JP7033923B2 - Carrier for nucleic acid synthesis using an inorganic porous body produced by the sol-gel method - Google Patents

Description

The present invention relates to a carrier for nucleic acid synthesis using an inorganic porous body having through holes, which is produced by a sol-gel method or the like.

The solid phase carrier used for RNA / DNA synthesis accounts for 1/4 to 1/3 of the raw material cost. In addition, four different chemical reactions are required to extend one base on the solid phase, and the carrier is washed with a large amount of organic solvent before and after each reaction. Therefore, it is desired to develop a carrier having high reaction efficiency.

Patent Document 1 discloses solid-phase synthesis in microwells.

JP 2012-507513

As a result of diligent research, the present inventors have achieved that by developing a carrier having high reaction efficiency, the amount of reagents and the amount of solvent used can be reduced, and the amount of synthesis per carrier unit volume can be significantly increased. As a solution to this problem, an inorganic porous body having through holes (integral type or particle type) was used as a base material. Conventionally, as a nucleic acid synthesis carrier, an inorganic porous body having through holes has not been used for the purpose of synthesizing on a large scale, especially for the purpose of synthesizing pharmaceutical products, but in the present invention, it is quick to increase the scale and height. The problem of mechanical limitation due to the pressure required to flow the solution was solved by adopting an inorganic porous body having through holes, particularly an integral type or a particle type.

For the inorganic porous body with through holes, we also made efforts to refine the optimum conditions on each side. Among the inorganic porous bodies having through holes, the monolith type means an integrated porous body. In the present invention, a continuous porous block having a length of about 1 mm or more on one side of the three sides of the inorganic porous body having through holes that can be defined by width, depth, and height is used as a carrier, and more specifically. In particular, the shape (pellet or powder), pore size, linker type, etc. were examined, and the optimum combination thereof was able to achieve high yield synthesis.

In addition, a particle type also exists in the inorganic porous body having through holes. Typically, the inorganic porous body having through holes has a particle type having a diameter of about 1 mm or less, so that it can be scaled up regardless of the size of the column, and mass synthesis is possible. In the microwell type used conventionally, since the porous body is filled in the minute space, the size of the column is limited and mass synthesis by increasing the size is difficult. The problem was also solved. Therefore, the present invention typically provides:

(1) A carrier for producing a nucleic acid or an analog thereof, which comprises an inorganic porous body having through holes.
(2) The carrier according to item (1), wherein the nucleic acid or an analog thereof can be synthesized by a phosphoramidite method or an H-phosphonate method.
(3) The carrier according to item (1) or (2), which has a binding linker for binding the nucleic acid or an analog thereof or a raw material thereof.
(4) The carrier according to item (3), wherein the binding linker contains at least one selected from the group consisting of an amino group, a hydroxyl group, and a thiol group.
(5) The carrier has a binding mode in which the carrier and the nucleic acid portion can be cleaved after the production, under the condition that the nucleic acid or its relatives, their binding compounds or the bonds linking the nucleic acids or their relatives are not decomposed. The carrier according to any one of items (1) to (4), which has a linker (cleavable linker).
(6) The carrier according to item (5), wherein the binding mode comprises a bond selected from the group consisting of esters, amides, imides, thioesters and disulfide bonds.
(7) The bound linker contains at least one group selected from the group consisting of an amino group, a hydroxyl group, a phosphoric acid group and a thiol group, and the group is from the group consisting of an ester, an amide, an imide, a thioester and a disulfide bond. The carrier according to any one of items (3) to (6), which binds to the inorganic porous body via a selected bond.
(8) The carrier according to any one of items (1) to (7), wherein the inorganic porous body further has pores.
(9) The carrier according to any one of items (1) to (8), wherein the inorganic porous body is an integral type or a particle type.
(10) The carrier according to any one of items (1) to (9), wherein the inorganic porous body contains silica gel or silica glass.
(11) The carrier according to any one of items (1) to (10), wherein the inorganic porous body has a pellet structure.
(12) The carrier according to item (11), wherein the pellet structure is rod-shaped, plate-shaped or lump-shaped.
(13) The carrier according to item (11) or (12), wherein the pores of the pellet structure are about 100 nm or more.
(14) The inorganic porous body has a continuous skeleton in a sub-micrometer size to micrometer-sized three-dimensional network, and penetrates the gaps between the skeletons in three-dimensional continuous sub-micrometer size to micrometer size. Items (1) to (13), which have pores and nanometer-sized pores continuously existing in the silica skeleton, or have a mesh-like skeleton in which the pore diameter is set to 0 and only through holes are present. The carrier according to any one item.
(15) The through hole has a size of about 0.1 to about 20 μm and is smaller than the size or particle size of the inorganic porous body.
The pores, if present, are from about 2 nm to about 300 nm and are less than the diameter of the through hole. The carrier according to item (14).
(16) The bonded linker contains an amino group, and the length from the silicon atom on the surface of the carrier is about 1 or more and less than about 20 in the linear direction including the carbon atom, the nitrogen atom and the oxygen atom. , The carrier according to any one of items (3) to (15).
(17) The carrier according to item (16), wherein the length is about 7 to about 14 in the linear direction including carbon atoms, nitrogen atoms and oxygen atoms.
(18) The carrier according to any one of items (3) to (15), wherein the binding linker is contained in a loading amount of about 10 to about 250 μmol / g.
(19) The carrier according to any one of items (3) to (15), wherein the binding linker is contained in a loading amount of about 30 to about 75 μmol / g.
(20) The carrier according to any one of items (5) to (15), wherein the cleaving linker is contained in a loading amount of about 30 to about 75 μmol / g.
(21) The carrier according to any one of items (1) to (20), which is produced by the sol-gel method.
(22) A nucleic acid or analog synthesizer comprising the carrier according to any one of items (1) to (21).
(23) The synthesizer according to item (22), wherein the synthesis is carried out by solid phase synthesis.
(24) The carrier carries about 30 to about 75 μmol / g of a raw material for the nucleic acid or an analog thereof (for example, a nucleoside or an analog thereof, and an organic compound (linker, dye derivative, etc.) that can be introduced). The synthesizer according to item (22) or (23), which is adjusted to.
(25) A method for synthesizing a nucleic acid or an analog thereof using the carrier according to any one of items (1) to (21) or the synthesizer according to any one of items (22) to (24).
(26) The method according to item (25), wherein the synthesis is carried out by solid phase synthesis.
(27) The carrier carries about 30 to about 75 μmol / g of a raw material for the nucleic acid or an analog thereof (for example, a nucleoside or an analog thereof, and an organic compound (linker, dye derivative, etc.) that can be introduced). The synthesis method according to item (25) or (26), which is adjusted to.

Although we do not want to be bound by theory, the inorganic porous body with through holes has a pore structure different from that of CPG normally used for nucleic acid synthesis, which improves the flow of reagents and improves the efficiency of synthesis. It is thought that it can be done.

The CPG used in the prior art has only pores (voids), but the inorganic porous body having through holes used in the present invention has good through holes for liquid passage (a fixed flow inside silica). Since it has a pathway), it is considered to be suitable for synthesis that extends DNA / RNA.

We have succeeded in developing a carrier with high reaction efficiency, and it has become possible to reduce costs by reducing the reagents and solvents used, and it has become possible to significantly improve the amount of nucleic acid synthesis per unit.

With conventional beads, the void ratio cannot be adjusted, the shape of the flow path is distorted and resistance is high, the pore diameter is non-uniform and the diffusion length is long, and the polymer carrier has no pores, so chemical modification is possible only on the surface. On the other hand, in the present invention, since an inorganic porous body having through holes is used, the void ratio can be controlled, the shape of the flow path is round and the resistance is small, the pore diameter is uniform, and diffusion is performed. The effect of short length is recognized, and the synthesis efficiency is dramatically improved.

In the present invention, among the inorganic porous bodies having through-holes, in particular, in the case of the particle type, the particles can be treated in the same manner as the conventional carriers such as silica gel and CPG, but (1) have continuous through-holes. Since a wider reaction field can be provided, (2) synthesis can be performed using a conventional synthesizer, and (3) a solution can be flowed at the same or lower pressure for liquid passage. The effect of reducing mechanical restrictions is achieved.

Further, in the present invention, among the inorganic porous bodies having through holes, especially in the case of the integrated type, (1) it can be cut into a desired size and shape from the same batch, so that it is used according to the machine. (2) Highly identical products can be produced, and (3) In the case of silica gel or CPG for column preparation, special machines and techniques are often required, but monolith type columns. The effect of being able to increase the size is achieved without the need for it.

FIG. 1 shows the observation results of the bare carrier before washing and the particle state after aminosilaneization with an optical microscope. FIG. 2 shows an example of the measurement result of the nucleoside loading amount. The vertical axis shows the absorbance, and the horizontal axis shows the wavelength (nm). FIG. 3 shows the correlation between the yield of the synthetic sample and the amount of DMT-dT carried on the carrier. The vertical axis shows the yield (mg), and the horizontal axis shows the amount of DMT-dT supported (μmol / g). FIG. 4 shows a typical example of actual filter paper insertion.

Hereinafter, embodiments of the present invention will be described in detail. The same contents will be omitted as appropriate in order to avoid repetitive complications. It should also be understood throughout the specification that the singular representation also includes its plural concept, unless otherwise noted. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the plural concept unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this specification (including definitions) takes precedence.

First, the terms used in the present invention and general techniques will be described.

As used herein, the term "inorganic porous body" refers to an inorganic material having innumerable fine pores inside. One of the features of the inorganic porous body used in the present invention is that it has through holes. Therefore, in one aspect, the present invention relates to an "inorganic porous body having through holes". The inorganic porous body having such through holes is typically produced by the sol-gel method. The inorganic porous body used in the present invention may further have pores.

As used herein, the term "through hole", also referred to as macropore or through-pore, refers to a large-sized pore in an inorganic porous body, which is submicron-sized to micro-sized, and typically has a diameter of about about. A hole with a size of 0.1 to about 20 μm.

As used herein, the term "pore" is also referred to as mesopore or mesopore, and typically refers to a hole smaller than a through hole having a diameter of about 1 to about 300 nm.

The inorganic porous body having through holes used in the present invention is further classified into "integral type" (monolith type) and "particle type" according to its shape. Usually, the integrated inorganic porous body has continuous through holes and pores, and the particle type inorganic porous body has only continuous through holes without the pore diameter, but is not limited to this. Some of the body-shaped inorganic porous bodies have a structure of only through holes, and some of them are particle-shaped and retain pores.

As used herein, the term "integral" or "monolith" is used interchangeably and is a continuous inorganic porous body having a length of one of the three sides of the structure of 1 mm or more. It is an integrated porous body with a characteristic structure in which mesh-like skeletons on the order of about micrometer are connected. Etymologically, it means a single stone, and it looks like a choke. When observed with an electron microscope, it has a structure with a series of jungle gym-like skeletons. Innumerable pores called through holes on a micron scale are opened around the gaps in the skeleton, and more preferably, holes called nanoscale pores are opened in the skeleton. The through hole and the pore are connected without being blocked, and it is said that it boasts a high porosity in which about 85% of the total volume is a hole. When nanoscale pores are present, they have a high specific surface area due to their innumerable presence.

In the present specification, "particle type", "grain (child) shape" or "non-integrated type" refers to the shape of an inorganic porous body, and is a general term for those other than "integrated type", and is "particle type". The inorganic porous body is an inorganic porous particle having all three sides of the inorganic porous body of 1 mm or less. The particle-type inorganic porous body is typically crushed particles obtained by crushing and classifying an integrated inorganic porous body, has a high porosity and a specific surface area, and has through holes peculiar to monoliths and. Since it has two-stage pores of pores, it is a particle with high-speed processing performance peculiar to monolith. The inorganic porous body can have a pellet structure.

In the present specification, the inorganic porous body is composed of silica or titania (titanium dioxide) (in this case, also referred to as silica monolith, titania monolith, etc., respectively), and the purity thereof is typically 99.9% or more. Monolith, which is a co-continuous porous body consisting of through holes and pores with two different sizes, can independently control the size of these two stages of pores when the material is silica (through holes (through holes (through holes (through holes)). (Also referred to as three-dimensional network pores) is typically about 0.1 to about 10 μm, and pores (also referred to as mesopores) are typically about 1 to about 200 nm. Larger ones, for example, about 300 nm can be manufactured), and each has a feature that the pore diameter can be optimized according to the purpose of use. Since silica monolith boasts a high porosity and specific surface area, it can be used as a low-pressure and high-performance filter or adsorption column, or can contain a large amount of liquid, so the drug can be slowly adsorbed from the monolith impregnated and adsorbed with medicinal properties. It is also used as a material to release, but it is not usually used for solid-phase synthesis.

As used herein, the term "nucleic acid" refers to any biopolymer in which a nucleotide consisting of a base, a sugar, and a phosphate (which is also a raw material for the synthesis of the present invention) is linked by a phosphodiester bond, for example, DNA, RNA, etc. Can be mentioned.

As used herein, the term "an analog" of nucleic acid and its raw material is chemically modified and chemically modified so as to be used in a solid phase synthesis method such as a phosphoromidite method or an H-phosphonate method. Nucleic acid (or nucleotide) analogs, typically nucleic acids in which the 2'-position hydroxyl group of sugar is modified or protected with an alkyl group, modified nucleic acid into which an F group is introduced, LNA or BNA called crosslinked nucleic acid, Includes PNA, UNA, other derivatives that can be introduced into nucleic acid sequences (such as linkers and dye derivatives), as well as other common organic compounds as long as they can be synthesized.

The bases of the nucleic acids or analogs thereof and their raw materials used herein include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Is included. Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthin, hypoxanthine, 2-aminoadenine and other synthetic and natural nucleobases, adenine and guanine 6-methyl. And other alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH ₃ ) uracil. And other alkynyl derivatives of cytosine and pyrimidine bases, 6-azouracil, cytosine and timine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl , And other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, Includes 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other modified nucleobases include phenoxadincytidine (1H-pyrimid [5,4-b] [1,4] benzoxazine-2 (3H) -on) and phenothiazinecytidine (1H-pyrimid [5,4-b]). ] [1,4] Tricyclic pyrimidines such as benzoothiazine-2 (3H) -on), substituted phenoxadin cytidines (eg, 9- (2-aminoethoxyl) -H-pyrimids [5,4-b] [1, 4] Benzoxazine-2 (3H) -on), carbazolecytidine (2H-pyrimid [4,5-b] indol-2-on), pyridoindolecytidine (H-pyrimidine [3', 2': 4,5) ] G-clamps such as Pyrrolo [2,3-d] pyrimidine-2-on) are included. Modified nucleobases may include purin or pyrimidine bases replaced with other heterocycles such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Other nucleobases include USP No. 3, 687, 808, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al.,, Angewandte Published in Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Including those disclosed by.

These few nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds synthesized in the present invention (oligomers, eg oligonucleotides, which are examples of nucleic acids of interest or their analogs of the invention). .. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substituents have been demonstrated to increase nucleic acid double-strand stability by 0.6-1.2 ° C (Sanghvi, YS Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pages 276-278), and a suitable base substituent, even more particularly suitable when combined with 2'-O-methoxyethyl sugar modification.

Representative USPs (US Pat. Nos.) Teaching the preparation of other modified nucleobases or analogs thereof as well as some of the above modified nucleobases include, but are not limited to: the above USPs. Not only 3,687,808 but also the following USPs; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941 and their respective contents are incorporated herein by reference.

Other modifying groups can also be made at other positions of the oligonucleotide, especially at the 3'position of the sugar at the 3'end nucleotide and at the 5'position of the 5'end nucleotide, if the oligomer is an oligonucleotide. For example, in the case of an oligonucleotide which is an example of an oligomer produced in the present invention, when a ligand is bound to one of the oligonucleotides, the other modifying group of the oligonucleotide is the activity, cell distribution or cell uptake of the oligonucleotide. May include the chemical linkage of one or more other non-ligon moieties or conjugates to the oligonucleotide. The moieties include, but are not limited to: lipid moieties such as cholesterol components (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86,6553), cholic acid (Manoharan et al., Bioorg). Med. Chem. Lett., 1994, 4,1053), thioether, eg, hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533), aliphatic chains, eg dodecanediol or undecyl residues (Saison-Behmoaras et). al., EMBO J., 1991, 10,111; Kabanov et al., FEBS Lett., 1990, 259,327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids, eg, dihexadecyl-rac-glycerol or triethyl Ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990 , 18, 3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantan acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), palmityl moieties (Mishra) et al., Biochim. Biophys. Acta, 1995, 1264,229), or octadecylamine Or the hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Nucleic acids targeted in the present invention include not only those bound by phosphodiester bonds but also those having amide bonds and other backbones (peptide nucleic acids (PNA)) as long as they contain the specific modified nucleic acids of the present invention. ) Etc.) are included. As described above, nucleic acids and their analogs include, for example, natural and artificial nucleic acids and may be nucleic acid derivatives, nucleic acid analogs, nucleic acid derivatives and the like. Such nucleic acid analogs are well known in the art and include, but are limited to, for example, phosphorothioates, phosphoamidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, peptide nucleic acids (PNAs). Not done. The skeleton of PNA may include a skeleton consisting of aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfonamide, polysulfone amide, or a combination thereof (Krutzfeldt, J. et al., Nucleic Acids Res. 35: 2885-2892; Davis, S. et al., 2006, Nucleic Acids Res. 34: 2294-2304; Boutla, A. et al., 2003), Nucleic Acids Res. 31: 4973-4980; Hutvagner, G. et. al., 2004, PLoS Biol. 2: E98; Chan, JA et al., 2005, Cancer Res. 65: 6029-6033; Esau, C. et al., 2004, J. Biol. Chem. 279: 52361- 52365; Esau, C. et al., 2006, Cell Metab. 3: 87-98).

As used herein, the term "raw material" for nucleic acids or analogs thereof refers to any material that is part of nucleic acids or analogs thereof. Examples of such raw materials include, but are not limited to, nucleosides and their analogs, and introduceable organic compounds (such as linkers and dye derivatives).

As used herein, the term "carrier" is also referred to as a carrier, and refers to a substance that serves as a base for fixing other substances. In the present invention, it is used in a production method such as solid phase synthesis. It is desirable that the carrier itself is chemically stable and does not interfere with the intended operation (typically, a synthetic reaction in the present invention).

As used herein, the term "binding linker" refers to a binding structure used to bind one substance to another. For example, an amino group, a hydroxyl group (including a phosphoric acid group and the like) and a thiol group can be mentioned. Silane coupling agents having these substituents are commercially available and can be appropriately used. With these substituents, the linker can be extended by a general organic reaction (condensation, etc.). The carrier of the present invention may preferably contain a binding linker, but is not always necessary. Examples of such a binding linker include a binding linker using a silane coupling agent, a phosphonic acid-based coupling agent (Dojindo, a phosphonic acid derivative sold as a surface treatment-related reagent; http://dominoweb.dojindo. Co.jp/goodsr7.nsf/ByChuInfo/08? See OpenDocument) is included, and as such treatment agents, for example, 3-aminopropyltrimethoxysilane and 3-aminopropyltrimethoxysilane, as examples of silane coupling agents. There are aminopropyltriethoxysilane, N-2- (aminoethyl) -3-aminopropyltrimethoxysilane, 3-acryloxypropyltrimethoxysilane, 3-mercaptopropyltrimethoxysilane, 3-isoxapropyltriethoxysilane, etc. Examples of the phosphonic acid system include, but are not limited to, 11-aminoundecylphosphonic acid, 11-hydroxylundesylphosphonic acid, 12-mercaptododecylphosphonic acid, 10-carboxydecylphosphonic acid and the like.

As for the size of the binding linker, any size can be used, but the length from the silicon atom of the carrier surface is about one or more in the linear direction including the carbon atom, the nitrogen atom and the oxygen atom. The number is less than 20, preferably about 7 to about 14.

The binding linker used in the present specification is preferably contained in a loading amount of about 10 to about 250 μmol / g, and more preferably contained in a loading amount of about 30 to about 75 μmol / g.

As used herein, the term "cleavable linker" refers to a binding mode in which a carrier and a nucleic acid moiety can be cleaved under conditions that do not decompose a nucleic acid or its analog, a binding compound thereof, or a bond linking the nucleic acid or its analog. Refers to a linker that has. Cleavable linkers include, but are not limited to, linkers containing, for example, ester bonds, amide bonds, imide bonds, thioester bonds and disulfide bonds. Examples of such a linker include a dicarboxylic acid derivative such as oxalylic acid, succinyl acid, and a hydroquinone-O, O-diacetic acid derivative, a compound having a 1,2-diol structure called a universal linker, and a carboxylic acid having a phthalimide group. Acids, disulfides and the like can be mentioned.

The cleavable linkers used herein are also preferably contained in a carrying amount of about 10 to about 250 μmol / g, more preferably about 30 to about 75 μmol / g.

The binding linker used herein comprises at least one group selected from the group consisting of an amino group, a hydroxyl group (including, for example, a phosphate group) and a thiol group, wherein the group is an ester, an amide. A bond selected from the group consisting of, imide, thioester and disulfide bonds is preferred, and any binding linker having these bonds can be used.

As used herein, the term "monolith-type silica" refers to silica having a monolithic structure. The monolith type silica may be used by itself (for example, a pellet type), or may be used as a particle type which is processed into a certain diameter by pulverization or the like and classified. It may be a powder. Examples of the monolith type silica and the particle type silica obtained by pulverizing the monolith type silica include those having through holes and pores, and those having no pores and having only through holes. Both silica monolith and CPG (controlled pore glass) are a type of porous silica, but unlike CPG, silica monolith is produced by the organic-inorganic hybrid method and has a network of pores in the network structure. It is characterized by having holes of two sizes, a through hole or a macro hole) and a pore in the skeleton thereof. Monolith type silica can be obtained by a sol-gel method. "Sol-gel method" refers to a wide range of methods for producing gels that can be dried into glassy particles or monoliths. The sol-gel process can use a precursor solution of the intended material (eg silica).

(Example of production of inorganic porous body)
The inorganic porous body used in the present invention can be produced by a known method such as a sol-gel method.

As used herein, the "sol-gel method" refers to a wide range of methods for producing gels that can be dried into glassy particles or monoliths. The sol-gel process procedure can be made using a precursor solution of the material of interest (eg silica). Examples of the procedure include the following. First, a solution or suspension of a material (for example, Si) is prepared, and when the material is Si, hydrolysis catalyzed by an acid or a base of the Si preparation is performed to form a Si—OH group. The reaction formula is as follows.

SiXn + nH ₂ O → Si (OH) n + nHX
The mixture obtained by this method is a solution or colloidal suspension called a "sol".

Next, polycondensation of Si—OH groups is performed by the following reaction.

Si-OH + Si-OH → Si-O-Si + H ₂ O
This stage results in an increase in viscosity and concomitant formation of a matrix called a "gel". Drying of the gel forms a porous monolithic structure. Drying can be done by controlled evaporation of the solvent to produce the xerogel or supercritical extraction of the solvent to produce the airgel. A colloidal (sol) solution is a mixture of one or more metal or metalloid oxide precursors (typically Si above) with water or water / alcohol in the presence of a catalyst such as an acid or base. Can be prepared. The metal or metalloid oxide may be an n-valent cation of an element belonging to Group 3, 4 or 5 of the Periodic Table, but may be Si, Ge, Ti, Al or any combination thereof. good. The X may be selected from the group comprising oxides, alkoxides, methoxys, tetramethoxys, ethoxys, propoxys or butoxys or any combination thereof. The hydrolysis step can be carried out at room temperature for about 5 minutes to 4 hours or more or until the hydrated cationic oxide forms a sol. A colloidal suspension of at least one existing cationic oxide may be added to the sol prior to gelation. For example, if a precursor containing a silicon oxide is utilized, a solution / suspension prepared by mixing water, optionally a solvent, fumed silica, acid or base can also be added to the sol. Gelation of the sol can be performed by incubating the sol, typically at a temperature below about 90 ° C., for at least a few minutes.

After gelation, the gel is washed with an organic solvent such as water or methanol so that the solvent in the gel is replaced by an aprotic solvent or water and methanol. The aprotic solvent can be selected from the group comprising acetone, dioxane, hydrofuran.

The gel thus obtained is then in a pressurized chamber purged with an inert gas at a pressure suitable for achieving a total pressure below the solvent critical pressure at temperatures below the critical temperature of the gel solvent. Can be dried in. Under these conditions, the temperature of the pressurized chamber is raised according to a predetermined program for the sol-gel reaction, i.e., the gel solvent evaporating to produce a dry sol-gel. The dried sol-gel undergoes vitrification and can be heated to temperatures above about 100 ° C to about 1650 ° C under normal atmospheric or inert gas pressure. The gas can be selected from the group consisting of nitrogen, argon, helium, oxygen, chlorine and the like. The dried sol-gel can be heated from about 10 minutes to a long time.

(Example of particle type manufacturing)
For the particle-type inorganic porous body, the integrated inorganic porous body produced by the above manufacturing method is crushed by a spray-drying method, crushed by a W / O emulsion method, and impacted or applied using a mortar or crusher. It can be manufactured by a method such as pulverization by pressure.

(Manufacturing of carrier for synthesizing nucleic acid or its analog and synthesis of nucleic acid or its analog)
(1. Bonding of linker and / or starting material (monomer, etc.) to inorganic porous body)
The inorganic porous carrier of the present invention can be subjected to surface modification to nucleoside addition reaction by the following procedure.
Reaction 1: Addition of a linker on the carrier surface (bare carrier (bare carrier) only)
Reaction 2: Carboxyl group introduction reaction 3: Silanol group capping reaction 4: Addition of 3'-terminal nucleoside (method: washing)
An unsurfaced bare carrier (referred to as a bare carrier; eg 2.9 g) is placed in a suitable container (eg Erlenmeyer flask), suspended in a suitable wash solution (eg 2M aqueous hydrochloric acid solution (100 mL)), and then very slowly and appropriately. The fine particles are washed by stirring for a long time (for example, about 1 hour). After allowing the container containing the carrier to stand, the supernatant solution is removed, and the suspended fine particles are removed. Subsequently, water (for example, 100 mL) is added, and the operation of standing and removing the solution is repeated (for example, washing three times). After that, aminosilaneization modification (reaction 1) is performed.

(Method: Introduction of linker (reaction 1))
The washed carrier is collected by filtration under reduced pressure (for example, washed with water or methanol), dried (for example, dried at 40 ° C., 60 ° C., 80 ° C. for 2 hours each, air-dried for 16 hours, and further dried at 100 ° C. for 2 hours. Let).

A suitable solvent (eg, toluene (40 mL)) and a suitable linker (eg, 3-aminopropyltriethoxysilane (MW.221.37, d = 0.946 g / mL, 10 mL)) on the dried carrier (eg 1.41 g). )) Is added and refluxed (eg at 110 ° C.) (eg 10 hours). It is then vacuum filtered, washed with a suitable wash solution (eg toluene, chloroform) respectively (eg 3 times), then air dried and then dried (eg for several hours under reduced pressure).

(Method: Introduction of carboxyl group (reaction 2))
A suitable solvent (eg 60 mL of water) and a suitable carboxylic acid reagent (eg 2 g of succinic anhydride) are added to the carrier (eg 1.4-2 g) and the reaction vessel is gently shaken without stirring (eg room temperature). Lower, manually stir the container about once every 30 minutes). The reaction solution is subsequently maintained at the appropriate pH (eg pH 4-5) by adding the appropriate base (eg 2M sodium hydroxide solution) and after the appropriate time (eg 3 hours) has elapsed the appropriate carboxylic acid reagent (eg anhydrous). 1 g of succinic acid) and a suitable base (eg 2M sodium hydroxide) were added. After performing the reaction for an appropriate time (eg 7 hours), the carrier is collected by filtration (eg under reduced pressure), washed (eg 3 times each with water and methanol) and the resulting carrier is air dried. Subsequently, the carrier is dried under appropriate conditions (eg, 70 ° C. under reduced pressure for 2 hours, 80 ° C. under normal pressure for 2 hours).

(Method: Capping of silanol group (reaction 3))
The inorganic porous carrier (eg 1-2 g) is suspended in a suitable solvent (eg anhydrous pyridine (50 mL)) and a suitable capping agent (eg chlorotrimethylsilane (5 mL)) is added (eg at room temperature) gently. Shake. The powder is taken down under reduced pressure (for example, after 2 hours) and air-dried (for example, washed with pyridine and chloroform three times each). Further, after drying under reduced pressure at room temperature (2 hours), the capped inorganic porous carrier is recovered. ..

(Method: Addition of nucleoside to inorganic porous carrier (Reaction 4))
For each condensation reaction, use an appropriate container (eg, a glass vial with a capacity of 5 mL), an inorganic porous carrier, a nucleoside (eg, 5'-dimethoxytrityldeoxythymidine (5'-DMT-dT)), a condensing agent, etc. After adding the solvent to the cap and capping, shake (for example, gently with the vial at room temperature using a shaker). After shaking for a period of time, the inorganic porous carrier is collected by filtration (eg, using a Kiriyama funnel), and the carrier is further washed with appropriate washing solutions (eg, pyridine and dichloromethane).

(Measurement of nucleoside loading amount)
After completion of the nucleoside condensation reaction (Reaction 4), the dried nucleoside-added inorganic porous carrier (eg, about 10 mg) is accurately weighed into a suitable measuring instrument (eg, volumetric flask) and the appropriate reagent (eg, perchloric acid (70)). % Aqueous solution) -ethanol mixed solution (3: 2) 10.0 mL) is added to desorb the dimethoxytrityl cation (DMT ⁺ , molar absorption coefficient: ε ₅₀₀ = 71,700). The nucleoside carrying amount (μmol / g) is calculated by diluting this solution with the above mixed solution (for example, further 10 times) and then measuring the absorbance (for example, at 500 nm).

(2. Solid phase synthesis)
When the carrier for synthesis is completed, solid-phase synthesis is performed thereafter. Nucleic acid or nucleic acid analogs are synthesized by conventional synthetic reactions such as H-phosphonate method, phosphor ester method, and solid phase phosphoramidite method. As a general description of solid-phase synthesis, Japanese Patent Application Laid-Open No. 2009-280544 or Japanese Patent Application Laid-Open No. 2008-511743 can be referred to. Solid-phase synthesis was carried out by the phosphoramidite method (for example, using a synthesizer nS-8 (manufactured by GeneDesign, Inc.)) using the prepared inorganic porous carrier for synthesizing oligonucleic acid (for example, 24 bases). Obtain RNA. The solid phase phosphoramidite method is preferably adopted because it has a high ability to synthesize oligomers such as oligonucleotides and provides oligomers such as high-purity oligonucleotides. This method typically includes the following four steps.
(1) Use a deprotecting agent such as dichloroacetic acid to remove the DMT group (dimethoxytrityl group; also abbreviated as DMTr group) which is the protecting group of 5'-OH of the nucleoside bonded to the functional group on the inner wall surface of the microchannel. Detrityling process,
(2) A coupling step in which a nucleoside phosphoramidite (amidine reagent) activated by tetrazole or the like is bound to the above-mentioned deprotected 5'-OH by a nucleophilic substitution reaction.
(3) A capping step of acetylating and protecting 5'-OH to which the amidite reagent did not bind with acetic anhydride or the like, and (4) oxidizing the phosphorous acid bond derived from the amidite reagent with an oxidizing agent such as iodine. The oxidation process to form a phosphate reagent.
An exemplary scheme of the principles of the phosphoramidite method is shown below.

Synthesis begins by removing the DMTr group (dimethoxytrityl group) on the carrier (with the first nucleoside) and freeing the 5'hydroxyl group terminal (step 1). An activator such as amidite and tetrazole is introduced and activated, reacted with the 5'hydroxyl group terminal on the carrier (step 2), and oxidized with an oxidizing agent such as I ₂ (step 3). In addition, the unreacted 5'hydroxyl group terminal is acetylated (capped). However, this operation may be omitted in the case of synthesis of short-chain DNA (step 4). These steps 1 to 4 are repeated to extend the chain length of the oligomer (for example, oligonucleotide).

