JP2010122071A - Material-immobilized carrier having material immobilized thereon, and method for preparing material-immobilized carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material-immobilized carrier on which an immobilization object material is immobilized in a mode wherein a characteristic of a material (immobilization object material) to be immobilized on the carrier can be shown properly, and a method for immobilizing the immobilization object material on the carrier by a specific and simple operation. <P>SOLUTION: It is found that the immobilization object material can be immobilized on the carrier specifically and simply through a bioorthogonal reaction point, by introducing the bioorthogonal reaction point into the immobilization object material. This material-immobilized carrier including a coupling structure by the bioorthogonal reaction point is expressed by a general formula X-S-R, wherein X is an optional carrier, S is a spacer, and R is a material for immobilization. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a substance-immobilized carrier on which a substance to be immobilized is immobilized, an immobilization carrier and an immobilization substance for producing the substance-immobilized carrier, and a method for producing the substance-immobilized carrier. Furthermore, the present invention relates to an array comprising a substance-immobilized carrier containing one or more substances to be immobilized, a chromatography gel, and an immobilization catalyst.

  In recent years, protein arrays and peptide arrays (protein chips and peptide chips) in which proteins and peptides are immobilized on carriers such as gels and substrates as substances to be immobilized on the carrier have been produced. Protein arrays enable rapid detection of interactions between proteins and proteins, proteins and nucleic acids, and proteins and low-molecular compounds, and are very useful for basic research, clinical diagnosis and drug discovery research. That is expected.

  In addition to the protein array, a DNA microarray (DNA chip), which is one of gene analysis tools, may be mentioned as a substance to be immobilized on a carrier. A DNA microarray is a substance that functions as a probe by immobilizing DNA, which is the substance to be immobilized, on a carrier such as a substrate. The interaction between the DNA on the carrier and the molecule to be analyzed (analyte) is comprehensive. Used for analysis purposes. In addition, sugar chain arrays (sugar chain chips) in which sugar chains are immobilized on a carrier have been developed as substances to be immobilized. Sugar chain arrays are used for the purpose of analyzing the interaction between sugar chains and analytes, and detecting bacteria and viruses. Furthermore, an array (chip) in which a low-molecular compound is immobilized on a carrier as a substance to be immobilized has been developed. This chip is used for searching for endogenous ligands of proteins and screening for agonists and antagonists. Arrays in which various substances to be immobilized are immobilized on a carrier are used for basic research, clinical diagnosis, drug discovery research, and the like.

  As a method for immobilizing a target substance such as protein or DNA on a carrier, physical adsorption, a method of covalently bonding a functional group on the carrier and a functional group of the target substance, or a photoreactive group A method of immobilizing a substance to be immobilized on a carrier by using it has been studied so far. For example, in order to immobilize a protein on a carrier, a method is known in which a lysine residue present on the protein surface is chemically reacted with the carrier nonspecifically. However, with this method, it is impossible to immobilize proteins with the same orientation, and when there are multiple types of proteins that often deactivate the protein to be immobilized, only the protein to be immobilized is specific. There was a problem that it could not be fixed.

In order to solve these problems, various methods for binding the substance to be immobilized to the carrier have been proposed. For example, Micklefield et al. Have reported a method in which a protein having a peptide tag sequence recognized by this enzyme and a carrier are mixed in the presence of an enzyme catalyst called Phosphopantetheinyl transferase. Patent Document 1). Also disclosed are a method by cyanating the SH group of a protein, a method utilizing the affinity between fluorine compounds, and a method utilizing an α-amino group present in the protein (Patent Documents 1 to 3).
Patent 2990217 JP 2007-187647 JP2008-266221 J. Am. Chem. Soc., 130, 12456 (2008)

  An object of the present invention is to fix a substance in which the substance to be immobilized is immobilized in such a manner that the characteristics of the substance to be immobilized on the carrier (also referred to as “immobilization object substance” in this specification) can be appropriately exhibited. It is to provide an immobilized carrier and a method for immobilizing the substance to be immobilized on the carrier by a specific and simple operation.

  As a result of intensive studies in view of the above problems, the inventors of the present application introduced a bioorthogonal reaction point into the immobilization target substance, thereby allowing the carrier to specifically and easily be bound to the carrier via the bioorthogonal reaction point. The inventors have found that it is possible to immobilize the substance to be immobilized, and have completed the present invention.

That is, this invention consists of the following.
1. A substance-immobilized carrier comprising at least one binding structure with bioorthogonal reaction points and represented by the general formula XSR, wherein X is an arbitrary carrier, S is a spacer, and R is an immobilizing substance. A substance-immobilized carrier.
2. 2. The substance-immobilized carrier according to item 1 above, wherein the binding structure based on the bioorthogonal reaction point is (i) present at least in the binding part between the immobilizing substance and the spacer, or (ii) contained in at least the immobilizing substance. .
3. 3. The substance immobilization carrier according to item 1 or 2, wherein the immobilization target substance in the immobilization substance is selected from the group consisting of proteins, peptides and low molecular weight compounds.
4). The bond structure by the bioorthogonal reaction point is a combination of a functional group having a carbon-carbon triple bond and an azide group, and a carbon-carbon double bond represented by the following chemical formula I and three 4. The substance-immobilized carrier according to any one of items 1 to 3, which is a nitrogen-containing five-membered ring structure composed of nitrogen atoms of
5. Bond structure by bioorthogonal reaction point
(A) When the substance to be immobilized is a protein or peptide, a functional group represented by the following chemical formula XV is bonded to an azide group attached to the protein or peptide; or
(A) When the substance to be immobilized is a low molecular compound, a functional group represented by the following chemical formula XV is bonded to an azide group in the low molecular compound;
5. The substance-immobilized carrier according to any one of items 1 to 4, wherein the binding structure based on the bioorthogonal reaction point is a structure represented by the following chemical formula XVI.
In compounds XV and XVI, R ₁ , R ₂ , R ₃ and R ₄ are each an arbitrary substituent, or R ₁ and R ₂ form a cyclic structure, and R ₃ and R ₄ are each an arbitrary substitution. R ₃ and R ₄ form a cyclic structure, and R ₁ and R ₂ are optional substituents, respectively, or R ₁ and R ₂ form a cyclic structure and R ₃ and R ₄ Form a ring structure.
6). Bond structure by bioorthogonal reaction point
(C) When the substance to be immobilized is a protein or peptide, a functional group represented by the following chemical formula XIII is bonded to an azide group attached to the protein or peptide; or
(D) When the substance to be immobilized is a low molecular compound, a functional group represented by the following chemical formula XIII is bonded to an azide group in the low molecular compound;
6. The substance-immobilized carrier according to any one of 1 to 5 above, wherein the binding structure based on the bioorthogonal reaction point is a structure represented by the following chemical formula XIV.
7). The immobilization target substance is a solidification target protein or peptide, the amino terminus of the solidification target protein or peptide is a basic amino acid, and an amino acid having a bioorthogonal reaction point is bound to the basic amino acid. 7. The substance-immobilized carrier according to any one of 1 to 6 above.
8). A carrier for immobilization represented by the general formula XS, wherein X is an arbitrary carrier, S is a spacer having a bioorthogonal reaction point,
A carrier for immobilization for immobilizing a substance for immobilization having a bioorthogonal reaction point that reacts with a bioorthogonal reaction point present in a spacer.
9. An immobilization protein or peptide immobilized on an immobilization carrier,
The carrier for immobilization is represented by the general formula XS, X is an arbitrary carrier, S is a spacer,
A protein or peptide for immobilization, wherein the protein or peptide for immobilization has a binding structure by bioorthogonal reaction points or a bioorthogonal reaction point.
10. 8. A method for producing the substance-immobilized carrier according to any one of 1 to 7 above, which comprises the following steps:
(A) preparing an immobilization carrier represented by the general formula XS;
(B) providing a bioorthogonal reaction point to the immobilization target substance to produce an immobilization substance;
(C) A step of producing a substance-immobilized carrier by bringing the immobilizing carrier into contact with the immobilizing substance.
11. 11. The method according to item 10 above, wherein the step (b) or the step (c) includes a bioorthogonal reaction, and the bioorthogonal reaction does not use a catalyst.
12 The substance to be immobilized is a protein or peptide, and the step (b) comprises immobilizing the protein or peptide to be immobilized, a non-natural amino acid having a bioorthogonal reactive site, tRNA, aminoacyl tRNA synthetase, and aminoacyl tRNA protein transferase. 12. The method according to 10 or 11 above, wherein the mixed solution is used.
13. An array comprising the substance-immobilized carrier according to any one of 1 to 7 above, which contains at least one substance to be immobilized.
14. A chromatography gel comprising the substance-immobilized carrier according to any one of 1 to 7 above, which contains at least one substance to be immobilized.
15. An immobilization catalyst comprising the substance-immobilized support according to any one of 1 to 7 above, which contains at least one substance to be immobilized.

  The substance-immobilized carrier of the present invention can appropriately exhibit the characteristics of the substance to be immobilized, such as catalytic action, on the carrier. Further, according to the immobilization carrier, the immobilization substance, and the method for producing the substance immobilization carrier of the present invention, the immobilization target substance can be immobilized on the carrier specifically and in a uniform orientation, A substance-immobilized carrier on which the substance to be immobilized is immobilized at a high density can be obtained. Furthermore, any substance to be immobilized can be immobilized on a carrier as long as it is a substance to be immobilized that can introduce bioorthogonal reaction points. Since specific immobilization is possible, for example, the immobilization operation of the substance to be immobilized can be performed in a state in which various contaminating proteins such as a cell extract are mixed, and a substance-immobilized carrier can be easily prepared. it can.

The present invention relates to a substance-immobilized carrier represented by the general formula XSR. In the formula, X represents an arbitrary carrier, S represents a spacer, and R represents an immobilizing substance. The substance-immobilized carrier of the present invention is characterized by including a binding structure with bioorthogonal reaction points. The immobilization substance (R) includes the immobilization target substance. In the substance-immobilized carrier, the substance to be immobilized is immobilized in such a manner that it can exhibit characteristics. Examples of the characteristics of the substance to be immobilized include a function as a probe and a catalytic function.
Furthermore, the present invention extends to an immobilization carrier (general formula XS) and an immobilization substance (general formula R) for producing a substance immobilization carrier.

