JP2021059518A - Nucleotide-modified solid-phase carriers, method for producing dna chain and rna chain-modified carriers, and methods for producing dna and rna - Google Patents

Abstract

To provide a nucleotide-modified solid-phase carrier that can be used to separate extended RNA or DNA from a fixed carrier under mild conditions rather than harsh conditions, such as ammonia treatment (treatment under basic conditions).SOLUTION: Provided is a nucleotide-modified solid-phase carrier comprising a nucleic acid attached to an aromatic group, which is attached to a solid-phase carrier via an ether group, a silyl group, an alkyl group, an amide group, or an ester group, by a nitro group and a phosphate group.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for producing a nucleotide-modified solid-phase carrier, a DNA chain and RNA chain-modified carrier, and a method for producing DNA and RNA. It relates to a synthesis method which does not require the processing in.

Although various modified nucleic acids are used for nucleic acid drug development, existing nucleic acid synthesis methods have been developed for the purpose of natural DNA / RNA synthesis, and there are restrictions on the functional groups that can bind to DNA or RNA. is there. In the current nucleic acid synthesis method, separation from the fixed carrier after synthesis is performed under severe conditions such as treatment with concentrated ammonia water, so it is not possible to bind a functional group (acyl group, etc.) that is unstable to a base. Can not.

Against this background, the development of a synthetic method that does not require ammonia treatment (treatment under basic conditions) for separation from the fixed carrier is underway. So far, it has been reported that nucleic acids bound to a fixed carrier are separated by light irradiation (for example, Non-Patent Document 1). However, the method of separating the nucleic acid bound to the fixed carrier by light irradiation has a problem that the functional group of the base having a functional group may be eliminated, and it is difficult to obtain a modified nucleic acid. .. Therefore, there is a demand for a synthetic method that does not require ammonia treatment (treatment under basic conditions) to separate nucleic acids having a base having a functional group from a fixed carrier.

C. Dell'Aquila, et al. , Tetrahedron Lett. , 38, 30, 5289-5292 (1997).

The present invention has been made in view of the above circumstances, and is an RNA strand extended from a fixed carrier under mild conditions rather than harsh conditions such as ammonia treatment (treatment under basic conditions). It is an object of the present invention to provide a nucleotide-modified solid phase carrier that can be used for separating a DNA strand, a method for producing a DNA strand and an RNA strand-modified carrier, and a method for producing DNA and RNA.

The present inventors have made extensive studies to solve the above-mentioned problems. As a result, it is possible to use a nucleotide-modified solid phase carrier having a specific structure, extend a DNA chain or RNA chain from the nucleotide-modified solid phase carrier, and then dissociate RNA or DNA from the solid phase carrier at pH 6 to 8. RNA or DNA can be separated under mild conditions using possible reducing reagents, and by thus separating RNA or DNA, the RNA or DNA strand has a chemically unstable functional group. Even if it is, RNA or DNA can be obtained in a state where the functional group is not accompanied by a chemical change. Therefore, it has been found that an unprecedented chemically modified RNA or DNA can be produced, and the present invention has been completed. Specifically, the present invention provides the following.

（Ｂは修飾されていてもよい塩基であり、ＸはＨ又はＯＨ、ｎ＝１以上、ｐ＝１〜３、Ｒは炭素数１〜５のアルキル基、炭素数１〜５のアルコキシ基、炭素数１〜５のアルキルチオ基、−Ｓｉ（ＯＲ^１）_３、−ＳｉＲ^１（ＯＲ^１）_２、−ＳｉＲ^１ _２（ＯＲ^１）、−ＳｉＲ^１ _３、又は−ＮＲ^１ _２を表し、各Ｒ^１はそれぞれ独立に、炭素数１〜５のアルキル基であり、Ｙはエーテル基、シリル基、アルキル基、アミド基又はエステル基であり、Ｚ、Ｗはそれぞれ独立に、Ｈ又は保護基であり、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素又はアルキル基であり、ＳＰは固相担体を表す。） (1) The first invention of the present invention is a nucleotide-modified solid-phase carrier represented by the following general formula (1).

(B is a base that may be modified, X is H or OH, n = 1 or more, p = 1-3, R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon _{^{atoms, -Si (oR 1) 3,}} -SiR 1 (oR 1) 2, -SiR 1 2 (oR 1), - SiR 1 3, or represents ^-NR _{1 2,} each R ¹ is an alkyl group having 1 to 5 carbon atoms independently, Y is an ether group, a silyl group, an alkyl group, an amide group or an ester group, and Z and W are independent H or protecting groups, respectively. , ^Ra and R ^b are independently hydrogen or alkyl groups, respectively, and SP represents a solid phase carrier.)

