more specifically, to a porous material that has high nucleic acid synthesis efficiency and reduces the amount of impurities mixed into the synthesized nucleic acid. The present invention relates to a method for producing a carrier for nucleic acid synthesis.

(Prior Art) The solid-phase synthesis method, in which polymers with high molecular weight are obtained by repeated reactions on a carrier such as a solid powder, is currently known as the solid-phase synthesis method, which was developed by Merrifield (M.
It was proposed by John E. Errifield, 1962) and became widely used. The solid support initially used for this purpose was a copolymer of polystyrene and divinylbenzene with amino groups introduced, but when this support was applied to nucleic acid synthesis, crushed powders of silica gel and porous glass were used. Today, the common method is to bond alkylamino groups to the surface of porous glass with a pore diameter of about 500A using a silane coupling agent or the like. There is. However, today, when using carriers manufactured by conventional methods, the synthesis efficiency rapidly decreases at the stage where 5 or 15 nucleotides are linked (57- to 15-mer), and this results in a highly polymerized nucleic acid. Attempts to obtain this result tend to result in the contamination of large amounts of by-products.

This is generally due to the fact that on the carrier surface, reactive sites at the ends of nucleic acid molecules of such length come into contact with chemical structures on the carrier surface or with adjacent growing nucleic acid molecules, causing undesirable side reactions. It is thought that this may be the case. In an attempt to avoid this, a method has been implemented in which the amino group is bonded via a longer alkyl group, but the above-mentioned drawbacks have hardly been improved by this method.

This is the result of a lack of sufficient understanding of the chemical structure of the surface of the carrier used and insufficient attention to the behavior of the powdered carrier within the reaction vessel. Ta.

(Problems to be Solved by the Invention) In view of the above-mentioned circumstances, the present invention provides a carrier for nucleic acid synthesis that has high nucleic acid synthesis efficiency and is capable of carrying out a uniform reaction without scattering or adhesion of the carrier during the reaction. That is.

(Means for Solving the Invention) An object of the present invention is to use high-purity silica gel or porous glass that has been previously formed into a spherical shape, and to sufficiently dehydrate granular materials having a specific pore size and diameter. After applying
This is achieved by reacting with a silane coupling agent having an alkylamino group under conditions such that a specific amino group density is achieved.

That is, the gist of the present invention is that 95% or more of silicon dioxide,
It has a composition of 5% or less of boron oxide, 5% or less of aluminum oxide, and 5% or less of other elements, and has a pore size of 900 to 200O.
In A, silica gel molded into a spherical shape with a diameter of 30 to 200μ, or a spherical porous glass obtained by making it porous by phase separation treatment and subsequent acid elution treatment using the principle of spinodal decomposition. is sufficiently dehydrated in an organic solvent or in an air stream, and then reacted with a silane coupling agent having a flukylamino group to convert the alkylamino group into an alkoxysilane at a ratio of 0.3 to 2.5 μmol/Td. The present invention relates to a method for producing a carrier for nucleic acid synthesis, characterized in that the carrier is bonded to an isolated silanol group on the surface via a group.

The present invention will be explained in detail below.

The composition of the silica gel or porous glass targeted by the present invention is one with high purity of 95% or more silicon dioxide, 5% or less boron oxide, 5% or less aluminum oxide,
Contains 5% or less of other elements. Aluminum oxide has unfavorable effects as a carrier for nucleic acid synthesis. Therefore, in the present invention, porous glass that does not contain impurity elements such as aluminum or porous glass that contains aluminum, which is manufactured using high-purity silica gel or high-purity borosilicate glass as a base glass, is subjected to extraction treatment with acid or alkali. Porous glass from which most of the aluminum on the surface has been removed is used. Note that the method for preparing porous glass in the present invention is a method in which the glass is made porous by phase separation treatment and subsequent acid extraction treatment using the principle of so-called spinodal decomposition.

The above-mentioned silica gel or porous glass is formed into a spherical object having a diameter of 30 to 200 μm, preferably 50 to 100 μm during the manufacturing process.

Since automatic synthesis of nucleic acids is carried out in a small reaction vessel and a filtration process is required, whether the diameter of the spherical particles is too large or small is inconvenient when used as a carrier for nucleic acid synthesis in the reaction. However, when it is within the above range, a uniform reaction can be carried out. Porous glass may be made into a spheroid by, for example, a known method in which it is molded at the stage of forming the base glass. In addition, when the base glass is phase-separated by heat treatment after molding, even though the inside of the sphere is porous, the surface may be covered with a dense shell-like skin. It is preferable to remove it by cleaning with acid or caustic soda.

In addition, the pore diameter of silica gel or porous glass is 500~
200 OA, preferably 900 to 1800 Å, and the surface area is usually 10 to 100 TIt/q. In short, within this range, the nucleic acid yield is particularly high and it can work efficiently as a carrier, and it also has good strength as a carrier and can give a uniform reaction.