It is understood that the nucleic acid monomers used herein can also be modified or analogs using the phosphoramidite method or other methods. In the case of the H-phosphonate method, an acid chloride, a condensing agent (DCC or BOP-Cl) or the like can be used instead of the activator tetrazole or a weakly acidic substance. Oxidation to the phosphate ester is possible for each extension, but it is also possible to oxidize collectively at the end. In addition, as for the capping agent, it is common to use alkyl phosphite.

Confirmation of synthesis efficiency and the like can be confirmed by comparing the yield of the obtained nucleic acid with the synthesis by CPG as a reference.

(3. Cutting and purification after solid-phase synthesis)
After solid-phase synthesis, a suitable reagent (for example, 28% aqueous ammonia / 40% aqueous methylamine solution = 1/1 (hereinafter, may be abbreviated as AMA) 1 mL), a suitable temperature (for example, room temperature), a suitable time (for example). In 2h), the oligomer (for example, oligonucleotide) is cut out from the carrier, and the column is washed with an appropriate washing solution (for example, 50% ethanol).

After the solution has been dried (eg by SpeedVac ^TM (Thermo Scientific)), deprotection at the 2'position is a mixed solution of appropriate reagents (eg 3HF · TEA / TEA / DMSO (TEA: triethylamine, DMSO: dimethyl sulfoxide)). Perform under appropriate conditions (for example, 65 ° C. for 1.5 hours) or 55 ° C. for 17 hours in 0.3 mL), cool to an appropriate temperature (for example, room temperature), and then purify (for example, isopropanol precipitation). be able to.

After the sample is dried, it is dissolved in a suitable solvent (for example, a DNase-free buffer having a pH of about 7), and the absorbance (Abs) and liquid chromatography (LC) are measured.

(Preferable embodiment)
Hereinafter, preferred embodiments of the present invention will be described. It is understood that the embodiments provided below are provided for a better understanding of the invention and the scope of the invention should not be limited to the following description. Therefore, it is clear that a person skilled in the art can appropriately make modifications within the scope of the present invention in consideration of the description in the present specification. It is also understood that the following embodiments of the present invention may be used alone or in combination thereof.

(Inorganic porous carrier for solid phase synthesis)
In one aspect, the invention provides a carrier for producing a nucleic acid or an analog thereof, comprising an inorganic porous body having through-holes. Examples of the substance that can be synthesized by the present invention include nucleic acids and, as an analog thereof, any substance to which the solid-phase synthesis method of nucleic acids can be applied. For example, such nucleic acids or analogs thereof may be those that can be synthesized by the phosphoramidite method or the H-phosphonate method. Examples of such substances include crosslinked nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), unlocked nucleic acid (UNA), peptide nucleic acid (PNA), and crosslinked nucleic acid (BNA). In addition, nucleic acids in which the 2'-position hydroxyl group of sugar is modified or protected with an alkyl group or the like, modified nucleic acids into which an F group is introduced, and other derivatives (linker, dye derivative, etc.) that can be introduced into a nucleic acid sequence are described in the present specification. Nucleic acid or nucleic acid analogs, but are not limited to these.

In one embodiment, the carrier of the invention comprises a binding linker for binding a nucleic acid or an analog thereof or a source thereof (eg, a nucleoside or an analog thereof, and an introduceable organic compound (such as a linker or dye derivative)). Such a binding linker may be any linker capable of binding the carrier of the present invention to a nucleic acid or an analog thereof or a raw material thereof. Such a binding linker is preferable. , At least one selected from the group consisting of amino groups, hydroxyl groups and thiol groups. Silane coupling agents having these substituents are commercially available, and are commercially available from Sigma Aldrich, Shin-Etsu Chemical Industries, and Tokyo Kasei Kogyo Co., Ltd. Further, the phosphonic acid coupling agent can be obtained from Dojin Kagaku and Sigma Aldrich. Linkers can be introduced using these reagents. These substituents are generally used. It is considered that the linker can be extended by a specific organic reaction (condensation, etc.). More specifically, such a bound linker is an aminopropyl group, an aminopentylamidepropyl group, an aminohexylureidopropyl group, N-. (2-Aminoethyl) Aminopropyl group, aminohexylaminoethyl ether group, aminopentylamidepropyl group, N-piperazinylamidepropyl group, ureidopropyl group and the like can be mentioned, but the present invention is not limited thereto.

In a preferred embodiment, the carrier of the present invention further links a nucleic acid or an analog thereof, a binding compound thereof or a nucleic acid thereof or an analog thereof to be synthesized after performing solid phase synthesis using the carrier of the present invention. It may contain a "cleavable linker" which is a linker having a binding mode capable of cleaving the carrier and the nucleic acid moiety under the condition that the binding is not decomposed. In a specific embodiment, the binding mode used in such a cleavable linker includes, but is not limited to, a bond selected from the group consisting of esters, amides, imides, thioesters and disulfide bonds.

In a more preferred embodiment, the carrier of the invention comprises a binding linker and a cleavable linker, in which case one linker may function as a binding linker and a cleavable linker, wherein the binding linker may be used. Contains at least one group selected from the group consisting of amino groups, hydroxyl groups and thiol groups, the group via a bond or bonding mode selected from the group consisting of esters, amides, imides, thioesters and disulfide bonds. It is bound to the inorganic porous body of the present invention. By including a linker containing such a binding mode, the nucleic acid or its analog can be effectively bound to the inorganic porous body, and after synthesis, the nucleic acid or its analog polymer is not cleaved. It can be advantageously separated from the inorganic porous body.

In one embodiment, the inorganic porous body having through holes used in the present invention may further have pores. In another embodiment, the inorganic porous body used in the present invention may be an integral type or a particle type. In certain embodiments, the inorganic porous material used in the present invention comprises silica gel or silica glass. In such a case, for example, the inorganic porous body used in the present invention is monolith type silica (for example, pellet type) or particle type silica. Particle-type silica can be produced by processing and classifying it to a certain diameter by a method such as pulverization, and there are those having through holes and pores, and those having no pore diameter and having only through holes. The monolith type is premised on passing the liquid inside the block, and a method of passing the liquid outside the monolith can also be defined as a preferable form of the monolith type silica. In a specific embodiment, the monolithic silica may be pellet type or particle type.

The inorganic porous body used in the present invention has through holes and pores, or has no pore diameter and has only continuous through holes. Silica monolith and CPG (controlled pore glass) are both types of porous silica, but silica monolith is produced by the organic-inorganic hybrid method and has a mesh-like pore (through hole) in the network structure. And its skeleton is characterized by having pores of two sizes, but the CPG has no through holes.

In a preferred embodiment, the inorganic porous material used in the present invention has a submicrometer size (that is, a size of less than 1 μm and usually refers to about several tens to several hundreds of nm) to a micrometer size (usually, about several tens to several hundreds nm). It has a three-dimensional network-like continuous skeleton with a size of 1 μm or more and a range of less than 1 mm.) It has nanometer-sized pores existing in the silica skeleton, or has a mesh-like skeleton with only through holes with the pore diameter set to 0.

In one embodiment, the through-holes of the inorganic porous body used in the present invention have a size of about 0.1 to about 20 μm and are equal to or smaller than the size or particle size of the inorganic porous body. The pores of the inorganic porous body used in the invention, if present, are preferably, but are not limited to, about 2 nm to about 300 nm and less than the diameter of the through hole. The lower limit of the preferred size of the through hole is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about. 0.9 μm, about 1.0 μm, about 2.0 μm, about 3.0 μm, about 4.0 μm, about 5.0 μm and the like can be mentioned, and the upper limit of the preferable size as the through hole is, for example, inorganic porous. Any combination thereof can be mentioned, such as about 50 μm, about 45 μm, about 40 μm, about 35 μm, about 30 μm, about 25 μm, about 20 μm, about 15 μm, about 10 μm, etc., which are less than or equal to the body size or particle size. Can be used, but is not limited to these.

The lower limit of the preferred size of the pores is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm. Etc., and examples thereof include about 500 nm, about 450 nm, about 400 nm, about 350 nm, about 300 nm, about 250 nm, about 200 nm, about 150 nm, and about 100 nm, and any combination thereof can be used. It can be, but is not limited to these. It is said that the monolith can be easily controlled in the range of about 2 to about 200 nm, but it is possible to manufacture the monolith in a range other than this.

In one embodiment, the inorganic porous material used in the present invention can be pellet type. Such pellet-type structures can be, for example, rod-shaped, plate-shaped or lump-shaped. The pores of the pellet-type structure of the present invention are typically about 100 nm or more. It is understood that even in the case of the pellet type, the preferred size of the pores can be within the range of any of the above combinations.

The binding linker used in the present invention contains an amino group, and the length from the silicon atom of the carrier surface used in the present invention is about one in the linear direction including the carbon atom, the nitrogen atom and the oxygen atom. The number is less than about 20, preferably about 7 to about 14 in the linear direction including carbon atoms, nitrogen atoms, and oxygen atoms. The range of preferable lengths is not limited to these, and the lengths from the silicon atoms on the surface of the carrier, including carbon atoms, nitrogen atoms and oxygen atoms, are up to about 30 and about 29 in the linear direction. , Approx. 28, Approx. 27, Approx. 26, Approx. 25, Approx. 24, Approx. 23, Approx. 22, Approx. 21, Approx. 20, Approx. 19, Approx. 18, Approx. 17, Approx. 16 pieces, about 15 pieces, minimum about 0 pieces, about 1 piece, about 2 pieces, about 3 pieces, about 4 pieces, about 5 pieces, about 6 pieces, about 7 pieces, about 8 pieces, about 9 pieces , About 10 and it is understood that any combination of these ranges is used.

In one preferred embodiment, the carrier of the invention may be provided in powder form. Although not bound by theory, it has been shown to improve the efficiency and yield of solid-phase synthesis by providing it as a powder, and such effects are easily achieved by conventional techniques. It can be said that it was unpredictable.

In one embodiment, the bound linker used in the carrier of the present invention is contained in a loading amount of about 10 to about 250 μmol / g, preferably about 30 to about 75 μmol / g. Alternatively, in another embodiment, the cleavable linker used in the carrier of the present invention is contained in a loading amount of about 10 to about 250 μmol / g, preferably about 30 to about 75 μmol / g. As shown in the examples, the maximum reaction amount of the nucleoside addition reaction to the inorganic porous carrier is about 50 to about 75 μmol / g, which exceeds about 80 μmol. This is because there is a tendency to decrease. Further, it has been found that a reaction amount of a level that could not be achieved in the past is achieved when the amount is about 30 μmol or more, and therefore, a carrier having a loading amount of about 30 to less than about 80 μmol / g or about 75 μmol / g is achieved. Is understood to be efficiently used for solid phase synthesis. Since the amount of nucleoside added does not increase even if a carrier (> about 100 μmol / g) into which an amino group (or carboxyl group) is introduced at a high density is used, such a range is required for efficient use of the carrier. Alternatively, it may be preferable to use a carrier supported by a supported amount in the vicinity thereof.