（Ｂは修飾されていてもよい塩基であり、ＸはＨ又はＯＨ、ｎ＝１以上、ｐ＝１〜３、Ｒは炭素数１〜５のアルキル基、炭素数１〜５のアルコキシ基、炭素数１〜５のアルキルチオ基、−Ｓｉ（ＯＲ^１）_３、−ＳｉＲ^１（ＯＲ^１）_２、−ＳｉＲ^１ _２（ＯＲ^１）、−ＳｉＲ^１ _３、又は−ＮＲ^１ _２を表し、各Ｒ^１はそれぞれ独立に、炭素数１〜５のアルキル基であり、Ｙはエーテル基、シリル基、アルキル基、アミド基又はエステル基であり、Ｚ、Ｗはそれぞれ独立に、Ｈ又は保護基であり、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素又はアルキル基であり、ＳＰは固相担体を表す。ただし、ＮＯ_２基は、リン酸に結合した官能基に対してオルト又はパラ位に結合するものである。） (2) The second invention of the present invention is a nucleotide-modified solid-phase carrier represented by the following general formula (2).
A nucleotide for producing DNA or RNA using a reducing reagent capable of extending a DNA chain or RNA chain from the 5'end of the nucleotide-modified solid-phase carrier and then dissociating it from the solid-phase carrier at pH 6 to 8. It is a modified solid phase carrier.

(B is a base that may be modified, X is H or OH, n = 1 or more, p = 1 to 3, R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, Represents an alkylthio group having 1 to 5 carbon atoms, -Si (OR ¹ ) ₃ , -SiR ¹ (OR ¹ ) ₂ , -SiR ¹ ₂ (OR ¹ ), ^{-SiR 1} ₃ or -NR ¹ ₂ and each R ¹ is an alkyl group having 1 to 5 carbon atoms independently, Y is an ether group, a silyl group, an alkyl group, an amide group or an ester group, and Z and W are independently H or a protective group. , ^Ra and R ^b are independent hydrogen or alkyl groups, respectively, and SP represents a solid phase carrier, where the NO ₂ group is attached to the functional group attached to phosphoric acid at the ortho or para position. It is a thing.)

(3) The third invention of the present invention is the nucleotide-modified solid phase carrier according to the second invention, wherein the reducing reagent is a reagent that reduces a nitro group bonded to an aromatic ring to an amino group. Is.

(4) The fourth invention of the present invention is a nucleotide-modified solid-phase carrier according to the third invention, wherein the reducing reagent is titanium trichloride.

(5) A fifth invention of the present invention comprises a step of extending a DNA strand using the nucleotide-modified solid support according to the first or second invention, which comprises producing a DNA strand-modified carrier. The method.

(6) The sixth invention of the present invention can dissociate DNA from a solid phase carrier at pH 6 to 8 with respect to the DNA strand modified carrier obtained by the method for producing a DNA strand modified carrier according to the fifth invention. A method for producing DNA, which comprises a step of separating the DNA by using a reducing reagent.

(7) A seventh invention of the present invention comprises a step of extending an RNA strand using the nucleotide-modified solid support according to the first or second invention, which comprises producing an RNA strand-modified carrier. The method.

(8) In the eighth invention of the present invention, RNA can be dissociated from a solid phase carrier at pH 6 to 8 with respect to the RNA difference modified carrier obtained by the method for producing an RNA strand modified carrier according to the sixth invention. It is a method for producing RNA, which comprises a step of separating the RNA by using a reducing reagent.

(9) The ninth invention of the present invention is a reagent in which the reducing reagent reduces the nitro group of an aromatic ring bonded to the solid phase carrier via the Y site to an amino group in the seventh invention. It is a method for producing DNA, which is characterized by being present.

(10) The tenth invention of the present invention is a reagent in which the reducing reagent reduces the nitro group of an aromatic ring bonded to the solid phase carrier via the Y site to an amino group in the eighth invention. It is a method for producing DNA, which is characterized by being present.

(11) The eleventh invention of the present invention is the method for producing DNA according to the seventh or eighth invention, wherein the reducing reagent is titanium trichloride.

(12) The twelfth invention of the present invention is the method for producing DNA according to the ninth or tenth invention, wherein the reducing reagent is titanium trichloride.

According to the present invention, a primer-modified substrate having a specific structure capable of separating RNA or DNA under mild conditions using a reducing reagent capable of dissociating RNA or DNA from a solid-phase carrier at pH 6 to 8 is provided. Can be provided. Further, by using a reducing reagent capable of dissociating RNA or DNA from a solid-phase carrier at pH 6 to 8, RNA or DNA can be separated under mild conditions, so that a functional group that undergoes a chemical change in conventional ammonia treatment RNA or DNA can be obtained.

HPLC chart of 5'-G ^iBu- TTTTTTTTTTTT-X-SP. 5'-C ^Ac- TTTTTTTTTTTTTT-X-SP HPLC chart.

Hereinafter, specific embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be carried out with appropriate modifications within the scope of the object of the present invention.

As used herein, the term "nucleic acid" means a polymer of nucleotides, and the number thereof is not particularly limited. Nucleic acids include oligonucleotides in which 10 to 100 nucleotides are linked, and even longer polynucleotides. The nucleic acid includes not only DNA single strand or double strand, but also RNA single strand or double strand, DNA / RNA hybrid, DNA / RNA chimera and the like. Further, the nucleic acid may be composed of a natural base, a nucleotide and a nucleoside, or may partially contain a non-natural base, a nucleotide and a nucleoside. Nucleic acids include all DNAs and RNAs including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA and synthetic RNA, as well as artificial peptide nucleic acids, morpholino nucleic acids, methylphosphonate nucleic acids and S-oligonucleic acids. Contains synthetic nucleic acids. Further, it may be single-strand or double-stranded.

The "nucleotide-modified solid-phase carrier" is one in which a nucleotide or a nucleotide chain (oligonucleotide, polynucleotide, degree of polymerization is not limited) for extending an RNA chain or a DNA chain is bound to the solid-phase carrier. A nucleotide in which an RNA strand or a DNA strand can be extended from the 5'terminal site.

Further, "DNA strand" and "RNA strand" refer to a strand bound to a solid phase carrier by some chemical bond, and "DNA" and "RNA" are in a free state not bound to a solid phase carrier. Say the chain of.

≪1. Nucleotide-modified solid-phase carrier ≫
The nucleotide-modified solid-phase carrier according to the first embodiment of the present invention is a nucleotide-modified solid-phase carrier represented by the following general formula (1).

（Ｂは修飾されていてもよい塩基であり、ＸはＨ又はＯＨ、ｎ＝１以上、ｐ＝１〜３、Ｒは炭素数１〜５のアルキル基、炭素数１〜５のアルコキシ基、炭素数１〜５のアルキルチオ基、−Ｓｉ（ＯＲ^１）_３、−ＳｉＲ^１（ＯＲ^１）_２、−ＳｉＲ^１ _２（ＯＲ^１）、−ＳｉＲ^１ _３、又は−ＮＲ^１ _２を表し、各Ｒ^１はそれぞれ独立に、炭素数１〜５のアルキル基であり、Ｙはエーテル基、シリル基、アルキル基、アミド基又はエステル基であり、Ｚ、Ｗはそれぞれ独立に、Ｈ又は保護基であり、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素又はアルキル基であり、ＳＰは固相担体を表す。）

(B is a base that may be modified, X is H or OH, n = 1 or more, p = 1-3, R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon _{^{atoms, -Si (oR 1) 3,}} -SiR 1 (oR 1) 2, -SiR 1 2 (oR 1), - SiR 1 3, or represents ^-NR _{1 2,} each R ¹ is an alkyl group having 1 to 5 carbon atoms independently, Y is an ether group, a silyl group, an alkyl group, an amide group or an ester group, and Z and W are independent H or protecting groups, respectively. , ^Ra and R ^b are independently hydrogen or alkyl groups, respectively, and SP represents a solid phase carrier.)

The nucleotide-modified solid-phase carrier according to the second embodiment of the present invention is a nucleotide-modified solid-phase carrier represented by the following general formula (2).
A nucleotide for producing DNA or RNA using a reducing reagent capable of extending a DNA chain or RNA chain from the 5'end of the nucleotide-modified solid-phase carrier and then dissociating it from the solid-phase carrier at pH 6 to 8. It is a modified solid phase carrier.

（Ｂは修飾されていてもよい塩基であり、ＸはＨ又はＯＨ、ｎ＝１以上、ｐ＝１〜３、Ｒは炭素数１〜５のアルキル基、炭素数１〜５のアルコキシ基、炭素数１〜５のアルキルチオ基、−Ｓｉ（ＯＲ^１）_３、−ＳｉＲ^１（ＯＲ^１）_２、−ＳｉＲ^１ _２（ＯＲ^１）、−ＳｉＲ^１ _３、又は−ＮＲ^１ _２を表し、各Ｒ^１はそれぞれ独立に、炭素数１〜５のアルキル基であり、Ｙはエーテル基、シリル基、アルキル基、アミド基又はエステル基であり、Ｚ、Ｗはそれぞれ独立に、Ｈ又は保護基であり、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素又はアルキル基であり、ＳＰは固相担体を表す。ただし、ＮＯ_２基は、リン酸に結合した官能基に対してオルト又はパラ位に結合するものである。）

(B is a base that may be modified, X is H or OH, n = 1 or more, p = 1 to 3, R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, Represents an alkylthio group having 1 to 5 carbon atoms, -Si (OR ¹ ) ₃ , -SiR ¹ (OR ¹ ) ₂ , -SiR ¹ ₂ (OR ¹ ), ^{-SiR 1} ₃ or -NR ¹ ₂ and each R ¹ is an alkyl group having 1 to 5 carbon atoms independently, Y is an ether group, a silyl group, an alkyl group, an amide group or an ester group, and Z and W are independently H or a protective group. , ^Ra and R ^b are independent hydrogen or alkyl groups, respectively, and SP represents a solid phase carrier, where the NO ₂ group is attached to the functional group attached to phosphoric acid at the ortho or para position. It is a thing.)

In the above general formula (1) or (2), B represents a base that may be modified, more specifically a nucleic acid base, as described above. Here, the "nucleobase" refers to all nucleic acid bases and nucleic acids found in naturally occurring nucleic acids such as chromosomal DNA, plasmid DNA, messenger RNA, ribosomal RNA, transfer RNA, and nuclear small molecule RNA of an organism. Includes all heteroaromatic rings that may have substituents that can be used in synthesis. This includes those not found in nature. Typical nucleobases include, but are not limited to, adenine, guanine, cytosine, uracil, thymine and the like. In addition, those having a functional group at the position of the nucleobase are also included.

Z is a protecting group to prevent unwanted reactions other than the chain extension reaction at 5'-OH of the nucleoside. Such protecting groups are not eliminated by each chemical treatment in the chain extension reaction of DNA / RNA synthesis, but are easily eliminated by the deprotection process at the time of DNA / RNA excision after the completion of the chain extension reaction. The one is selected. Examples of such protecting groups include a trityl group, a p-methoxyphenyldiphenylmethyl group (MMTr), a di (p-methoxyphenyl) phenylmethyl group (DMTr), and the like, among which MMTr and DMTr are trityl. It is preferably used because it is easier to deprotect with an acid than a group, but it is not limited to this.

W is a protecting group to prevent unwanted reactions in phosphoric acid. Such protecting groups are not eliminated by each chemical treatment in the chain extension reaction of DNA / RNA synthesis, but are easily eliminated by the deprotection process at the time of DNA / RNA excision after the completion of the chain extension reaction. The one is selected. As such a protecting group, a cyanoalkyl group is preferably used, and among them, a cyanoethyl group is more preferably used, but the present invention is not limited thereto.

In the above general formula (1) or (2), each R independently has an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, and −Si (OR). ¹ ) Represents ₃ , -SiR ¹ (OR ¹ ) ₂ , -SiR ¹ ₂ (OR ¹ ), ^{-SiR 1} ₃ or -NR ¹ _2. Examples of the alkyl group having 1 to 5 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group and the like. Examples of the alkoxy group having 1 to 5 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a tert-butoxy group, and a pentoxy group. Examples of the alkylthio group having 1 to 5 carbon atoms include a methylthio group, an ethylthio group, a propylthio group, an isopropylthio group, a butylthio group, an isobutylthio group, a tert-butylthio group and a pentylthio group. Here, each R ¹ is independently an alkyl group having 1 to 5 carbon atoms. Further, n is 1 or more and p is 1 to 3.

In the above general formula (1) or (2), the upper limit of n may be appropriately designed in consideration of the method for synthesizing the nucleotide-modified solid phase carrier and the required properties, and may be appropriately designed, for example, 100. Hereinafter, it may be 1000 or less and 10000 or less.

As shown above, each R in the general formula (1) or (2) is an electron donating group. In the nucleotide-modified solid-phase carrier of the present embodiment, this electron-donating group is bonded to an aromatic ring together with a nitro group. The following Scheme 1 embodies the above general formula (1) or (2) as an example for explanation.

As shown in Schema 1 below, the nucleotide-modified solid phase carrier of the present embodiment is reduced to convert a nitro group into an amino group, and when the nitro group is converted into an amino group, a lone electron pair on a nitrogen atom contained in the converted amino group Moves to the aromatic ring and breaks the bond between the oxygen atom and the methylene carbon. At this time, the nitrogen atom of the amino group is positively charged, but the electron-donating group is bonded to the aromatic ring to which the amino group is bonded, so that the positively charged structure is stabilized. Will be done. Then, as a result, the reaction of this amino group becomes easy to proceed. As can be seen by referring to Schema 1 below, this reaction involves a nitro group (reduced to an amino group) to a carbon atom 3 atoms away from the carbon atom on the aromatic ring, which is bonded to the methylene carbon contained in the leaving group. If) is combined, it proceeds. Further, as can be seen by referring to Schema 2 below, a nitro group is bonded to a carbon atom 1 atom away (that is, adjacent) from the carbon atom on the aromatic ring, which is bonded to the methylene carbon contained in the leaving group. The reaction will proceed in the same way. In the following Scenes 1 and 2, the aromatic ring is a benzene ring, but any structure of the aromatic ring can be similarly provided with a resonance structure by satisfying such conditions. Therefore, for example, Even if the aromatic ring is a furan ring, a thiophene ring, or an imidazole ring, the dissociation reaction (deprotection reaction) is similarly promoted by the electron-donating group bonded to the aromatic ring.

The following general formula (1a) can be given as an example in which the nucleotide-modified solid-phase carrier of the present embodiment represented by the general formula (1) or (2) is more embodied. Of course, the present invention is not limited to this.

Next, in order to explain the method for synthesizing the nucleotide-modified solid-phase carrier of the present embodiment, the method for synthesizing the solid-phase carrier is shown in Scheme 3 below as an example. First, a nucleotide-modified solid-phase carrier is obtained using commercially available 3-Hydroxy-4-nitrovenzaaldehyde as a starting material.

Next, in order to explain the method for synthesizing the DNA strand or RNA strand modified carrier of the present embodiment, the method for synthesizing the solid phase carrier is shown in Scheme 4 below as an example. First, a carrier typified by Scheme 3-6 is used as a starting material, and a DNA strand-modified carrier is obtained by the method described below.

Here, T means a nucleic acid containing thymine. However, nucleic acids containing bases other than thymine can be used without problems. For example, nucleic acids containing bases such as adenine (A), guanine (G), cytosine (C), and uracil (U) can be mentioned. ^The structure of ^{G iBu} and ^{C Ac} is a nucleic acid having a base shown below.

[Solid phase carrier]
The solid-phase carrier according to the present embodiment is not particularly limited as long as it is a solid capable of retaining a DNA chain or an RNA chain by a chemical bond. For example, a glass-based porous carrier, or a porous resin carrier such as a polystyrene-based porous carrier and an acrylamide-based porous carrier can be mentioned. In particular, it is preferable to use a porous resin carrier, and it is even more preferable to use a polystyrene-based porous carrier.

The "polystyrene-based porous carrier" is a porous carrier mainly composed of a resin composed of structural units of styrene or a derivative thereof, and among them, a polystyrene-based porous carrier having an amino group and / or a hydroxy group is preferable. .. Examples of the polystyrene-based porous carrier include a porous carrier composed of styrene-hydroxystyrene-divinylbenzene-based copolymer particles (Japanese Patent Laid-Open No. 2005-079545, JP-A-2005-325272 and JP-A-2006-342245). (See Japanese Patent Application Laid-Open), or a porous carrier made of a styrene- (meth) acrylonitrile-hydroxystyrene-divinylbenzene-based copolymer (see Japanese Patent Application Laid-Open No. 2008-074979).

The "acrylamide-based porous carrier" is a porous carrier mainly composed of a resin composed of structural units of acrylamide or a derivative thereof, and among them, an acrylamide-based porous carrier having an amino group and / or a hydroxy group is preferable. , An acrylamide-based porous carrier having a hydroxy group is preferable.

Further, as the solid phase carrier, as disclosed in Japanese Patent No. 5479828, a carrier composed of a first aromatic monovinyl compound-divinyl compound- (meth) acrylonitrile-second aromatic monovinyl compound-based copolymer. Can be mentioned. The "first aromatic monovinyl compound" means styrene or a substitute thereof. "Divinyl compound" means an aromatic divinyl compound, a di (meth) acrylic acid ester or a substitute thereof. "(Meta) acrylonitrile" means "acrylonitrile" or "methacrylonitrile", or "both acrylonitrile and methacrylonitrile". The "second aromatic monovinyl compound" means styrene or a substitute thereof, which is characterized by containing a "functional group capable of binding to a carboxyl group by a dehydration condensation reaction".

Specific examples of these solid-phase carriers include particles, filters, bags, dishes, reaction vessels, and the like, but are not particularly limited. Of these, the particle form is more preferable in consideration of handleability. The particle size in that case is not particularly limited, but may be 0.05 to 500 μm, preferably 1 to 100 μm, and particularly preferably 1 to 10 μm.

[Reducing reagent]
The reducing reagent used in the reducing operation for separation from the solid phase carrier is not particularly limited as long as it is a reducing reagent capable of reducing a nitro group to an amino group. Specifically, examples of such a reducing reagent include titanium trichloride (TiCl ₃ ), adithionic acid, samarium iodide, Zn dust, and the like, and among them, titanium trichloride can be preferably mentioned. Further, this reduction treatment can be carried out not only by the reducing reagents mentioned above but also by an intracellular enzyme such as nitroreductase in the presence of NADH (reduced nicotinamide adenine dinucleotide). Therefore, for example, an RNA derivative in which the hydroxyl group at the 2'position is protected is synthesized from the nucleoside derivative of the present embodiment, and this is administered to the human body as a nucleic acid drug to deliver it to desired cells, and then RNA is produced by an intracellular enzyme. It can also be used to deprotect and express its function. In particular, the above-mentioned deprotection progresses rapidly in cells placed in a reducing environment such as cancer cells.

According to the above-mentioned nucleotide-modified solid-phase carrier, RNA or DNA can be dissociated from the solid-phase carrier at pH 6 to 8 after the RNA chain or DNA chain is extended to the nucleotide-modified solid-phase carrier. RNA or DNA can be separated under mild conditions using reagents. Further, by using a reducing reagent capable of dissociating RNA or DNA from a solid-phase carrier at pH 6 to 8, RNA or DNA can be separated under mild conditions, so that a functional group that undergoes a chemical change in conventional ammonia treatment RNA or DNA can be obtained. Then, in such a production method, it is expected that an unprecedented chemically modified RNA or DNA having a functional group that causes a chemical change in, for example, conventional ammonia treatment can be obtained. Therefore, the above-mentioned nucleotide-modified solid-phase carrier is expected to be particularly applied in the field of nucleic acid medicine.

≪2. Method for producing DNA strand or RNA strand modified carrier ≫
The method for producing a DNA strand or RNA strand modified carrier according to the present invention is characterized by comprising a step of extending the DNA strand or RNA strand using the above-mentioned nucleotide modified solid phase carrier. Specifically, various known synthetic methods can be applied. That is, an extended oligonucleotide is obtained by sequentially binding a nucleotide to a nucleoside, a nucleotide or an oligonucleotide bound on a solid-phase carrier. Examples of the nucleic acid synthesis reaction include an H-phosphonate method, a phosphoester method, and a solid phase phosphoramidite method. For example, a method for synthesizing a DNA strand or RNA strand modified carrier based on the phosphite method is disclosed in detail on pages 36 to 45 of, for example, "Chemical Synthesis Method for DNA" (Hirokawa Shoten, Chemistry and Biology, Experimental Line 22). ..

≪3. DNA or RNA production method ≫
In the method for producing DNA or RNA of the present invention, DNA or RNA is separated from the above-mentioned DNA strand or RNA strand modified carrier by using a reducing reagent capable of dissociating DNA from the solid phase carrier at pH 6 to 8. By doing so, it is characterized in producing DNA or RNA.

Hereinafter, the present invention will be described in more detail by showing examples, but the present invention is not limited to the following examples. The compound number or carrier number in the following synthesis example corresponds to the number assigned to the compound or carrier in Schemes 3 and 4 above.

-Synthesis of compound 2

Commercially available 3-Hydroxy-4-nitrobenzaldehyde (2.87 g, 17 mmol) was dissolved in MeOH (50 mL) under an Ar atmosphere. Then NaBH ₄ (1.28 g, 34 mmol) was added and stirred at room temperature overnight. Acetone was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate, transferred to a separatory funnel, and washed once with 2M HCl and once with saturated brine. The organic layer was _{dried over anhydrous Na 2} SO ₄ , filtered through a cotton swab, and the filtrate was concentrated under reduced pressure, and the residue was obtained as compound 2 (yellow powdery solid). The yield was 99%. The signals of the NMR spectrum are as follows. ^{1 1} H NMR (600 MHz, CDCl ₃ ) δ = 10.66 (1H, s), 8.10 (1H, d, J = 8.3 Hz), 7.18 (1H, s), 6.98 (1H) , Dd, J = 8.2, 1.4 Hz), 4.77 (2H, d, J = 5.5 Hz), 1.89 (1H, t, J = 5.5 Hz)

-Synthesis of compound 3

Compound 2 was azeotropically dehydrated with Pyridine three times and then dissolved in Pyridine (20 mL). Then, DMTr-Cl (880 mg, 2.6 mmol) was added, and the mixture was stirred overnight at room temperature under an Ar atmosphere. An appropriate amount of H ₂ O was added to the reaction solution, and the reaction solution was concentrated under reduced pressure. The residue _{was washed with CHCl 3} into a separatory funnel and NaHCO ₃ aq. Twice with NaClaq. Washed once with. The organic layer was _{dried over anhydrous Na 2} SO ₄ , filtered through a cotton swab, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel C-60, hexane: EtOAc = 96: 4) to obtain Compound 3 (526 mg, 1.11 mmol, yield 55%, yellow viscous liquid). The signals of the NMR spectrum are as follows. ¹ H NMR (600 MHz, DMSO-D6) δ = 10.98 (1H, s), 7.84 (1H, d, J = 8.59 Hz), 7.42 (2H, d, J = 8.25) Hz), 7.33 (2H, t, J = 7.73 Hz), 7.29 (4H, d, J = 8.59 Hz), 7.24 (2H, t, J = 6.19 Hz) , 6.91 (4H, d, J = 8.59 Hz), 6.87 (1H, d, J = 8.59 Hz), 4.11 (2H, s), 3.73 (6H, s)

-Synthesis of compound 4

Compound 3 was dissolved in DMF (15 mL). Then, _{CsCO 3} (651 mg, 2 mmol) and ethyl bromoacetate (220 μL, 2 mmol) were added, and the mixture was stirred overnight at room temperature under an Ar atmosphere. The reaction solution was washed with ethyl acetate into a separating funnel, and NaClaq. Washed twice. The organic layer was _{dried over anhydrous Na 2} SO ₄ , filtered through a cotton swab, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel C-60, hexane: EtOAc = 90:10) to obtain Compound 4 (532 mg, 0.95 mmol, 95%, yellow viscous liquid). The signals of the NMR spectrum are as follows. ¹ H NMR (600 MHz, CDCl ₃ ) δ = 7.85 (1H, d, J = 7.90 Hz), 7.45 (2H, d, J = 7.56 Hz), 7.35 (4H, d) , J = 8.94 Hz), 7.31 (2H, t, J = 7.73 Hz), 7.23 (1H, t, J = 7.56 Hz), 7.03 (2H, d, J) = 9.28 Hz), 6.84 (4H, d, J = 8.94 Hz), 4.77 (2H, s), 4.24 (2H, s), 4.22 (2H, t, J) = 7.05 Hz), 3.79 (6H, s), 1.24 (3H, t, J = 7.22 Hz)

-Synthesis of compound 5

Compound 4 was dissolved in THF (15 mL). Then, a solution prepared by dissolving LiOH · H ₂ O (197 mg, 4.74 mmol) in H ₂ O (3 mL) was added, and the mixture was stirred overnight at room temperature. _{H 2} O (3 mL) was added to the reaction solution, adjusted to pH 6 with 10% citric acid, and washed with ethyl acetate in a separating funnel. Washed twice. The organic layer was _{dried over anhydrous Na 2} SO ₄ , filtered through a cotton swab, and the filtrate was concentrated under reduced pressure. The residue was compound 5. The yield was about 102% (pale yellow foamy solid).

-Synthesis of carrier 6

CPG having NH ₂ on the surface (hereinafter, may be abbreviated as "SP") ( _{-NH 2} 150 μmol / g, 100 mg, 15 μmol) is azeotropically dehydrated three times with Pyridine and then azeotroped three times with Toluene. It was removed and transferred to a vial. Add compound 5 which was azeotropically dehydrated 3 times with Pyridine and then azeotropically removed 3 times with Toluene, DMAP (17.5 mg, 150 μmol), and Dimethylformamide (21.5 μL, 150 μmol) dissolved in DMF (400 μL). , Stirred at room temperature for 3 days. A small amount of CPG was collected using Pasteur, washed with chloroform, and the ^{introduction rate was calculated by de-DMTr +} color development test and found to be 20%. It was filtered through a glass filter, washed 3 times with chloroform and dried. The mixture was transferred to a vial, Pyridine: Ac ₂ O = 9: 1 (v / v) (400 μL) and DMAP (9 mg, 75 μmol) were added, and the mixture was stirred for 1 day. It was filtered through a glass filter, washed 3 times with chloroform and dried. From the increase in mass of CPG, it was found that the carrier 6 had 30 μmol / g of compound 5 bound to it.

-How to determine the yield The carrier 6 (17.2 mg) was taken, 3% TCA in CH ₂ Cl ₂ (1 mL) for an automatic DNA synthesizer was added, and the mixture was sealed and allowed to stand for 5 minutes. After filtering with a cotton plug, the filtrate _{was scalpel-up to 10 mL using CH 2} Cl ₂ : MeOH = 7: 3 (v / v). The scalpel-up solution (100 μL) was weighed into a 2 mL Eppendorf tube and dried using a centrifugal concentrator. The introduction rate was calculated by dissolving in 600 μL of HClO ₄ : EtOH = 3: 1 (v / v) and measuring the absorbance at 498 nm.
The absorbance at 498 nm was 0.62. HClO ₄ : The absorbance ^{of DMTr +} at EtOH = 3: 1 (v / v) _{(ε 498 nm} 71.7 × 10 ³ ) was used.

-Synthesis of nucleotide-modified solid-phase carrier DNA strands were synthesized from carrier 6 (1 μmol) using the phosphoramidite method on a DNA / RNA automatic synthesizer to synthesize a modified thymidylate 12-mer (T12). .. The synthesis was completed with the DMTr group at the 5'-terminal left. The solid-phase carrier to which the DNA / RNA strand with the protecting group was bound was transferred to a vial, 3% TCA in CH _{2 Cl 2} (1 mL) was added, the stopper was sealed, and the mixture was allowed to stand for 5 minutes to deprotect the DMTr group. Then, the solid phase carrier was taken out, _{washed with CH 3} CN, followed by CH ₂ Cl ₂ , and dried with Ar gas. The solid-phase carrier was transferred to a vial _{, 1 mL of a mixed solution of CH 3} CN: DBU = 9: 1 was added, the mixture was sealed, and the mixture was allowed to stand at room temperature for 1 minute to deprotect the cyanoethyl group in the phosphoric acid diester section. The solid phase carrier was removed and washed with _{CH 3} CN followed by CH ₂ Cl _2. In this way, nucleotide-modified solid-phase carriers (ON1 to ON2) having the sequences shown in Table 1 were synthesized.

１）表中Ｘは以下の化学構造を有するものである。

1) X in the table has the following chemical structure.

-Transfer the nucleotide-modified solid phase carrier of the present invention bound to T12 to a separation vial of modified DNA / RNA _{, add 1 mL of a solution of titanium trichloride (TiCl 3} ) (50 mM TiCl ₃ , 4.5 M titrate buffer, pH 6), and add 1 mL. It was stirred slowly at room temperature. After 1 hour, the solid phase carrier was filtered to obtain a filtrate. The yield was 50 nmol and the yield was 20%. The filtrate was analyzed by HPLC. The results are shown in FIGS. 1 and 2. FIG. 1 shows the change over time when ON1 was separated from the solid-phase carrier by titanium trichloride ( _{after adding the titanium trichloride (TiCl 3} ) solution, the filtrate was stirred for 0 hours, 1 hour, and 1 hour after stirring. It is an HPLC chart which shows the refined product which was purified by HPLC fractionation. In addition, FIG. 2 shows the change over time when ON2 was separated from the solid-phase carrier by titanium trichloride ( _{after the addition of the titanium trichloride (TiCl 3} ) solution, stirring 0 hours, 1 hour, and stirring 1 hour later. It is an HPLC chart which shows the refined product) which purified the liquid by HPLC preparative. The analysis conditions for HPLC are as follows.