  The chemical formulas in the present invention and the structural formulas shown in the drawings are intended to include all isomers even if only a single isomer is described, unless otherwise specified.

  In the present invention, the binding structure by the bioorthogonal reactive site is (i) present at least in the binding portion of the immobilizing substance (R) and the spacer (S), or (ii) at least in the immobilizing substance (R). It is preferably included.

  In the present invention, a “bioorthogonal reactive site” is a functional group that allows a compound containing a bioorthogonal reactive site to be not misrecognized by any other compound, and is inherent in the organism. It means a functional group that reacts only with an artificially designed and introduced functional group without reacting with the functional group (such as lysine amino group or cysteine thiol group). Bio-orthogonal reaction points exhibit bio-orthogonality with each other due to the presence of a pair of bio-orthogonal reaction points, and the reaction can be performed. For example, functional groups having carbon-carbon triple bonds and azide groups (Ning et al., Angew. Chem. Int. Ed. Engl., 47, 2253 (2008); JA Codelli, CR Bertozzi et al, J Am Chem Soc. 2008 Aug 27; 130 (34): 11486-93), and tetrazine and transcyclooctene residues (Melissa L. Blackman et al, J Am Chem Soc. 2008; 130: 13518 ) And the like. The carbon-carbon triple bond is preferably contained in a cyclic structure having distortion such as an aromatic ring or a heterocyclic ring. The functional group containing a carbon-carbon triple bond may contain a fluorine atom.

In the present invention, the bonding structure by bioorthogonal reaction points is not particularly limited as long as the bioorthogonal reaction points to be paired are bonded to each other. The bond structure by the bioorthogonal reaction point is a carbon-carbon represented by the following chemical formula I, wherein a functional group having a carbon-carbon triple bond is bonded to an azide group contained in the immobilization substance. A nitrogen-containing five-membered ring structure composed of a double bond between them and three nitrogen atoms is preferable.

Furthermore, it is preferable that the bond structure by the bioorthogonal reaction point is a structure in which a cyclic structure is bonded to a nitrogen-containing five-membered ring structure represented by the following chemical formula VI.
In the formula, A represents an atomic group for completing a cyclic structure together with carbon atoms that are double-bonded in a nitrogen-containing five-membered ring structure. The ring formed may be any ring system and may form a condensed ring with another ring. The A atom group may further contain any functional group such as an alkyl group as long as it does not interfere with the bioorthogonal reaction. A is preferably a cyclic structure having strain.

Preferably, the bond structure by the bioorthogonal reaction point is a structure represented by the following chemical formula XVI.
In compound XVI, R ₁ , R ₂ , R ₃ , R ₄ are each an arbitrary substituent, or R ₁ and R ₂ form a cyclic structure, and R ₃ and R ₄ are each an arbitrary substituent. R ₃ and R ₄ form a cyclic structure, and R ₁ and R ₂ are optional substituents, or R ₁ and R ₂ form a cyclic structure, and R ₃ and R ₄ are cyclic Form a structure. The arbitrary substituent is any substituent containing a hydrogen atom, and may be, for example, two fluorine atoms.

More preferably, the bond structure based on the bioorthogonal reaction point is any one structure included in the following chemical formula X.

Furthermore, the binding structure based on the bioorthogonal reaction point is preferably any one of structures represented by the following chemical formula V.

In the present invention, the immobilization carrier comprises a carrier (X) and a spacer (S) that serve as a foundation when the substance to be immobilized is immobilized.
As the carrier (X), a wide variety of materials such as polymers such as silica, glass, polyolefin, polypropylene, polystyrene, agarose, and acrylamide, and hydrogel can be used. In addition, the carrier of the present invention includes any particulate carrier, monolith type carrier, plate-like or sheet-like substrate, etc., as long as they are insoluble and capable of immobilizing substances. As a commercially available carrier, Ultralink Biosupport (Pierce), protein gel hydrogel slide (Nexterion (registered trademark) Slide H, SCHOTT, # 1070936), and the like can be used.

  In the present invention, the spacer (S) has a function of binding the immobilizing substance to the carrier by being bound to the carrier and further bound to the immobilizing substance. The spacer is formed by attaching a reaction point (B1) for binding the immobilizing substance to the spacer compound. That is, the function of the spacer (the function of binding the immobilization substance to the carrier) is achieved by having a reaction point (B1) for binding the immobilization substance.

  The reaction point (B1) is not particularly limited as long as it allows the bonding between a substance and a substance. The reaction point (B1) may be a bio-orthogonal reaction point, or a known one such as avidin or streptavidin can be used. These reaction points (B1) bind to reaction points (B2) present in the immobilization substance, such as bioorthogonal reaction points or biotin, respectively. When the reactive site (B1) of the spacer is a bioorthogonal reactive site, specific examples include a functional group having a carbon-carbon triple bond, a tetrazine residue, or a transcyclooctene residue. The functional group having a carbon-carbon triple bond may contain a fluorine atom.

