Solid support having ligand immobilized thereon by using photocleavable linker

• US 7,456,022 B2
• Filed: 07/28/2005
• Issued: 11/25/2008
• Est. Priority Date: 07/30/2004
• Status: Active Grant

• Current Assignee: Kobe Chemical Genetics, Co., Ltd.
• Original Assignee: Reverse Proteomics Research Institute Company Limited
• Inventors: Tanaka, Akito, Haramura, Masayuki
• Primary Examiner(s): Warden; Jill
• Assistant Examiner(s): Mui; Christine T
• Application Number:
  US11/572,972
• Publication Number:
  US 20070212731A1
• Time in Patent Office: 1,216 Days
• Field of Search: 436/136, 436/6, 536/23.1, 435/7.1, 435/287.2
• US Class Current: 436/136
• CPC Class Codes: B01J 20/261 obtained by reactions only involving carbon to carbon unsaturated bonds macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds per se C08F
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/289 bonded via a spacer
• CPC Class Codes: B01J 20/321 consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3251 comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
• CPC Class Codes: B01J 20/3253 comprising a cyclic structure not containing any of the heteroatoms nitrogen, oxygen or sulfur, e.g. aromatic structures
• CPC Class Codes: B01J 20/3255 comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y10T 436/207497 Molecular oxygen

Abstract

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.

Claims

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):

3. The method of claim 2, wherein

R₂ is a hydrogen atom, an optionally substituted alkyl group or a optionally substituted alkoxy group;

R₃ is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group;

R₄ is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group; and

R₅ 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:

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):

7. The method of claim 6, wherein

R₂ is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group;

R₃ is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group;

R₄ is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted alkoxy group; and

R₅ 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: