Solid support having ligand immobilized thereon by using photocleavable linker
View Patent ↗The present invention provides a method of analyzing the interaction between a ligand and a target molecule, which uses a solid support wherein the ligand is immobilized via a linker cleavable by photoirradiation, particularly a method of searching and purifying a target molecule. By intervening a linker specifically cleavable by photoirradiation between the ligand molecule and the solid support, release and elution of the target molecule from the solid support without using a ligand or salt at a high concentration can be enabled, and suppression of release and elution of a nonspecific protein can be enabled.
1. A method of searching a target molecule, which comprises
(1) immobilizing a ligand on a solid support via a linker cleavable by photoirradiation,
(2) mixing a solid support with the ligand immobilized thereon, which is obtained in the above-mentioned (1), and a sample wherein a target molecule of the ligand is or is not contained,
(3) cleaving a ligand from the solid support by irradiation of light in the presence of 2-mercaptoethanol and/or hydrazine, and
(4) confirming binding of the target molecule to the ligand.
2. The method of claim 1 , wherein the solid support with a ligand immobilized thereon is represented by the following formula (I) or (II):
wherein
X is a ligand, which is cleavable by photoirradiation;
Y is a single bond or an optionally substituted alkylene group;
A is a group usable for binding to a solid support;
Z is a solid support; Q is NH, O or S;
R 1 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group;
R 2 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group;
R 3 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group;
R 4 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group; and
R 5 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group.
3. The method of claim 2 , wherein
R 2 is a hydrogen atom, an optionally substituted alkyl group or a optionally substituted alkoxy group;
R 3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group;
R 4 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group; and
R 5 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group.
4. The method of claim 2 , wherein the solid support is represented by the following formula:
wherein each symbol is as defined above.
5. A method of purifying a target molecule, which comprises
(1) immobilizing a ligand on a solid support via a linker cleavable by photoirradiation,
(2) mixing a solid support with the ligand immobilized thereon, which is obtained in the above-mentioned (1), and a sample wherein a target molecule of the ligand is contained,
(3) cleaving a ligand from the solid support by irradiation of light in the presence of 2-mercaptoethanol and/or hydrazine, and
(4) recovering the target molecule bound to the ligand.
6. The method of claim 5 , wherein the solid support with a ligand immobilized thereon is represented by the following formula (I) or (II):
wherein
X is a ligand, which is cleavable by photoirradiation;
Y is a single bond or an optionally substituted alkylene group;
A is a group usable for binding to a solid support;
Z is a solid support;
Q is NH, O or S;
R 1 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group;
R 2 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group;
R 3 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group;
R 4 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group; and
R 5 is a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted alkoxy group, a halogen atom or a cyano group.
7. The method of claim 6 , wherein
R 2 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group;
R 3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group;
R 4 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group; and
R 5 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group.
8. The method of claim 6 , wherein the solid support is represented by the following formula:
wherein each symbol is as defined above.