Inorganic porous carrier and method for producing nucleic acids using same

Patent metadata

Field	Value
Publication number	US-12371451-B2
Application number	US-202017599297-A
Country	US
Kind code	B2
Filing date	Feb 28, 2020
Priority date	Mar 29, 2019
Publication date	Jul 29, 2025
Grant date	Jul 29, 2025

Abstract

An inorganic porous carrier having pore distribution where a pore diameter is 0.04 μm or more, and including a linker of formula (1) [where a bond * represents a bond to an oxygen atom of a silanol group in an inorganic porous substance. R 1 and R 2 represent each independently an alkyl group containing 3 to 10 carbon atoms, or a phenyl group. L represents a single bond; an alkylene group containing 1 to 20 carbon atoms; or an alkylene group containing 2 to 20 carbon atoms containing —CH 2 -Q-CH 2 — group wherein any group Q selected from a group consisting of —O—, —NH—, —NH—CO—, and —NH—CO—NH— is inserted into at least one of —CH 2 —CH 2 — group constituting the alkylene group. A carbon atom of the methylene group bound to the group Q does not bond to another group Q at the same time.]; and a method for preparing nucleic acids using the same.

First claim

The invention claimed is:

1. An inorganic porous carrier, comprising:
an inorganic porous substance and a linker bonded to the inorganic porous substance and represented by the following formula (1), wherein the inorganic porous substance has a pore distribution in which a pore size, which is a mode diameter, is at least 0.04 μm:

wherein, * represents a bond to an oxygen atom of a silanol group in the inorganic porous substance;
R 1 and R 2 represent each independently an alkyl group comprising 3 to 10 carbon atoms optionally substituted with a substituent selected from the group consisting of an alkoxy group and a fluorine atom; or a phenyl group optionally substituted with a substituent selected from the group consisting of an alkyl group, an alkoxy group and a fluorine atom; and
L represents a single bond; an alkylene group containing 1 to 20 carbon atoms; or an alkylene group containing 2 to 20 carbon atoms which contains —CH 2 -Q-CH 2 — group wherein any group Q selected from a group consisting of —O—, —NH—, —NH—CO— and —NH—CO—NH— is inserted into at least one of —CH 2 —CH 2 — group constituting the alkylene group; providing that a carbon atom of a methylene group bound to the group Q does not bond to another group Q at the same time.

2. The inorganic porous carrier according to claim 1 , wherein a surface area per volume of the inorganic porous substance is from 0.1 m 2 /mL to 100 m 2 /mL.

3. The inorganic porous carrier according to claim 1 , wherein a pore volume per volume of the inorganic porous substance is from 0.05 mL/mL to 0.6 mL/mL.

4. The inorganic porous carrier according to claim 1 , wherein the inorganic porous substance has a porosity of at least 50%.

5. The inorganic porous carrier according to claim 1 , wherein the inorganic porous substance has a particle size, which is a median diameter, of from 1 μm to 1000 μm.

6. The inorganic porous carrier according to claim 1 , wherein the inorganic porous substance is silica, silica gel, zeolite, or glass.

7. An inorganic porous carrier, comprising:
an inorganic porous substance and a linker bonded to the inorganic porous substance and represented by the following formula (2), wherein the inorganic porous substance has a pore distribution in which a pore size, which is a mode diameter, is at least 0.04 μm:

wherein * represents a bond to an oxygen atom of a silanol group in the inorganic porous substance;
R 1 and R 2 represent each independently an alkyl group comprising 3 to 10 carbon atoms optionally substituted with a substituent selected from the group consisting of an alkoxy group and a fluorine atom; or a phenyl group optionally substituted with a substituent selected from the group consisting of an alkyl group, an alkoxy group and a fluorine atom;
L represents a single bond; an alkylene group comprising 1 to 20 carbon atoms; or an alkylene group comprising 2 to 20 carbon atoms which comprises —CH 2- Q-CH 2 — group wherein the group Q selected from the group consisting of —O—, —NH—, —NH—CO— and —NH—CO—NH— is inserted into at least one of —CH 2 —CH 2 — group constituting the alkylene group; provided that a carbon atom of a methylene group bound to the group Q does not bond to another group Q at the same time;
R b represents a nucleoside or nucleotide in which a reactive group is protected or deprotected; and
L l represents a divalent group bound to an oxygen atom of a primary or secondary hydroxyl group in R b .