Next, a silane coupling agent having an alkylamino group is applied to the surface of the spherical silica gel or porous glass, but the present invention requires that the silica gel or porous glass be sufficiently dehydrated before this. It is something. By carrying out this dehydration, it is possible to finally obtain a carrier having good properties as a carrier for nucleic acid synthesis.

The dehydration method usually involves using an organic solvent such as toluene, for example.
After drying with a dehydrating agent such as calcium hydride or calcium chloride to a water content of 1100 pl or less, silica gel or porous glass is immersed in this and heated under reflux. Examples include a method in which silica gel or porous glass is placed in a stream of air, nitrogen, or oxygen dried with calcium chloride, or a combination thereof, and heated and dried at a temperature of 400 to 700°C. In this dehydration treatment, the absorption in the infrared absorption spectrum of silica gel or porous glass (measured as a thin plate by compressing a spherical object) is usually 3.
It is desirable to carry out the experiment until a sharp peak is obtained at 750 cii-'. That is, in the case of silica gel or porous glass, the surface chemical structure required for bonding with the silane coupling agent is 3750 ct in the infrared spectrum.
There is only a hydroxyl group called an isolated silanol group that gives absorption to n-', and other water! ! ! Groups or attached moisture have an unfavorable effect on the reaction. In the case of ordinary silica gel or porous glass, for example, there are two or more adjacent hydroxyl groups and water attached to them, and the presence of these shows a broad absorption of 3700 ctn'' or less in the infrared spectrum. Due to the influence of impurity elements such as aluminum contained in the glass, there may be active hydroxyl groups and adhering water.The presence of this water has a negative effect on the reaction with the silane coupling agent, and the final Therefore, it becomes difficult to obtain a carrier suitable for nucleic acid synthesis.

The reaction with the alkylaminating agent can then usually be carried out in a dry organic solvent such as toluene. When dehydrating silica gel or porous glass in an organic solvent,
A silane coupling agent can be added to the mixture after the dehydration treatment, and the dehydration treatment can be carried out subsequently. The reaction temperature in this reaction is, for example, 80-120°C, preferably 100-120°C.
Although the temperature is 10° C., it is usually preferable to carry out the reaction at a temperature near the boiling point of the solvent, that is, the reflux temperature. Moreover, the reaction time is usually about 6 to 12 hours.

Examples of the silane coupling agent having an alkylamino group include lower alkoxysilanes substituted with lower alkyl amino acids of 01 to 4, such as aminopropyltriethoxysilane, aminopropyltrimethoxysilane, and aminoethyltrimethoxysilane. It will be done. Further, in the present invention, it is preferable to use a silane coupling agent that does not have reactive properties such as an alkylamino group in combination. This silane coupling agent may be, for example, a known one such as methyltrimethoxysilane. The usage ratio of this silane coupling agent is usually as follows:
It is 10 to 70 mol%, preferably 20 to 60 mol%. In the present invention, the above reaction conditions and the proportion of the silane coupling agent used are adjusted to introduce a predetermined amount of alkylamino groups, which will be described later, and the amino group density (
The ratio of the alkylamino group bonded to the isolated silanol group via the alkoxysilane group is controlled. Further, the amount of the silane coupling agent to be used can be selected depending on the amount of amino groups to be introduced. The surface amino group density of the silica gel or porous glass in the present invention is 0.3 to 2.5 μmol/m21, preferably 0.5 to 5 μmol/m21.
It is necessary that the amount is 1.5 μmol/end, and in this case, the most excellent effect as a carrier for nucleic acid synthesis is exhibited.

The surface amino group density was measured by B.F. Gisin in 1972 on aminated polystyrene in the Analytical Stain Power Acta magazine (B.F. Gisin, A.
nal iticaChimica Acta. 58
248-249, 1972). That is, after adsorbing picric acid dissolved in dichloromethane onto a porous material sample of about 30 cm, it is substituted and desorbed with diisopropylethylamine, and the amount of desorbed picric acid is quantified by the absorption intensity of ultraviolet light with a wavelength of 358 nm. . The upper limit of the surface amino group density of 1 to 2 μmol/bottom, which was recognized as the optimum here, corresponds to the density when the entire glass surface is covered with silanol groups, and the amino group density in the present invention is The optimum value of the group density is based on the value obtained by reacting all the glass surfaces in an orderly manner to alkylaminate.
It can be considered to be slightly smaller than that.

The carrier for nucleic acid synthesis of the present invention obtained by the above-mentioned reaction can be separated, washed, dried, and recovered by conventional methods.