-HPLC analysis conditions HPLC: SHIMADZU, LC-20AT
HPLC analysis condition column: GL Sciences, Intersill ODS-3 4.6 mm I. et al. D x 250 mm
Column temperature: Room temperature Mobile phase A: 5% acetonitrile in0.1M Triethylamine acetate buffer Mobile phase B: 50% acetonitrile in0.1M Triethylamine acetate buffer Mobile phase feed: Mobile phase A: B = 95: 5 (0 minutes) ), A: B = 65:35 (20 minutes), A: B = 50:50 (25 minutes)
Detector: SIMADZU, SPD-10A (measurement wavelength: 260 nm)

As shown in FIG. 1, in ON1, a signal of modified DNA / RNA separated from the solid-phase carrier was confirmed after 1 hour. The same was true for FIG. Table 2 shows that the signals of the modified DNA / RNA separated from the solid-phase carrier are 5'-G ^iBu- TTTTTTTTTTTTTT (hereinafter referred to as ON3) and 5'-C ^Ac- TTTTTTTTTTTTTT (hereinafter referred to as ON4), respectively. It is also clear from the measurement results of MOLDI-TOF-MASS of the refined product shown in.

As shown above, it was found that the nucleotide-modified solid-phase carrier of the present invention can dissociate nucleotide chains (DNA chains, RNA chains) under the conditions of pH 6 to 8 using a reducing reagent. Such a nucleotide-modified solid-phase carrier is further subjected to DNA / RNA synthesis, which is a conventionally known method, to extend an RNA chain or DNA difference of an arbitrary length, and then a reducing reagent under conditions of pH 6 to 8. It is possible to produce DNA or RNA of any length by dissociating with.

According to the present invention, since modified RNA or modified DNA can be produced while leaving a modifying group for any base, it is possible to produce various modified RNA or modified DNA. Further, since various modified RNAs or modified DNAs can be produced, the range of application as a nucleic acid drug is widened, and various pharmacological effects can be expected. This is a very significant invention in that it greatly expands the possibilities of nucleic acid medicine.

Claims (12)

A nucleotide-modified solid-phase carrier represented by the following general formula (1).

(B is a base that may be modified, X is H or OH, n = 1 or more, p = 1-3, R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon _{^{atoms, -Si (oR 1) 3,}} -SiR 1 (oR 1) 2, -SiR 1 2 (oR 1), - SiR 1 3, or represents ^-NR _{1 2,} each R ¹ is an alkyl group having 1 to 5 carbon atoms independently, Y is an ether group, a silyl group, an alkyl group, an amide group or an ester group, and Z and W are independent H or protecting groups, respectively. , ^Ra and R ^b are independently hydrogen or alkyl groups, respectively, and SP represents a solid phase carrier.) A nucleotide-modified solid-phase carrier represented by the following general formula (2).
A nucleotide for producing DNA or RNA using a reducing reagent capable of extending a DNA chain or RNA chain from the 5'end of the nucleotide-modified solid-phase carrier and then dissociating it from the solid-phase carrier at pH 6 to 8. Modified solid phase carrier.

(B is a base that may be modified, X is H or OH, n = 1 or more, p = 1 to 3, R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, Represents an alkylthio group having 1 to 5 carbon atoms, -Si (OR ¹ ) ₃ , -SiR ¹ (OR ¹ ) ₂ , -SiR ¹ ₂ (OR ¹ ), ^{-SiR 1} ₃ or -NR ¹ ₂ and each R ¹ is an alkyl group having 1 to 5 carbon atoms independently, Y is an ether group, a silyl group, an alkyl group, an amide group or an ester group, and Z and W are independently H or a protective group. , ^Ra and R ^b are independent hydrogen or alkyl groups, respectively, and SP represents a solid phase carrier, where the NO ₂ group is attached to the functional group attached to phosphoric acid at the ortho or para position. It is a thing.) The nucleotide-modified solid phase carrier according to claim 2, wherein the reducing reagent is a reagent that reduces a nitro group bonded to an aromatic ring to an amino group. The nucleotide-modified solid-phase carrier according to claim 3, wherein the reducing reagent is titanium trichloride. A method for producing a DNA strand-modified carrier, which comprises a step of extending a DNA strand using the nucleotide-modified solid-phase carrier according to claim 1 or 2. The DNA is separated from the DNA strand-modified carrier obtained by the method for producing a DNA strand-modified carrier according to claim 5 using a reducing reagent capable of dissociating DNA from the solid-phase carrier at pH 6 to 8. A method for producing DNA, which comprises a step. A method for producing an RNA strand-modified carrier, which comprises a step of extending an RNA strand using the nucleotide-modified solid-phase carrier according to claim 1 or 2. The RNA is separated from the RNA strand-modified carrier obtained by the method for producing an RNA strand-modified carrier according to claim 6 using a reducing reagent capable of dissociating RNA from the solid-phase carrier at pH 6 to 8. A method for producing RNA, which comprises a step. The method for producing DNA according to claim 8, wherein the reducing reagent is a reagent that reduces the nitro group of an aromatic ring bonded to the solid phase carrier via the Y site to an amino group. The method for producing RNA according to claim 9, wherein the reducing reagent is a reagent that reduces the nitro group of an aromatic ring bonded to the solid phase carrier via the Y site to an amino group. The method for producing DNA according to claim 7 or 8, wherein the reducing reagent is titanium trichloride. The method for producing RNA according to claim 9 or 10, wherein the reducing reagent is titanium trichloride.

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