8. The inorganic porous carrier according to claim 7 , wherein L 1 in the formula (2) includes a succinyl group as a functional group.

9. The inorganic porous carrier according to claim 7 , wherein the linker has a density of from 0.1 μmol/m 2 to 5.0 μmol/m 2 with respect to a specific surface area per mass of the inorganic porous substance.

10. The inorganic porous carrier according to claim 7 , wherein a surface area per volume of the inorganic porous substance is from 0.1 m 2 /mL to 100 m 2 /mL.

11. The inorganic porous carrier according to claim 7 , wherein a pore volume per volume of the inorganic porous substance is from 0.05 mL/mL to 0.6 mL/mL.

12. The inorganic porous carrier according to claim 7 , wherein the inorganic porous substance has a porosity of at least 50%.

13. The inorganic porous carrier according to claim 7 , wherein the inorganic porous substance has a particle size, which is a median diameter, of from 1 μm to 1000 μm.

14. The inorganic porous carrier according to claim 7 , wherein the inorganic porous substance is silica, silica gel, zeolite, or glass.

15. A method for preparing a nucleic acid, using an inorganic porous carrier, wherein R b in the following formula (2) represents a nucleoside or nucleotide in which a hydroxyl group as a reactive group is protected,

wherein * represents a bond to an oxygen atom of a silanol group in an inorganic porous substance;
R 1 and R 2 represent each independently an alkyl group comprising 3 to 10 carbon atoms optionally substituted with a substituent selected from the group consisting of an alkoxy group and a fluorine atom; or a phenyl group optionally substituted with a substituent selected from the group consisting of an alkyl group, an alkoxy group and a fluorine atom;
L represents a single bond; an alkylene group comprising 1 to 20 carbon atoms; or an alkylene group comprising 2 to 20 carbon atoms which comprises —CH 2- Q-CH 2 — group wherein the group Q selected from the group consisting of —O—, —NH—, —NH—CO— and —NH—CO—NH— is inserted into at least one of —CH 2 —CH 2 — group constituting the alkylene group; provided that a carbon atom of a methylene group bound to the group Q does not bond to another group Q at the same time; and
L 1 represents a divalent group bound to an oxygen atom of a primary or secondary hydroxyl group in R b ,
the method comprising:
(A) deprotecting a protecting group of the hydroxyl group at a 5′ position of the nucleoside;
(B) subjecting the hydroxyl group at the 5′ position of the nucleoside produced in (A) to a condensation reaction with an amidite compound having a second nucleoside base to produce a phosphite;
(C) oxidizing the phosphite produced in (B) to produce a nucleotide; and
(D) deprotecting a protecting group of a hydroxyl group at a 5′ position of the nucleotide produced in (C).

16. The method according to claim 15 , further comprising:
(B′) subjecting a product produced in (D) to a condensation reaction with an amidite compound having a nucleoside base to produce a phosphite;
(C′) oxidizing the phosphite produced in (B′) to produce an oligonucleotide; and
(D′) deprotecting a protecting group of a hydroxyl group at a 5′ position in an end of an oligonucleotide strand produced in (C′).

17. The method according to claim 16 , further comprising:
(E) repeating (B′), (C′) and (D′) m times, wherein m is an integer of 1 or more, to react a number of m of amidite compounds; and
cleaving an elongated nucleic acid.

Assignees

• Sumitomo Chemical Co

Inventors

• Yamazaki Kanako
• Kitahara Masaki
• Hara Takashi
• Yoshioka Takeshi

Classifications

• C01P2006/14

  Pore volume · CPC title

• C01P2006/12

  Surface area · CPC title

• C01P2004/61

  Micrometer sized, i.e. from 1-100 micrometer · CPC title

• C01P2004/32

  Spheres · CPC title

• C01B33/14

  Colloidal silica, e.g. dispersions, gels, sols · CPC title