The obtained particles can be dried into a powder or a dispersion liquid dispersed in an appropriate organic solvent such as acetonitrile. In some cases, the obtained particles are classified to remove fine particles, coarse particles, aggregated particles, foreign substances and the like contained in the particles. In this way, a solid phase carrier for synthesizing nucleic acids or nucleic acid analogs according to the present invention can be obtained.

(Nucleic acid or nucleic acid analog synthesizer)
In one aspect, the invention provides a synthesizer of nucleic acids or analogs thereof, comprising the carrier of the invention. Here, the nucleic acid of the present invention or an analog synthesizer thereof can use any form known in the art, and the carrier of the present invention is used instead of the carrier (typically CPG) used therein. By using it, the yield and reaction efficiency can be dramatically improved. In particular, regarding the synthesizer and the synthesis method, the present invention can use the existing synthesis system and method as they are, and if a crushed inorganic porous carrier is used, the apparatus can be changed even in a large system. One of the features of this case is that it can be handled in the same way as when an existing carrier is used and good results can be obtained without doing so. As the carrier used in the synthesis method of the present invention, it is understood that any embodiment described in (inorganic porous carrier for solid phase synthesis) described in the present specification can be used.

The synthesizer of the present invention may be divided into a reaction part, a flow part and a storage part. The reactor has a capacity of about 0.2 to about 2 ml, is made of fluororesin, glass, stainless steel, or the like and has a tubular structure having an upper and lower filters, and the carrier of the present invention is sealed inside and subjected to the reaction. There are two types of reactors, one is a repetitive type that weighs and fills the nucleosided carrier for each synthesis, and the other is a disposable type that replaces the reactor filled with the carrier. be. The main function of the flow section is the valve system. In the raw material / reagent / solvent supply section, most of the devices these days are built in the machine. In addition to this, a control system is usually provided, which consists of hardware and software. In a typical embodiment, a solid-phase carrier carrying a nucleoside linker is placed in a reaction vessel of an automatic nucleic acid synthesizer, and acetonitrile is poured as a solvent. Then, the nucleic acid automatic synthesizer according to the synthesis program, the deprotection reaction of the 5'-OH group of the nucleoside, the coupling reaction of the nucleoside phosphoramidite to the 5'-OH group, and the unreacted 5'-OH group. An oligomer such as an oligonucleotide having a target sequence is synthesized by repeating a cycle consisting of a capping reaction and an oxidation reaction of the produced phosphite. The finally synthesized oligomer such as oligonucleotide is hydrolyzed from the linker using ammonia or the like and cut out from the solid phase carrier. As a solid-phase carrier used for synthesizing such oligomers such as oligonucleotides, particle carriers such as polystyrene-based resin beads and glass-based porous beads are known, but in the present invention, they are inorganic instead. By adopting a porous body, we succeeded in providing an efficient and high-yield synthesizer. Compared with the case of synthesizing DNA, when synthesizing RNA, it is necessary to synthesize using phosphoramidite in which a protecting group is bound to a 2'-OH group, so that the coupling efficiency of amidite is lowered, and the coupling efficiency of amidite is lowered. As a result, there is a problem that the purity of the obtained RNA is lowered. In order to synthesize RNA without reducing the purity as much as possible, it is necessary to reduce the amount of the nucleoside linker bound on the solid phase carrier that is the starting point of the synthesis, and the present invention has also solved this problem.

The column reaction vessel for solid phase synthesis that can be used in the reactor can be formed by ordinary injection molding. As an example of the shape, a cylindrical column reaction vessel for solid-phase synthesis can be mentioned, but since the reaction of solid-phase synthesis is carried out using the inner wall surface of the microchannel as a reaction field, the outer shape of the column reaction vessel for solid-phase synthesis May have any shape as long as it has a shape suitable from the viewpoint of connectivity with the pipe to be connected and handleability. In addition to the cylindrical shape, it may be a polygonal column such as a quadrangular prism. The cross-sectional shape of the microchannel is not particularly limited as long as injection molding is not difficult, and may be a polygon such as a quadrangle or a hexagon as well as a circle. Reaction reagents, washing solutions, etc. for synthesizing nucleic acids or their analogs are sent to the microchannel, but from the viewpoint of reagent utilization efficiency, the ratio of the inner circumference of the channel to the cross-sectional area of the microchannel is It is desirable that it be formed so as to be large. That is, the inner diameter of the microchannel is about 1 mm or less, preferably about 1 to about 100 μm. Although it is composed of a single microchannel, as another embodiment, it can be synthesized at once by providing a plurality of microchannels and integrally molding the plurality of microchannels together. It is possible to increase the amount of oligomers such as oligonucleotides. It is preferable that each microchannel is formed so as not to intersect each other from the inlet to the outlet on the way. Solid-phase synthesis involves hydroxy group (-OH), amino group (-NH ₂ ), carboxyl group (-COOH), methoxy group (-OCH ₃ ), and thiol group (-SH) present on the inner wall surface of the microchannel. It has been clarified that it is preferable to make the total area of the inner wall surface of the microchannel occupying the volume of the column reaction vessel for solid-phase synthesis as large as possible because it is carried out via a linker that is bound to a functional group such as. Specifically, the total number of microchannels per unit area is increased so that the total inner circumference of the microchannel per cross-sectional area of the column reaction vessel for solid-phase synthesis orthogonal to the microchannel is large. Is preferable. When a plurality of microchannels are integrally molded, the cross-sectional shape of each microchannel is preferably polygonal. In particular, for solid-phase synthesis, a structure having a large number of axially penetrating flow holes partitioned by a partition wall is formed, such as a honeycomb structure used as a catalyst carrier for purifying exhaust gas from an internal combustion engine or a boiler. It is preferable to use a column reaction vessel. Examples of the resin constituting the plurality of integrally molded microchannels include acrylic resin (for example, polymethylmethacrylate, etc.), polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyimide, polyester, polyvinyl chloride, and the like. .. In particular, acrylic resins (eg, polymethylmethacrylate, etc.), polystyrene and polyethylene terephthalate are preferred.

In one embodiment, the synthesis performed in the present invention is solid phase synthesis. Solid-phase synthesis is one of the chemical (synthetic experiment) techniques, in which molecules are linked on particles and placed in a solution of a reaction reagent to carry out a synthetic reaction step by step. The building block molecule protects all reactive functional groups. Then, only the two mutual functional groups involved in the expected reaction of the building block molecule and the reaction liquid molecule are controlled so as to be deprotected.

In another embodiment, the carrier of the present invention carries about the amount of the raw material of the nucleic acid to be synthesized or an analog thereof (such as a nucleoside or an analog thereof, and an organic compound that can be introduced (such as a linker or a dye derivative)). Adjusted to 30-about 80 μmol / g, this range is, for example, up to about 70 μmol / g, about 75 μmol / g, about 80 μmol / g, about 85 μmol / g, about 90 μmol / g, about 95 μmol / g, about 100 μmol. It may be / g, and the lower limit may be about 20 μmol / g, about 25 μmol / g, about 30 μmol / g, about 35 μmol / g, about 40 μmol / g, about 45 μmol / g, about 50 μmol / g. ..

(Synthesis method)
The present invention provides a method for synthesizing a nucleic acid or an analog thereof using a synthesizer of the carrier of the present invention or the nucleic acid of the present invention or an analog thereof. As the carrier or synthesizer used in the synthesis method of the present invention, any embodiment described in the present specification (inorganic porous carrier for solid phase synthesis) or (nucleic acid or nucleic acid analog synthesizer) can be used. It is understood that it can be used.

In one embodiment, one of the features of the method for synthesizing nucleic acids or analogs thereof of the present invention is that the synthesis is carried out by solid phase synthesis. The synthetic method of the present invention can be carried out by substituting the carrier in known methods, including any of the methods described elsewhere herein.

In another embodiment, the carrier of the present invention carries about 30 raw materials for the nucleic acid to be synthesized or its analogs (such as nucleosides and their analogs, and introduceable organic compounds (such as linkers and dye derivatives)). It is adjusted to about 80 μmol / g.

(General technology)
The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art.

In the present specification, oligonucleotides are used by replacing the support with the carrier of the present invention by a standard method known in the art, for example, in an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). It is also possible to synthesize oligomers such as. For example, by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), oligomers such as phosphorothioate oligonucleotides can be synthesized, and a regulated pore glass polymer support (Sarin) can be synthesized. By using the carrier of the present invention in the method described in et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451), oligomers such as methylphosphonate oligonucleotides are prepared. It is also possible.

For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A. Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc. These are incorporated herein by reference in the relevant parts.

As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When specified as "within the range of two values" in the present specification, the range also includes the two values themselves. The expression "about" has a tolerance of 10% unless otherwise specified, and if it is a measured value, it can be obtained by rounding off one significant digit or one digit below the displayed digit. The numerical value in the range of.

References such as scientific literature, patents, and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as they are specifically described.

The present invention has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present invention will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present invention. Accordingly, the scope of the invention is not limited to the embodiments or examples specifically described herein, but only by the claims.

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.

Hereinafter, oligomers such as nucleoside analogs, oligonucleotides and oligonucleotide analogs related to the present invention were synthesized according to the following synthetic scheme. These synthesis will be described in more detail in the examples. The carrier can also be produced by the following production procedure, and the carrier can be produced, for example, by R. Miyamoto, Y. Ando, C. Kurusu, H. Bai, K. Nakanishi, M. Ippommatsu, J. Sep. . Sci. 2013, 36 (12): Can be manufactured based on 1890-1896.

(Example 1: Preparation of inorganic porous carrier for oligonucleic acid synthesis)
1. 1. Outline of carrier preparation for oligonucleic acid synthesis In order to use a solid-phase carrier for oligonucleic acid synthesis, a carrier to which a 3'-terminal nucleoside is bound must be prepared. Since the synthesized oligonucleic acid needs to be excised from the carrier under basic conditions, the 3'end nucleoside is generally immobilized by an ester bond. It is known that the yield of oligonucleic acid synthesis is affected by the density of nucleosides immobilized on the carrier, the distance from the solid phase surface, and the like. In order to investigate whether the inorganic porous carrier can be applied to oligonucleic acid synthesis, surface modification of the powdered inorganic porous carrier to addition reaction of 3'-terminal nucleoside are performed, and it is possible to use it as a carrier for oligonucleic acid synthesis. I checked the sex.

1-1. The used inorganic porous carrier (bare) without surface treatment and three types of inorganic porous carriers (GRAmino191, 192, 196) to which an amino group was added in advance were used (Table 1). Two types of amino groups, an aminopropyl group and an aminopentylamidepropyl group, were to be verified, and the bare carrier was subjected to the aminosilanelation reaction.

(Manufacturing of barrier carrier)
Add 0.8 g of polyethylene oxide to 10 mL (milliliter) of diluted acetic acid aqueous solution, add 5 mL of tetramethoxysilane under ice cooling, stir for 30 minutes, transfer the obtained uniform solution to a resin container, and incubate at a constant temperature of 40 ° C. The mixture was allowed to stand for 24 hours and gelled to obtain a wet gel. The obtained gel was immersed in ammonia water for 24 hours and aged, then immersed in distilled water and an alcohol solution and allowed to stand for 24 hours for washing, and the wet gel was taken out and air-dried to obtain a dry gel. The dried gel was heat-treated at 500 ° C. in an electric furnace to obtain a bare carrier.

(Manufacturing of modified carriers such as aminopropyl groups)
The bare carrier was dehydrated by heating in a constant temperature bath at 100 ° C. for 8 hours or more, immersed in a toluene solution containing 3-aminopropyltriethoxysilane, and heated to reflux for 8 hours. Subsequently, the treated carrier was immersed in an alcohol solution and allowed to stand for 24 hours for washing, and the wet gel was taken out and air-dried to obtain a modified carrier such as an aminopropyl group.

1-2. Procedure of surface modification The procedure from the surface modification performed on the above-mentioned inorganic porous carrier to the addition reaction of nucleoside is shown in Scheme 1.

反応１：担体表面への一級アミノ基の付加（アミノシラン化）（ｂａｒｅ担体のみ）
反応２：コハク酸の結合
反応３：シラノール基のキャッピング
反応４：３’末端ヌクレオシドの付加

Reaction 1: Addition of a primary amino group to the surface of the carrier (aminosilane formation) (bare carrier only)
Reaction 2: Succinic acid binding reaction 3: Silanol group capping reaction 4: Addition of 3'terminal nucleoside

2. 2. Surface treatment 2-1. Cleaning and aminosilanetion of the bare carrier If the carrier used for oligonucleic acid synthesis contains extremely fine particles, it may cause clogging of the synthetic filter. Therefore, such non-uniform fine particles need to be removed before surface treatment. After washing the bare carrier under the following conditions, non-uniform fine particles floating without precipitation were removed.

(Method: Cleaning)
The bare carrier (2.9 g) was placed in an Erlenmeyer flask, suspended in a 2 M aqueous hydrochloric acid solution (100 mL), and then stirred very slowly for about 1 hour to wash the fine particles. After allowing the flask to stand, the supernatant solution was removed and suspended fine particles were removed. Subsequently, water (100 mL) was added, and the operation of standing and removing the solution was repeated (washing 3 times).

Cleaning and removal of fine particles were repeated, but the presence of suspended fine particles did not subside and could not be completely removed. As the fourth washing, water was added and left for 16 hours, but suspended fine particles were confirmed. This indicates that very small particles are present in the initial sample. The complete removal of the fine particles was abandoned, and the bare carrier after washing was dried by the following method, and then aminosilanelation modification (reaction 1) was performed.

(Method: Aminosilaneization (Reaction 1))
The washed carrier was collected by filtration under reduced pressure (washed with water and methanol) and dried (40 ° C, 60 ° C, 80 ° C, 2 hours each). At this time, an attempt was made to dry under reduced pressure, but it was difficult to dry under reduced pressure due to the presence of fine particles. Therefore, after air-drying for 16 hours, the mixture was further dried at 100 ° C. for 2 hours.

Toluene (40 mL) and 3-aminopropyltriethoxysilane (MW.221.37, d = 0.946 g / mL, 10 mL) were added to the dried carrier (1.41 g), and the mixture was refluxed at 110 ° C. (10). time). Subsequently, it was filtered under reduced pressure, washed with toluene and chloroform three times, air-dried, and then dried under reduced pressure for several hours. When a part of the carrier was collected and subjected to a ninhydrin color development test, it was confirmed that an amino group was added because it showed a dark blue color (it was colorless in the bare carrier before modification).

Here, the particle state of the carrier was observed using an optical microscope (Fig. 1). Comparing the state of the particles after aminosilaneization with the bare carrier before washing, it was found that the bare carrier before washing contained ultrafine fine particles in addition to large particles, and the shape of the large particles was also non-uniform. Was confirmed. In addition, the aminosilaneized carrier had a large proportion of ultrafine particles. We do not want to be bound by theory, but we attribute this to the crushing of the carrier in a reflux environment.

2-2. Bonding of succinic acid A carrier having an amino group added to the surface was subjected to a binding reaction with succinic anhydride. This step was performed on each of the four types of powdered inorganic porous carriers shown in Table 1.

(Method: Succinic acid treatment (reaction 2))
Water (60 mL) and succinic anhydride (2 g) were added to the carrier (1.4-2 g). Originally, succinic anhydride is added to the suspension solution in which the solution is slowly stirred, but as described above, it was suggested that the inorganic porous carrier is extremely vulnerable to physical impact, so the reaction vessel was not stirred. Was gently shaken to carry out the reaction (at room temperature, the container was manually stirred once every 30 minutes). Subsequently, a 2M sodium hydroxide solution was added to maintain the reaction solution at pH 4 to 5, and after 3 hours, succinic anhydride (1 g) and 2M sodium hydroxide were added. After the reaction for 7 hours, the carrier was collected by filtration under reduced pressure, washed with water and methanol (3 times each), and the obtained carrier was air-dried. Subsequently, the carrier was dried at 70 ° C. under reduced pressure for 2 hours and at 80 ° C. under normal pressure for 2 hours, and then a ninhydrin color development test was performed using a part of the carrier to confirm that the primary amino group was not present on the carrier. did.

2-3. Capping of silanol groups Unreacted silanol groups remain on the carrier on which the carboxyl group is formed on the surface. Since they are capable of hydrogen bonding and have acidity, they may inhibit the reaction of the amidite on the carrier and reduce the yield of nucleic acid synthesis. Therefore, an unreacted silanol group was capped and inactivated. This step was performed for all carriers.

(Method: Capping (reaction 3))
The inorganic porous carrier (1-2 g) was suspended in anhydrous pyridine (50 mL), chlorotrimethylsilane (5 mL) was added, and the mixture was gently shaken at room temperature. After 2 hours, the powder was taken down under reduced pressure (washed with pyridine and chloroform 3 times each) and air-dried. Further, the capped inorganic porous carrier was recovered after drying under reduced pressure (2 hours) at room temperature.

3. 3. Addition of nucleosides to an inorganic porous carrier 3-1.3'Examination of conditions for terminal nucleoside addition (condensation) reaction Next, regarding the deoxynucleoside monomer addition reaction to an inorganic porous carrier, the optimum conditions for the condensation reaction were determined. To clarify, a study was conducted using a succinic acid-modified carrier prepared from a bare carrier (Scheme 2). Deoxythymidine in which the 5'-hydroxyl group is protected with a dimethoxytrityl group (DMT group or DMTr group) is used as the nucleoside monomer, and it is generally used as a dehydration condensing agent in the modification reaction of a normal controlled pore glass (CPG) resin. 1-Ethyl-3- (3-dimethylaminopropyl) Carbodiimide Hydrochloride (EDC / HCl) (Damha, M, J., et al., Nucleic Acids Res. 1990, 18, 3813-3821; Walsh, AJ , Et al., Tetrahedron Lett. 1997, 38, 1651-1654.), And O- (benzotriazole-1-yl) -N, N, N', N'-tetramethyl, which is widely used as a peptide condensing agent. Reactions were carried out with uronium hexafluorophosphate (HBTU) and 1H-benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (Kim, MH, and Patel, DV Tetrahedron Lett. 1994, 35). , 5603-5606; Albericio, F., Bofill, et al., J. Org. Chem. 1998, 63, 9678-9683; Pon, RT, and Yu, S. Tetrahedron Lett. 1997, 38, 3331-3334; Pon, RT, et al., Bioconjugate Chem. 1999, 10, 1051-1057). After the reaction, the condensation efficiency was evaluated by comparing the amount of nucleoside supported by the obtained modified carrier.

(Method: Addition of nucleoside (Reaction 4))
A glass vial with a capacity of 5 mL was used for each condensation reaction, and a succinic acid-modified inorganic porous carrier (prepared from a bare carrier), 5'-dimethoxytrityldeoxythymidine (5'-DMT-dT (5'-DMTr)). -DT)), and after capping by adding a solvent to a condensing agent or the like, the whole vial was gently shaken at room temperature using a shaker. Since the supported amount of the amino group or the carboxyl group was not measured in the inorganic porous carrier prepared from the bare carrier, the equal amount and the like were calculated assuming that the supported amount was about 100 μmol / g. After shaking for a certain period of time, the inorganic porous carrier was collected by filtration using a Kiriyama funnel, and the carrier was washed with pyridine and dichloromethane. The obtained modified inorganic porous carrier (dT-modified inorganic porous carrier) is dried under reduced pressure at room temperature for 4 hours, and then the DMT group (also referred to as DMTr group) derived from deoxythymidine bound to the carrier is quantified. Therefore, the amount of nucleoside supported was determined. The condensation reaction conditions examined below are shown below.

(Measurement of nucleoside loading amount)
After completion of the nucleoside condensation reaction (reaction 4), the dried nucleoside-added inorganic porous carrier (dT-ized carrier) (about 10 mg) is accurately weighed in a volumetric flask, and a mixture of perchloric acid (70% aqueous solution) and ethanol is used. 10.0 mL of the solution (3: 2) was added to desorb the dimethoxytrityl cation (DMT ⁺ , molar absorption coefficient: ε ₅₀₀ = 71,700). This solution was further diluted 10-fold with the above mixed solution, and then the absorbance at 500 nm was measured to calculate the nucleoside carrying amount (μmol / g) (Scheme 3). The result of reaction condition 1 is shown in FIG. 2 as an example of measuring the amount of nucleoside supported.

(Result of condensation reaction)
Table 6 shows the carrier yield and the amount of nucleoside supported for each reaction in the condition study.

The highest nucleoside loading was obtained when PyBOP (condensation conditions 3 and 4) was used as the condensing agent. It was found that the loading amount increased slightly due to the extension of the reaction time (2 hours to 24 hours), but a sufficient loading amount could be obtained even after the treatment for 2 hours (condensation condition 3). When HBTU was used as the condensing agent, a slight decrease in the amount of nucleoside supported was confirmed (condensation condition 2). On the other hand, under the condition using EDC, which is widely used in the modification reaction of the CPG carrier, the supported amount was about 3/4 of that of PyBOP even after the treatment for 24 hours (condensation condition 1). From these results, it was shown that PyBOP is suitable for the nucleoside addition reaction to the inorganic porous carrier.

In each of the reactions, the yield of the obtained nucleoside-modified inorganic porous carrier was lower than that at the start of the reaction. I do not want to be bound by the theory, but this is because the shape of the inorganic porous carrier used in this reaction is very fine particles, and the inorganic porous carrier fine particles are adsorbed on the filter paper during filtration recovery. It is presumed that there is.