The functional group having a carbon-carbon triple bond is preferably a functional group having a carbon-carbon triple bond present in a cyclic structure represented by the following chemical formula IX.
In the formula, A represents an atomic group for completing a cyclic structure together with a carbon atom having a triple bond. The ring formed may be any ring system and may form a condensed ring with another ring. The A atom group may further contain any functional group such as an alkyl group as long as the reaction by the reaction point (B1) is not hindered. A is preferably a cyclic structure having strain.

Preferably, the bond structure by the bioorthogonal reaction point is a structure represented by the following chemical formula XV.

In Compound XV, R ₁ , R ₂ , R ₃ and R ₄ are each an arbitrary substituent, or R ₁ and R ₂ form a cyclic structure, and R ₃ and R ₄ are each an arbitrary substituent. R ₃ and R ₄ form a cyclic structure, and R ₁ and R ₂ are optional substituents, or R ₁ and R ₂ form a cyclic structure, and R ₃ and R ₄ are cyclic Form a structure. The arbitrary substituent is any substituent containing a hydrogen atom, and may be, for example, two fluorine atoms.

The functional group having a carbon-carbon triple bond existing in a strained cyclic structure includes a functional group having a carbon-carbon triple bond in cycloalkyne represented by the following chemical formula XI.

In addition, the functional group having a carbon-carbon triple bond present in a strained cyclic structure is particularly preferably a functional group represented by the following chemical formula II or chemical formula XII.

Any spacer compound can be used as long as it does not interfere with the function of the substance to be immobilized. The spacer compound has the function of [1] directing the reaction point (B1) attached to the carrier to the outside and promoting the solvation of the reaction point (B1), or [2] bioorthogonal reaction during the reaction This is thought to alleviate steric hindrance between points. Examples of the spacer compound include compounds composed of various amino acids, polyethylene glycol, polypeptides, or other various polymers. By adjusting the type, length, molecular weight, etc. of the spacer compound, the distance between the substance to be immobilized and the surface of the carrier can be adjusted. Furthermore, it is possible to prepare an environment (for example, a hydrophilic environment) around the substance to be immobilized. Thereby, for example, the interaction between the immobilization target substance and another substance (for example, an analyte) is appropriately performed, and the analysis or the like can be performed more accurately and efficiently. Commercially available spacer compounds include Boc diaminopropanoic acid (Bachem), O- (2-Aminoethyl) -O '-[2- (Boc-amino) ethyl] decaethylene glycol (Fluka # 77090), H ₂ N- (CH ₂ CH ₂ O) ₄₄ —CH ₂ CH ₂ —NH ₂ (manufactured by Fluka, # 14501) can be used.

  The reaction point (B1) can be imparted to the spacer compound by a method known per se.

  In the present invention, the immobilization substance (R) includes the substance to be immobilized, and includes a bioorthogonal reaction point imparted to the binding structure by the bioorthogonal reaction point or the substance to be immobilized. The immobilization substance (R) includes a reaction point (B2), and the reaction point (B2) has reactivity with the reaction point (B1) imparted to the spacer.

  The bioorthogonal reaction point imparted to the immobilization target substance has a function of binding to the spacer, and may function as the reaction point (B2) itself. Alternatively, the bioorthogonal reaction point imparted to the immobilization target substance may have a function of imparting the reaction point (B2) to the immobilization target substance. That is, in this case, the bioorthogonal reaction point given to the immobilization target substance reacts with the bioorthogonal reaction point in the compound having the reaction point (B2), and binds to the compound having the reaction point (B2). In other words, it means that there may be a binding structure with bioorthogonal reaction points.

The immobilization target substance is expected to exhibit the characteristics of the immobilization target substance in a state of being immobilized on the carrier. The characteristic of the substance to be immobilized means, for example, a function as a probe or a catalytic function.
The substance to be immobilized is not particularly limited as long as it is a substance capable of imparting a bioorthogonal reaction point, and examples thereof include nucleic acids, proteins, peptides, low molecular compounds, and high molecular compounds. The substance to be immobilized is preferably a substance selected from the group consisting of proteins, peptides, and low molecular compounds. In the present invention, nucleic acid means DNA or RNA, protein means a peptide having a molecular weight of 5000 or more, and peptide means one having an amino acid sequence containing two or more amino acids. A low molecular weight compound is a compound having a molecular weight of 10,000 or less, preferably 3,000 or less, and means a compound excluding nucleic acids, proteins and peptides. The high molecular compound is a compound having a molecular weight larger than 10,000, and means a compound excluding nucleic acid and protein. The substance to be immobilized may be natural or non-natural, for example, those obtained by isolation from the living body of various organisms, known per se such as organic synthesis and genetic engineering techniques. The produced product can be used using a technique.

  In the present invention, a bioorthogonal reaction point is given to the immobilization target substance.

First, the case where the bioorthogonal reaction point given to the substance to be immobilized functions as the reaction point (B2) itself in the substance for immobilization will be described. The bioorthogonal reaction point imparted to the immobilization target substance only needs to have bioorthogonality with the spacer reaction point (B1). When the reaction point (B1) of the spacer is a functional group having a triple bond contained in a strained cyclic structure, the reaction point (B2) of the substance to be immobilized is preferably an azide group. When the immobilization target substance is a protein or peptide, the azide group is added as a functional group represented by the following chemical formula III. When the immobilization target substance is a low molecular compound, the azide group is represented by the following chemical formula IV. It is preferable that it is provided as a functional group.
In addition, when the reaction point (B1) of the spacer is a tetrazine residue or a transcyclooctene residue, the reaction point (B2) given to the substance to be immobilized is a transcyclooctene residue or a tetrazine residue. preferable.

  When a protein or peptide is used as the immobilization target substance, the bioorthogonal reaction point may be added to one place such as the amino terminus or carboxyl terminus of the immobilization target protein or peptide, or the interior of the protein or peptide. That's fine. It is preferred that a bioorthogonal reactive site is attached to the amino terminus.

  In order to give a bioorthogonal reactive site to the protein or peptide to be immobilized, it is possible to introduce a bioorthogonal reactive site itself, for example, a functional group itself containing an azide group into the protein or peptide, It is also possible to introduce an unnatural amino acid having a reactive site into the protein or peptide to be immobilized. Examples of non-natural amino acids having bioorthogonal reactive sites include those containing an azide group, tetrazine residue or transcyclooctene residue, and more specifically, an azide group without an azidophenylalanine or phenyl group. And unnatural amino acids (Link, AJ; Tirrell, DAJ Am. Chem. Soc. 2003, 125, 11164-11165, etc.).

  A case where the bioorthogonal reaction point assigned to the immobilization target substance has a function of imparting the reaction point (B2) to the immobilization target substance will be described. The reaction point (B2) may be a bioorthogonal reaction point or other than the bioorthogonal reaction point, and may be any one having reactivity with the spacer reaction point (B1). For example, when the reaction point (B1) of the spacer is avidin or streptavidin, the reaction point (B2) of the immobilization substance is preferably biotin, and biotin is immobilized on the substance to be immobilized via the bioorthogonal reaction point. Join.

For example, as shown in the following chemical formula VIII, when biotin and a functional group having a triple bond contained in a distorted cyclic structure are covalently bonded, an azide group is added to the substance to be immobilized. The bio-orthogonal reaction sites bind to each other, so that biotin is bound to the substance to be immobilized. For example, it is preferable that biotin is bound to the substance to be immobilized with a binding structure represented by the following chemical formula VII.

  Giving bio-orthogonal reaction points to the immobilization target substance and bio-orthogonal reaction points to the compound containing the reaction point (B2) can be performed by a method known per se. For example, provision of a bioorthogonal reaction point to the immobilization target substance can be achieved by binding a compound having a bioorthogonal reaction point to the immobilization target substance.

  When the immobilization target substance is an immobilization target protein or peptide, the structure of the immobilization target protein or peptide is not particularly limited, but is specified according to the method for imparting bioorthogonal reaction points to the immobilization target protein or peptide. May require structural features such as the presence of a specific amino acid at position. Specifically, when the method described in Japanese Patent Application No. 2008-181303 invented by the present inventors (also referred to as the Nexta method) is used, the amino terminus of the protein or peptide to be immobilized is lysine or arginine, histidine, Leucine and methionine are preferred. In the method described in Japanese Patent Application No. 2008-181303, particularly when leucyl / phenylalanyl protein transferase is used as an aminoacyl tRNA protein transferase, a basic amino acid (eg, (Lysine or arginine, histidine) is preferably present. In this case, an amino acid having a bioorthogonal reaction point can be bound to a basic amino acid of the protein or peptide to be immobilized, thereby providing a bioorthogonal reaction point to the protein or peptide to be immobilized.

  The present invention further extends to a method for producing a substance-immobilized carrier. The method for preparing the substance-immobilized carrier is a step of preparing an immobilization carrier represented by the general formula XS; (b) providing a bioorthogonal reaction point to the substance to be immobilized, and producing the substance for immobilization A step; (c) a step of producing a substance-immobilized carrier by bringing the immobilization carrier into contact with the substance for immobilization.

  In the step (a), the immobilization carrier can be prepared by producing by a method known per se. For example, it can be prepared by adding a spacer compound to a carrier and further giving a reaction point (B1), or obtaining a carrier to which a spacer compound has been given in advance and giving a reaction point (B1).