When carrying out nucleic acid synthesis using the carrier of the present invention, various deoxyribonucleoside-3'-0
(N,N-diisopropylamino)-β-cyanoethylphosphoramidite (cyanoethyl-phosphoramidite)
or deoxyribonucleoside-3'-0 (N, N
-diisopropylamino> It can be carried out by a method using methylphosphoramidite. These methods are edited by the Japanese Biochemical Society, Biochemistry Experiment Course 1, Gene Research Pope, Nucleic Acid Chemistry and Analysis Techniques, Chapter 1 Chemical Synthesis of DNA, 1
~33 pages, Tokyo Kagaku Doujin (1986), or M, J.
Gate ed. Oligonucleotide Synthesis, Chapter 3 (E
Dited by M, J, Galt,” Qlig
onucleodide 5ynthesis, aE)
"ractical approach", Chapter
er 3.5 solid-phase 3ynthe
sis of Oligodeoxyribonucle
eot-ides by the Phosphit
etriester Method.

by T, Atkinson and M, Sm1
th, I RLPress, Oxford, 1
It can be carried out according to the method described in 984, ). However, the principles of this invention can of course be applied to carriers used in other nucleic acid synthesis methods such as the phosphotriester method.

(Effects of the Invention) According to the carrier for nucleic acid synthesis of the present invention, nucleic acid synthesis efficiency is very good. This is done by selecting a raw material, silica gel or porous glass, with a specific pore size and diameter, and after thorough drying, a silane coupling agent with an alkylamino group and a silane without an amino group are combined. This is because the carrier has the most suitable density of surface amino groups to react with a coupling agent and perform nucleic acid synthesis. That is, as a result, a carrier having excellent carrier physical properties, less side reactions, and a specific amount of amino group density can be effectively produced.

(Example) Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to the description of the following examples unless it exceeds the gist thereof.

Example 1 (Production of carrier for nucleic acid synthesis using silica gel)
Obtained by a manufacturing method using an alkaline silicate solution and mineral acid as raw materials, the pore size is 1195, the average diameter is 100μ, and the surface area is 25.
4Td/(j spherical silica gel (SiOz 95% or more,
1q of other elements (5% or less) was immersed in 100 ml of toluene dried over calcium hydride to a water content of 10 ppm or less, and boiled under reflux for 7 hours to dehydrate the spherical silica gel. When the infrared absorption spectrum of the spherical silica gel at this time was measured, there was a peak at 3750 m-1, and almost no peaks below 3700 ca-' were observed.

Next, the supernatant toluene was removed, and a mixture of 5 d of 7minoprobyltriethoxysilane and 15 m of methyltriethoxysilane was added to another 30 mm of dry toluene, and the mixture was heated under reflux for 12 hours. The reaction was carried out.

Then, the spherical silica gel after the reaction is dried under reduced pressure with an aspirator via a desiccant, and then vacuum dried.
A carrier for nucleic acid synthesis of the present invention was obtained.

The amount of amino groups determined by picric acid determination of this carrier is 52.1
μmol/q, and the surface amino group density is 2.1μ
It was mol/Td. This was treated with 3' p-nitrophenylsuccinate of 5' dimethoxytrityladenosine to bond adenosine to the aminated silica gel. The dimethoxytrityl group was removed with trichloroacetic acid, and the amount of adenosine bound was determined from the amount of removal, which was 18 μmol per gram, and 0.7 μmol per square meter.

Example 2 (Production of carrier for nucleic acid synthesis using porous glass with high-purity borosilicate glass as the base glass) Glass particles obtained by forming borosilicate glass into spherical droplets due to surface tension in a hot air flow Heat treatment to separate the phases, elute this with hydrochloric acid to obtain a porous body with a dense shell-like skin, and then wash this with 1% hydrofluoric acid to obtain an average particle size of 100μ and a pore size. 960A, surface area 3
A porous glass of 9.6 butts/g was produced.

After sufficiently dehydrating this in a heated dry air stream, dry toluene (water content ml ooppm or less) 30
A mixed solution containing 10-aminopropyltriethoxysilane and 10rIi1 of methyltriethoxysilane was poured into the solution, and the mixture was heated and reacted under reflux for 12 hours. Next, the carrier of the present invention was obtained by suction filtration, drying under reduced pressure with an aspirator, and further drying under vacuum.

The amount of amine groups in this carrier determined by picric acid determination is 36.4
(μmol/CI), and the surface amino group density is 0.9(
μmol/e).

In addition, 3 of this and 5'-dimethoxytrityladenosine
' After reacting with p-nitrosuccinate to convert amine groups into nucleosides, the amount of nucleosides carried was determined from the amount of dimethoxytrityl groups released, which was 14.8 μmol per gram, which was 0.4 μmol per square meter.
It was a mole.

Comparative Example 1 Spherical porous glass manufactured according to the method of Japanese Patent Publication No. 62-25618, that is, having the following composition obtained by phase separation and elution treatment with acid, pore diameter 1095A, pore volume 0.63cm3/(Ill, surface area 23TIt/
1 g of glass containing 1 g of aluminum was immersed in 40 d of 1N sulfuric acid, subjected to extraction treatment under reflux at a temperature of 110° C. for 7 hours, and then washed with acid until the washing liquid became neutral. Dry at a temperature of °C. (This elution process resulted in a weight loss of approximately 5%.) Table 1 The porous glass was heated to 600'C in a stream of dry air over silica gel and phosphorus pentoxide and dehydrated for 6 hours. processed. When the infrared absorption spectrum of the porous glass was measured at this time, it was found to be 3750 cm
”A peak was observed.

A mixed solution containing 30 ml of dry toluene, 3 d of aminopropyltriethoxysilane and 9 d of methyltriethoxysilane was poured into this porous body, heated, and heated under reflux for 12 ml.
After reacting for a period of time, the mixture was suction filtered, dried under reduced pressure using an aspirator, and further vacuum dried using an oil rotary vacuum pump to obtain a carrier. The amount of amino groups of this carrier determined by picric acid quantification was 27.7 μmol/q, and the surface amine group density was 1.2 μmol/TIi. In addition, the amount of nucleosides bound to this carrier is 4.0 micromol per gram, and the surface density is 0.17 μmol per square meter.
It was a mole.

Comparative Example 2 A solution of 3-7 minopropyltriethoxysilane 15d dissolved in purified toluene 35- was added to a spherical porous body 1o made of high-purity borosilicate glass with a pore diameter of 960A and an average particle diameter of 100μ, and under reflux, After reacting at a temperature of 110° C. for 30 minutes, 0.1 d of water was added, followed by continuous distal flow for 11 hours, followed by cooling, filtration, washing, and drying to produce a carrier. When the amine groups on this carrier were quantified, it was found to be 414.9 μmol/g, and the surface amino group density was 10.5 μmol/Td.

After bonding an adenine nucleoside whose 5' hydroxyl group is protected with a dimethoxytrityl group, the amount of nucleoside supported is measured by the amount of dimethoxytrityl group released, and it is 47.9 μmol per gram, and the surface density is 1.2 μm per square meter. It was a mole.

Synthesis example of nucleic acid> Synthesis example 1 Automatic DNA synthesizer GenetAII manufactured by Nippon Zeon
5' dimethoxytrityl β-cyanoethyl phosphoramidite was bonded to the aminated silica gel, which is the carrier of the present invention prepared in Example 1, using tetrazole as a condensing agent, and oxidized with iodine to form a phosphole group. A 15-mer oligonucleotide was synthesized by a conventional method using the nucleotide as the nucleotide. The base sequence used at this time was 5'GGCTGCTACTACTGA3'.

The amount of dimethoxytrityl groups recovered in each step is shown in FIG. 1, with the amount of nucleoside bound on the silica gel being 100. The final yield of 157- was 77%.

Synthesis Example 2 When a 15-mer having the same base sequence as in Synthesis Example 1 was synthesized using the porous glass carrier of the present invention produced in Example 2 above as a carrier, the final yield of the 15-mer was 87.
%Met. Further, the synthesis progress is shown in FIG. 2.

Synthesis Example 3 After extracting as much aluminium as possible from the porous glass containing aluminum obtained in Comparative Example 1 with a hot acid, synthesis was performed using a carrier having an alkylamino group bonded to the surface by the method of the present invention. A nucleic acid having the same base sequence as in Example 1 was synthesized in the same manner, and the synthesis progress was as shown in FIG. 3, with a final yield of 76%, which was quite good.

Synthesis Example 4 A nucleic acid having the same base sequence as Synthesis Example 1 was synthesized using the carrier obtained in Comparative Example 2 to which aminoalkyl groups were bound in excess, and the synthesis progress was as shown in FIG. The yield decreased rapidly.

[Brief explanation of drawings]

FIGS. 1 to 4 are graphs showing the synthesis progress of each nucleic acid synthesis in Synthesis Examples 1 to 4.

Claims (1)

[Claims] (1) 95% or more silicon dioxide, 5% or less boron oxide,
Utilizing the principle of silica gel or spinodal decomposition, which has a composition of 5% or less of aluminum oxide and 5% or less of other elements, and is molded into a spherical shape with a pore diameter of 900 to 2000 Å and a diameter of 30 to 200 μ, The porous glass obtained by making it porous through phase separation treatment and subsequent acid elution treatment is sufficiently dehydrated in an organic solvent or in a dry air stream, and then reacted with a silane coupling agent having an alkylamino group. A method for producing a carrier for nucleic acid synthesis, characterized in that alkylamino groups are bonded to isolated silanol groups on the surface via alkoxysilane groups at a rate of 0.3 to 2.5 μmol/m^2.