3-2. Preparation of nucleoside-added inorganic porous carrier Since the optimum conditions for the nucleoside addition reaction were found by examining the above conditions, four types of inorganic porous carriers were modified using these conditions (Table 7). As described above, the carboxyl group-modified inorganic porous carrier prepared from the bare carrier was in the form of very fine fine particles, but the GRAmino 191 and 192, 196 carriers were inorganic with a particle size of 63 to 212 μm. A porous carrier was used.

4. Summary First, with the inorganic porous carrier prepared from the bare carrier, a carrier having a high nucleoside loading amount of 72.3 μmol / g could be prepared (condensation reaction 1). Since the amount of nucleoside supported by a carrier made from ordinary glass resin (CPG resin) is about 25 to 40 μmol / g, a carrier having a carrier amount 1.8 to 3 times higher than that of an inorganic porous carrier can be used. It became clear that it could be produced. Even in the inorganic porous carrier (GRAmino191, condensation reactions 2 and 3) in which the aminopropyl group is introduced at a high density, the nucleoside loading amount is 68 to 76 μmol / g, which is the same as that derived from the bare carrier. A carrier having an amount could be obtained. However, this value was a very low yield with a modification rate of 25 to 28% as compared with the amount of aminopropyl group supported before the condensation reaction (270 μmol / g). On the other hand, in the medium-density aminopropyl group-modified inorganic porous carrier (GRAmino192, condensation reactions 4 and 5), a modified carrier having a nucleoside loading amount of 55 to 57 μmol / g was obtained. Although this value is slightly reduced as compared with the supported amount on the GRAmino 191 carrier, the modification rate is 64 to 66% as compared with the carried amount of aminopropyl groups before modification (87 μmol / g), and the GRAmino 192 carrier has a modification rate of 64 to 66%. It was found that the condensation reaction on the surface of the carrier proceeded with high efficiency. Although not bound by theory, it is said that such a decrease in nucleoside condensation efficiency due to an increase in the modification density of the carrier surface is associated with a steric inhibitory effect on the surface of the inorganic porous carrier. Guessed. That is, in a low to medium density inorganic porous carrier, the influence between adjacent carboxyl groups (amino groups oxidized by shaving) on the surface of the carrier during the condensation reaction is small, so that the nucleoside condensation reaction proceeds efficiently. (Scheme 4).

However, in the carrier modified with high density, it is considered that the condensation reaction with the nucleoside monomer was inhibited by the steric bulkiness of the adjacent carboxyl groups (Scheme 5).

From these results, the maximum reaction amount of the nucleoside addition reaction to the inorganic porous carrier is about 50 to 75 μmol / g, and the carrier (>) in which the amino group (or carboxyl group) is introduced at high density. It was suggested that the amount of nucleoside addition did not increase even when 100 μmol / g) was used.

On the other hand, in the modification reaction of the inorganic porous carrier modified with the aminopentylamide propyl group (GRAmino196, condensation reaction 6), compared with the results of the aminopropyl group-modified inorganic porous carrier (condensation reactions 4 and 5). No significant difference was observed, and a carrier having a good nucleoside loading amount (54.2 μmol / g, modification rate 54%) was obtained. Although not bound by theory, this result indicates that the chain length of the amino group introduced into the inorganic porous carrier does not significantly affect the introduction of the nucleoside monomer.

By the way, it has been clarified that the inorganic porous carrier is easily crushed by a physical stimulus such as friction, and as a result, a fine particle-like carrier is produced. Mixing of such fine carriers causes clogging of the filter during the synthesis of oligomers such as oligonucleotides. Therefore, in order to suppress the crushing of the inorganic porous carrier, two stirring methods, a method of gently stirring manually (stirring condition A) and a method of gently stirring using a shaker (stirring condition B), are condensed. The reaction was carried out. As a result, it was found that in the method of manually stirring (stirring condition A), pulverization of the carrier was hardly observed and the modified carrier could be recovered almost quantitatively. On the other hand, in the method of stirring using a shaker (stirring condition B), a slight suspension occurred in the reaction solution, suggesting partial pulverization of the carrier. Therefore, under the stirring condition B, the fine particles generated by removing and washing the suspended solution portion before recovering the inorganic porous carrier are removed. Therefore, the amount of the modified carrier recovered (carrier yield) is slightly reduced. The condensation reaction carried out under stirring condition B has a slightly higher nucleoside loading amount.

(Example 2: Evaluation of carrier)
A synthetic test was performed and the yield of the resulting nucleic acid was compared to the synthesis on the reference CPG. The synthesis test was performed according to the following procedure.
1. 1. An inorganic porous carrier for oligonucleic acid synthesis prepared by the method of Example 1 or a method of reacting a nucleoside-succinic acid conjugate prepared in advance in methanol to prepare one step (see below). Using the synthesizer nS-8, 24-base RNA was synthesized (0.1 M RNA (TBDMS) amidite / MeCN) (TBDMS: tert-butyldimethylsilyl).
2. 2. After the synthesis, the oligomer (oligonucleotide in this case) was cut out from the carrier at 1 mL of 28% aqueous ammonia / 40% aqueous methylamine solution (hereinafter abbreviated as AMA) and room temperature of 2 hours, and the column was charged with 50% ethanol. Cleaning was performed.
3. 3. After the solution was dried by SpeedVac ^TM , deprotection at the 2'position was performed in 0.3 mL of a mixed solution of 3HF / TEA / TEA / DMSO at 65 ° C. for 1.5 hours or 55 ° C. for 17 hours, and the mixture was cooled to room temperature as it was. Later, isopropanol precipitation was performed.
4. After the sample was dried, it was dissolved in 2.0 M triethylamine-acetate buffer (TEAA), and the absorbance (Abs) and liquid chromatography (LC) were measured.

(One-step manufacturing method)
The method for producing the above-mentioned nucleotide-succinic acid conjugate prepared in advance by reacting it in methanol in one step is as follows.

(1) Synthesis of nucleoside-succinic acid conjugate (DMT-dT-Suc) DMT-dT 845 mg (1.55 mmol), succinic anhydride 282 mg (2.8 mmol), and 480 μl of triethylamine in dichloromethane (10 ml) with stirring 17 Time reacted. The reaction mixture was separated with triethylamine-phosphate buffer, and the aqueous layer was washed with dichloromethane. Sodium sulfate was added to the collected organic layer, dried, and then concentrated to dryness to obtain a white solid. Yield 921 mg, yield 92.1%.

(2) Reaction of DMT-dT-Suc with aminosilanelated inorganic porous carrier DMT-dT-Suc and condensing agent 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4- A mixture of methylmorpholinium chloride (DMT-MM) in methanol (0.1 M each) at a ratio of 1: 2 was added to an inorganic porous carrier, immersed in the carrier, and shaken for 17 hours. At that time, the amount of DMT-dT charged was between 0.4 and 3 equivalents with respect to the amount of amino groups of the inorganic porous carrier. After completion of the reaction, the carrier was washed with methanol and acetonitrile and then dried under reduced pressure to obtain a DMT-dT-modified inorganic porous carrier. The amount of DMT-dT supported on the carrier was determined by quantification of trityl groups using a mixture of perchloric acid and ethanol.

(result)
-Pellet-shaped sample The results of the pellet-type inorganic porous carrier examined below are shown. Each column type corresponds to the linker structure shown in Scheme 6 below. Items showing values exceeding the reference CPG in Table 8 are shown in gray.

From the above data, the following points could be confirmed.
-In many cases, the yield is low for pore sizes, especially those with small macropores (eg GRAmino 13, 15, etc.).
-As for the types of linkers, the shorter ones (linker 1) and the longer ones (linker 2) had higher yields, and some were higher than CPG per 1 μmol. However, since many of the carriers of Linker 2 have a low DMT-dT loading amount, it is difficult to identify the cause from this data alone.

With the pellet-shaped carrier, there was a large variation in production in each lot, and in some cases, the results varied even in the same lot. Considering the need to evaluate in a uniform state, quality assurance of carriers produced in the future, and ease of handling, we conducted a study using a powdered carrier made by crushing an inorganic porous carrier. I changed the direction to go.

-Particle-type sample For the particle-type inorganic porous body, the integrated inorganic porous body is crushed by a spray-drying method, a method using a W / O emulsion, or crushing by impact or pressure using a mortar or crusher. By manufacturing.

Regarding the particle type carrier, the pore size, particle size, type of linker, etc. were examined. The results of the study are shown in Table 9. Similar to Table 8, those showing values exceeding CPG are shown in gray.

The mesopore size is considered to be better at 100 nm than the pellet type 50 nm or less, and this pore size is close to the CPG pore size.

The particle size was examined with GRAmino95-96 and GRAmino125-131 at several particle sizes, but no significant difference was observed. It was possible to synthesize as long as the particle size did not hinder the passage of liquid.

In the examination of the linker type, the yields of the relatively long linkers 2 and 5 tended to be slightly better than those of the short linker 1. In particular, GRAmino 122 showed a high yield.

Based on the above, the carrier pore size and particle size were prepared to be equivalent to those of CPG, and the following studies were carried out.

・ Examination by changing the amount of amino group supported Particle-type inorganic porous material having the same pore size (1.7 μm, 100 nm) and particle size (100 to 150 nm) in order to consider the correlation between the amount supported and the yield in more detail. The study was carried out using particles modified with amino groups with different loading amounts on the body. The linkers were examined with structures 1 and 5. (Some carriers are measured up to the amount of trityl). The results are shown in Table 10.

When the amount of DMT-dT supported was large (from 80 μmol / g), the yield tended to decrease. The same tendency was seen regardless of whether the linker was long or short.

The carrier modified with linker 5 showed better yield than the carrier of linker 1.

It was found that the amount of DMT-dT carried had a great influence on the yield, and that the amount carried too high led to a decrease in yield, and in addition, the contribution of the linker length also existed.

Liquid passage test of the sample prepared from the bare carrier In the DMT-dT modified carrier prepared from the barrier carrier, the particles were considerably finer due to stirring in the preparation process and the like. Therefore, the back pressure may increase during the synthesis process, so a liquid flow test was conducted.

Liquid flow test-Conditions-
A carrier was placed in a 1 μmol synthesis column for the synthesizer nS-8, acetonitrile was passed through the column, and the time was measured.
AIST-03 (supported amount 72.3 μmol / g) was used as the carrier, and the test was performed under the following conditions (LCAA CPG was used by subjecting the Prime Synthesis carrier to an acetyl cap treatment).
Sample 1: AIST-03 1 μmol (13.8 mg)
Sample 2: AIST-03 1 μmol (13.8 mg) Insert a filter paper in front of a normal filter (see Fig. 4)
Sample 3: AIST-03 1 μmol (13.8 mg) mixed with capped LCAA CPG 13 mg Sample 4: AIST-03 1 μmol (13.8 mg) mixed with capped LCAA CPG 26 mg Actual A typical example of inserting the filter paper is shown in FIG.
--Liquid flow test results

When the filter paper was sandwiched in front of the filter, the time required for passing the liquid was slightly shortened (Sample 2).

When LCAA CPG was added, the liquid passing time was 2/3 of the original when doubled by weight (Sample 4).

Although subjective, it seemed that the particles were pressed above the column rather than clogged with the filter due to the passage of liquid, and the gap disappeared, resulting in an increase in back pressure and a decrease in flow velocity.

It is considered that mixing large particles into the carrier this time is effective, but there is a problem that the apparent amount of dT supported decreases as the mixing ratio increases.

Synthesis test Consider synthesis using a column sandwiching the filter paper of the above sample. The sequence, synthesis conditions, etc. are the same as the tests performed so far (24mer siRNA, using TBS amidite) (TBS: tert-butyldimethylsilyl).

Synthesis test of carriers with different amounts of DMT-dT supported Carriers were modified with different loading amounts of carriers of the same lot as shown below, and comparisons were made. The synthesis and post-treatment were performed in the same way as before.
In case of introduction of aminopropyl group (191, 192)
GRAmino191 (Amino group carrier amount 270 μmol / g)
GRAmino192 (Amino group carrier amount 87 μmol / g)
DMT-dT loading amount of the prepared carrier 191A 76.2 μmol / g
192A 57.0 μmol / g
191B (3eq.) 124.6 μmol / g
192B1 (3eq.) 95.4 μmol / g
192B2 (0.7eq.) 52.9 μmol / g
192B3 (0.4eq.) 24.3 μmol / g
The charging ratio referred to in this example refers to the amount of DMT-dT-suc added to the amount of amino groups present on the surface. If it was 3 eq, 3 times (excess) DMt-dT-suc was added to the amino group. The amount carried may vary from lot to lot due to the difference in reactivity that seems to be derived from the degree of congestion of amino groups.

In 192A, 192B2 and 192B3, yields equivalent to those of the CPG to be compared were obtained. These are carriers having a low loading amount, and the yield tends to decrease with other high-supporting amounts alone.

Similar to the studies conducted so far, the results show that the yield decreases as the loading amount increases. This suggests that when the pore size is 100 nm, the limit of the supported amount is around 50 to about 70 μmol / g. Condensation efficiency showed the same value as CPG for all carriers. FIG. 3 shows a graph in which the crude yield is plotted against the amount of DMT-dT supported.

Linker extension carrier (196)
The charging ratio was changed to 3 equivalents, 0.7 equivalents, 0.4 equivalents, and 0.3 equivalents.

The synthesis test and post-processing were performed in the same way as before.

DMT-dT loading amount of the prepared carrier 196A 54.2 μmol / g
192B1 (3eq.) 107.6μmol / g
192B2 (0.7eq.) 45.7μmol / g
192B3 (0.4eq.) 37.9μmol / g
192B4 (0.3eq.) 29.6μmol / g
It was manufactured by the same manufacturing method at each manufacturing site.

Compared with the cases of 191 and 192 in the previous section, the amount of DMT-dT introduced is slightly lower with the same amount of charge.

As a result of the synthesis test, the yield was low at 196B1, but the yield was equal to or higher than that of CPG in other cases (carrying amount was about 30 to about 60). In particular, at 196 A, the unit weight and the yield per unit volume are also high. In addition, the average condensation efficiency was almost the same as that of CPG.

When the results of the study on the presence or absence of the linker extension were combined, it was found that the presence or absence of the linker had an effect, but the degree of surface congestion had a greater effect on the yield.

By adjusting a carrier with an appropriate loading amount, an oligomer can be obtained with a yield exceeding CPG, and particularly when the loading amount of the carrier is about 50 to about 70, the yield per unit volume is more than twice that of CPG. ..

Summary An RNA synthesis test was performed and evaluated using an inorganic porous carrier having a porosity pattern different from that of the CPG carrier.

We investigated optimization of various parameters such as pore size, linker type, and the amount of the first base nucleoside supported on the inorganic porous carrier, and the amount of nucleoside supported was about 50 to about 70 μmol / g with good yield. It was found that it is the upper limit of the product that can be obtained, and that if the loading amount is higher than that, the yield will decrease. It was also found that the length of the linker connecting the carrier particles and the nucleoside does not affect as much as the amount carried, but the product can be obtained in a higher yield when extended. For example, a carrier having a short linker length (linker 1) and a carrier having a first base loading of 52.9 μmol / g has a crude nucleic acid yield of 2.62 mg / μmol, a yield of 138.6 mg per 1 g of the carrier, and a carrier having a long linker (linker 1). In the linker 5), the crude nucleic acid yield was 2.88 mg / μmol and the yield per 1 g of the carrier was 156.1 mg for the carrier having the first base carrier amount of 52.9 μmol / g. The results showed that the yield per μmol was equal to or higher than that of CPG (supported amount 28 μmol / g, crude yield 2.47 mg / μmol, yield 69.2 mg per 1 g of carrier), and the yield per unit weight was also higher. With the carrier optimized for various parameters, the yield per unit weight was more than twice that of CPG.

Although the references are shown below, they are used to refer to the information necessary in the present specification, and this description does not indicate the prior art to the present invention, and the present invention may be publicly known or easily conceived. I add that it does not admit that.

(Reference)
Damha, M, J., Giannaris, PA, Zabarylo, SV An improved procedure for derivatization of controlled-pore glass beads for solid-phase oligonucleotide synthesis. Nucleic Acids Res. 1990, 18, 3813-3821.
Walsh, AJ, Clark, GC, and Fraser, W. A direct and efficient method for derivatization of solid supports for oligonucleotide synthesis. Tetrahedron Lett. 1997, 38, 1651-1654.
Kim, MH, and Patel, DV “BOP” as a reagent for mild and efficient preparation of ester. Tetrahedron Lett. 1994, 35, 5603-5606.
Albericio, F., Bofill, JM, Elfaham, A., and Kates, SA Use of onium salt-based coupling reagents in peptide synthesis. J. Org. Chem. 1998, 63, 9678-9683.
Pon, RT, and Yu, S. Rapid automated derivatization of solid-phase supports for oligonucleotide synthesis using uronium or phosphonium coupling reagents. Tetrahedron Lett. 1997, 38, 3331-3334.
Pon, RT, Yu, S., Sanghvi, YS Rapid esterification of nucleosides to solid-phase supports for oligonucleotide synthesis using uranium and phosphonium coupling reagents. Bioconjugate Chem. 1999, 10, 1051-1057.

As described above, the present invention has been exemplified by using the preferred embodiment of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of claims. Patents, patent applications and other documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Is understood. This application claims priority to Japanese Patent Application No. 2016-2834 filed in Japan on January 8, 2016, the entire contents of which are incorporated herein by reference in their entirety.

The present invention is useful in the industry for the manufacture of nucleic acids or analogs thereof.

Claims (25)

A carrier for producing nucleic acids, chemically modified nucleic acids or nucleotides having through-holes with a diameter of about 0.1 to about 20 μm and containing an inorganic porous body containing silica gel or silica gel , said inorganic. The porous body is a carrier, which is monolithic silica or particle silica . The carrier according to claim 1, wherein the nucleic acid, chemically modified nucleic acid or nucleotide can be synthesized by a phosphoramidite method or an H-phosphonate method. The carrier according to claim 1 or 2, wherein the carrier has a binding linker for binding the nucleic acid, a chemically modified nucleic acid or nucleotide, or a raw material thereof. The carrier according to claim 3, wherein the binding linker comprises at least one selected from the group consisting of an amino group, a hydroxyl group, and a thiol group. After the production of the carrier, the carrier and the nucleic acid portion are cleaved under the condition that the nucleic acid, the chemically modified nucleic acid or nucleotide, the binding compound thereof or the binding connecting the nucleic acid, the chemically modified nucleic acid or the nucleotide is not decomposed. The carrier according to any one of claims 1 to 4, which has a linker having a capable binding mode (cleavable linker). The carrier of claim 5, wherein the binding mode comprises a bond selected from the group consisting of esters, amides, imides, thioesters and disulfide bonds. The binding linker comprises at least one group selected from the group consisting of amino groups, hydroxyl groups, phosphate groups and thiol groups, the group being selected from the group consisting of esters, amides, imides, thioesters and disulfide bonds. The carrier according to claim 3, which binds to the inorganic porous body via a bond. The carrier according to claim 1, wherein the inorganic porous body further has pores. The carrier according to claim 1, wherein the inorganic porous body has a pellet structure. The carrier according to claim 9 , wherein the pellet structure is rod-shaped, plate-shaped or lump-shaped. The carrier according to claim 9 , wherein the pores of the pellet structure are about 100 nm or more. The inorganic porous body has a sub-micrometer-sized to micrometer-sized three-dimensional network-like continuous skeleton, and sub-micrometer-sized to micrometer-sized three-dimensional continuous through holes in the gaps between the skeletons. The carrier according to claim 1, wherein the carrier has nanometer-sized pores that are continuously present in the silica skeleton, or has a mesh-like skeleton in which the pore diameter is set to 0 and only through holes are present. The through hole is smaller than the size or particle size of the inorganic porous body and is smaller than the size or particle size of the inorganic porous body.
The pores, if present, are from about 2 nm to about 300 nm and are less than the diameter of the through hole. The carrier according to claim 12 . The bonded linker contains an amino group, and the length from the silicon atom on the surface of the carrier is about 1 or more and less than about 20 in the linear direction including the carbon atom, the nitrogen atom and the oxygen atom. The carrier according to any one of 3 to 13 . The carrier according to claim 14 , wherein the length is about 7 to about 14 in the linear direction including carbon atoms, nitrogen atoms and oxygen atoms. The carrier according to claim 3, wherein the binding linker is contained in a loading amount of about 10 to about 250 μmol / g. The carrier according to claim 3, wherein the binding linker is contained in a loading amount of about 30 to about 75 μmol / g. The carrier according to claim 5, wherein the cleaving linker is contained in a loading amount of about 30 to about 75 μmol / g. The carrier according to any one of claims 1 to 18 , wherein the carrier is produced by a sol-gel method. A nucleic acid, chemically modified nucleic acid or nucleotide synthesizer comprising the carrier according to any one of claims 1 to 19 . The synthesizer according to claim 20 , wherein the synthesis is performed by solid phase synthesis. The synthesizer according to claim 21 , wherein the carrier is adjusted so that the amount of the raw material of the nucleic acid, the chemically modified nucleic acid or the nucleotide is carried to about 30 to about 75 μmol / g. A method for synthesizing a nucleic acid, a chemically modified nucleic acid or a nucleotide using the carrier according to any one of claims 1 to 19 or the synthesizer according to any one of claims 20 to 22 . 23. The method of claim 23 , wherein the synthesis is performed by solid phase synthesis. The synthetic method according to claim 23 or 24 , wherein the carrier is adjusted to carry an amount of a raw material of the nucleic acid, a chemically modified nucleic acid or a nucleotide as a raw material of about 30 to about 75 μmol / g.

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