Ultralink Biosupport (Pierce) is used as a commercially available carrier, and Boc diaminopropanoic acid (Bachem) or O- (2-Aminoethyl) -O '-[2- (Boc-amino) is used as a commercially available spacer compound. An example of using ethyl] decaethylene glycol (Fluka # 77090) is shown. Boc diaminopropanoic acid (Bachem) or O- (2-Aminoethyl) -O '-[2- (Boc-amino) ethyl] decaethylene glycol (Fluka # 77090), Ultralink Biosupport, dimethyl sulfoxide (DMSO), dimethyl By mixing formamide (DMF), diisopropylethylamine and the like and mixing them at 50 ° C. for 7 hours, a spacer compound can be imparted to the carrier.
Alternatively, a hydrogel slide for protein chip (Nexterion (registered trademark) Slide H, manufactured by SCHOTT, # 1070936) is used as a commercially available carrier, and H ₂ N— (CH ₂ CH ₂ O) ₄₄ is used as a commercially available spacer compound. An example of using —CH ₂ CH ₂ —NH ₂ (from Fluka, # 14501) will be described. Spot DMSO solution containing H ₂ N- (CH ₂ CH ₂ O) ₄₄ —CH ₂ CH ₂ —NH ₂ (Fluka, # 14501) on a hydrogel slide and incubate in the chamber at room temperature and overnight. Thus, a spacer compound can be imparted to the carrier.

  A method of imparting a bioorthogonal reactive site as a reactive site (B1) to the spacer compound applied to the carrier can be carried out by a method known per se. As a bioorthogonal reaction point, compound 6 (Carbonic acid 7, 8- didehydro-1,2: 5,6- is used to give a functional group having a triple bond contained in a distorted cyclic structure to the spacer compound. dibenzocyclooctene-3-yl ester 4-nitrophenyl ester) can be used (see Example 1 (2)). When using compound 6, for example, first, compound 6 is synthesized from a commercially available compound (phenylacetaldehyde, Aldrich) by synthesizing 6 steps, and compound 6 or dimethylformamide is placed on a gel or slide having a spacer compound. (DMF), triethylamine, etc. are brought into contact with each other and mixed overnight at 30 ° C. (or room temperature) to prepare an immobilization carrier to which a bioorthogonal reaction point is imparted.

  A method of imparting a reaction point other than the bioorthogonal reaction point, for example, streptavidin, as the reaction point (B1) to the spacer compound imparted to the carrier can also be carried out by a method known per se. A carrier such as streptavidin-agarose commercially available from each company may also be used. For example, Streptavidin-Agarose from Streptomyces avidinii (Sigma-Aldrich # S1638).

  In the step (b), the step of producing a material for immobilization by imparting a bioorthogonal reaction point to the material to be immobilized can be performed by a method known per se. For example, a bioorthogonal reaction point can be imparted to the immobilization target substance by binding a compound having a bioorthogonal reaction point to the immobilization target substance. The bioorthogonal reaction point of the immobilizing substance may itself function as a reaction point (B2) or may have a function of binding a compound having the reaction point (B2).

  When a protein or peptide is used as an immobilization target substance, a bioorthogonal reaction point is assigned to one location such as the amino terminus or carboxyl terminus of the immobilization target protein or peptide, or the interior of the immobilization target protein or peptide. What should I do? It is preferred that a bioorthogonal reactive site is attached to the amino terminus.

In order to impart a bioorthogonal reactive site to the protein or peptide to be immobilized, it is possible to introduce the bioorthogonal reactive site itself, for example, a functional group itself containing an azide group, into the protein or peptide to be immobilized. It is also possible to introduce an unnatural amino acid having a bioorthogonal reactive site into the protein or peptide to be immobilized. It is preferable to impart a bioorthogonal reaction point to the immobilization target substance by binding an unnatural amino acid having a bioorthogonal reaction point to the protein or peptide to be immobilized.
Examples of non-natural amino acids having bioorthogonal reactive sites include those containing an azide group, tetrazine residue or transcyclooctene residue, and more specifically, an azide group without an azidophenylalanine or phenyl group. And unnatural amino acids (Link, AJ; Tirrell, DAJ Am. Chem. Soc. 2003, 125, 11164-11165, etc.).

  The introduction of non-natural amino acids having bioorthogonal reactive sites into the protein or peptide to be immobilized is a non-natural amino acid mutation method (Alexander Deiters et al, J Am Chem Soc. 2003; 125: 11782; Link, AJ; Tirrell, DAJ Am. Chem. Soc. 2003, 125, 11164-11165); using leucyl / phenylalanylprotein transferase and tRNA aminoacylated with an unnatural amino acid Thus, a method of enzymatically binding an unnatural amino acid to the amino terminus of a protein or peptide to be immobilized (M. Taki, M. Sisido, Biopolymers: Peptide Science, 88, 263 (2007); RE Conner, DA Tirrell et al. ChemBioChem, 9, 366 (2008)); method described in Japanese Patent Application No. 2008-181303 (method using reaction of tRNA, aminoacyl tRNA synthetase and aminoacyl tRNA protein transferase: also referred to as Nexta method); When synthesizing a protein or peptide to be immobilized using the phase synthesis method, a method in which only one unnatural amino acid having an azide group is introduced into the terminal or inside of the protein or peptide to be immobilized in the course of synthesis. ,It can be carried out.

  When introducing a non-natural amino acid having a bioorthogonal reactive site into a protein or peptide to be immobilized by the method of Japanese Patent Application No. 2008-181303, leucyl / phenylalanyl protein transferase is used as an aminoacyl tRNA protein transferase. If used, an unnatural amino acid having a bioorthogonal reactive site can be introduced at the amino terminus of the peptide or protein to be immobilized. Note that the contents of Japanese Patent Application No. 2008-181303 are all described in this application by reference.

  When using the method described in Japanese Patent Application No. 2008-181303 (also referred to as Nexta method), it is preferable to use a protein or peptide to be immobilized whose amino terminus is lysine or arginine, histidine, leucine, methionine or the like. In the method described in Japanese Patent Application No. 2008-181303, particularly when leucyl / phenylalanyl protein transferase is used as an aminoacyl tRNA protein transferase, a basic amino acid (eg, (Lysine or arginine, histidine) is preferably present.

  Using the Nexta method described in Japanese Patent Application No. 2008-181303, a method for adding an azidophenylalanine, an unnatural amino acid, to a peptide or protein to be immobilized is, for example, tRNA, buffer, ATP (Na) -KOH, immobilization The protein or peptide to be activated, azidophenylalanine, aminoacyl tRNA synthetase and aminoacyl tRNA protein transferase are mixed and incubated. The incubation temperature is about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., and the incubation time may be about 15 minutes to 120 minutes, but may be several minutes (about 5 minutes). For example, incubation may be performed at 37 ° C. for 60 minutes.

  The case where a low molecular weight compound is used as the substance to be immobilized is exemplified. TAMRA-OSu (Sigma; official name: (5 (6) -carboxytetramethylrhodamine N-hydroxysuccinimide ester) By mixing -3,6,9-trioxaundecan-1-amine (Fluka) and incubating overnight at room temperature, a bioorthogonal reactive site can be imparted to the low molecular weight compound.

  The case where the bioorthogonal reaction point assigned to the immobilization target substance has a function of binding the compound having the reaction point (B2) will be described. Hereinafter, when the compound having the reaction point (B2) is biotin, biotin is given a functional group having a carbon-carbon triple bond contained in a distorted cyclic structure, and the immobilizing substance has an azide group An example will be described.

  First, a compound in which a bioorthogonal reaction point is imparted to biotin is obtained. Such compounds can be synthesized, purified and identified according to the method of Ning et al., Angew. Chem. Int. Ed. Engl., 47, 2253 (2008), for example.

  Next, a binding reaction between the immobilization target substance provided with the bio-orthogonal reaction point and a compound provided with bio-orthogonal reaction point for biotin is performed. In such a binding reaction, for example, a methanol solution containing a biotin-orthogonal compound-containing compound is mixed with biotin-orthogonal compound to be immobilized and incubated. In this coupling reaction, it is not necessary to further use a specific catalyst such as a copper catalyst, and it is preferable not to use it. If the carbon-carbon triple bond is contained in a strained cyclic structure, the bioorthogonal reaction can proceed rapidly without using a copper catalyst. The incubation time, temperature, and the like can be appropriately set. For example, the incubation may be performed at room temperature to about 37 ° C. for about 20 minutes to 1 hour.

  In the step (c), the substance-immobilized carrier can be produced by bringing the immobilizing carrier into contact with the immobilizing substance.

When the reaction point (B1, B2) is a bioorthogonal reaction point, the reaction may be performed by incubating the immobilization carrier and the immobilization substance in contact with each other, and a specific catalyst or the like may be further added. It is not necessary to add. For example, when the immobilization carrier and the immobilization substance are bonded by a carbon-carbon triple bond and an azide group, a copper catalyst may be used, but it is preferably not used. If the carbon-carbon triple bond is contained in a strained cyclic structure, the bioorthogonal reaction can proceed rapidly without using a copper catalyst. Incubation time for contacting with the front Symbol carrier for immobilizing the immobilization material, the temperature, etc. can be appropriately set, for example at room temperature, it may be carried out for about 20 minutes to 1 hour.

  When the reaction point (B1, B2) is other than the bioorthogonal reaction point, the reaction conditions and the like can be determined according to the type of the reaction point. For example, when the reaction point (B1) is streptavidin and the reaction point (B2) is biotin, the reaction may be performed with reference to the method of J. N. Rybak et al., Proteomics, 4, 2296 (2004). In addition, when commercially available Streptavidin-agarose is used, the reaction may be performed according to the manufacturer's protocol.

  The present invention further extends to an array, a gel for chromatography, or an immobilization catalyst comprising a substance immobilization support containing one or more substances to be immobilized. An array means a protein array (also called a protein chip), a gel for chromatography is exemplified by a gel packed in a column for liquid chromatography, etc., an immobilized catalyst is exemplified by immobilized enzyme, etc. Specifically, an enzyme immobilized on a microreactor can be mentioned.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these.

(Example 1) Preparation of immobilization carrier (1) Addition of spacer compound to carrier Gel 7 derived from commercially available acrylamide (Ultralink Biosupport, manufactured by Pierce), gel 7 having a short spacer compound and long spacer Gel 8 with a compound was synthesized (FIG. 2). Gels with spacer compounds are Boc diaminopropanoic acid (Bachem) or O- (2-Aminoethyl) -O '-[2- (Boc-amino) ethyl] decaethylene glycol 180 μmol, Ultralink Biosupport 0.1 g (30 μmol reaction point It was prepared by mixing 800 μL of dimethyl sulfoxide (DMSO), 800 μL of dimethylformamide (DMF), and 200 μL of diisopropylethylamine and mixing them at 50 ° C. for 7 hours to carry out the reaction. The supernatant was removed, 800 μL of 1.5 M Tris-HCl buffer solution (pH 8.8) was added, and the mixture was mixed at 50 ° C. for 30 minutes to crush unreacted functional groups on the gel. The supernatant was removed and the gel was washed with water. Next, trifluoroacetic acid (1 mL) was added on ice and left on ice for 30 minutes. The trifluoroacetic acid was removed and the gel was washed with diethyl ether and then dried with a vacuum pump.

(2) Synthesis of a compound having bioorthogonal reaction points In order to give the gel a bioorthogonal reaction point, a 6-step organic synthesis was performed from a commercially available compound (phenylacetaldehyde, Aldrich) to obtain compound 6 (Carbonic acid 7, 8- didehydro-1,2: 5,6-dibenzocyclooctene-3-yl ester 4-nitrophenyl ester) was synthesized. Synthesis and identification of the compound by NMR and the like were performed according to the method of Ning, Angew. Chem. Int. Ed. Engl., 47, 2253 (2008) (Scheme 1 below). Compounds 1-6 were all purified by silica gel column chromatography and identified by NMR and MS spectra.

(3) Giving a bioorthogonal reactive site to the carrier Compound 6 (4.6 mg; 12 μmol) against a dry gel with a spacer compound (10 mg; probably containing about 3 μmol of amino groups (reactive sites)) ) And dimethylformamide (DMF) 200 μL triethylamine (0.9 μL; 9 μmol) were mixed and reacted overnight at 30 ° C. The next day, the supernatant was removed and the gel was washed three times with dimethyl sulfoxide (DMSO) and then twice with water. As a result, gels 9 and 10 having strained triple bonds as bioorthogonal reaction points were prepared (FIG. 2).

(4) Determination of the number of moles of bioorthogonal reaction points on gels 9 and 10
TAMRA-OSu (Sigma; official name: (5 (6) -carboxytetramethylrhodamine N-hydroxysuccinimide ester; mixture of two isomers) and 11-Azido-3,6,9-trioxaundecan-1-amine (Fluka) Was used to synthesize a fluorescent compound 11 (red with the naked eye) having a bioorthogonal reactive site (azide group) according to the following scheme 2. Purification was performed using HPLC and identified by NMR and MS spectra. .
Compound 11 was dissolved in water and mixed with gel 9 or 10 to confirm the reactivity.

  The results are shown in FIG. Immediately after mixing with gels 9 and 10, the functional groups that are bioorthogonal reaction points react with each other to stain the gel red (right side of FIG. 3 (gel 10); the same result is obtained with gel 9) Not shown), the red color of the solution disappeared and the gel was stained red. As a result of mixing the compound 7 with the strain 7 or 8 having no triple bond, the gel remained white and the color of the solution did not disappear (left of FIG. 3 (gel 8); the same applies to the gel 7). Results, but no pictures are shown). This indicates that no reaction occurred between gel 7 or 8 and compound 11.

  The results are shown in FIG. Immediately after mixing with gels 9 and 10, the functional groups that are bioorthogonal reaction points react with each other and the gel is stained red (the right result in FIG. 3 (gel 10), the same result is obtained with gel 9) Not shown), the red color of the solution disappeared and the gel was stained red. As a result of mixing the compound 11 with the strain 7 having no triple bond and the compound 11, the gel remained white and the color of the solution did not disappear (left of FIG. 3 (gel 8), the same with the gel 7). Results, but no pictures are shown). This indicates that no reaction occurred between gel 7 or 8 and compound 11.

  Further, the gels 9 and 10 were titrated with the compound 11, and the number of moles of the bioorthogonal reaction point (triple bond) on the gel was determined. It had orthogonal reaction points.

(Example 2) Preparation of peptide for immobilization (Nexta method)
0.1 OD / μl E. coli tRNA ^Phe 3 μl (0.3OD; 460 pmol), 5 × aat buffer (aminoacylation buffer; 50 mM MgCl ₂ , 5 mM Spermine, 250 mM Hepes buffer (pH 7.6), 100 mM KCl) 7.2 μl , 250 mM ATP (Na) -KOH (pH 7.0) 0.36 μl, 350 pmol / μl Fluorescent peptide Lys-Ala-AMC (Lys-Ala-7-amido-4-methylcoumarin dihydrochloride; Sigma # L8139) 6 μl (2.1 nmol) ), Azidophenylalanine 60 nmol, 33 μM E. coli PheRS (Ala294 → Gly) 3 μl (100 pmol), 26 μM L / F transferase (wild type) 3 μl (78 pmol) mixed solution (total amount to 64 μl with ultrapure water) Adjustment). Incubated at 37 ° C for 60 minutes.
As a result, a fluorescent peptide to which an azide group was added was obtained (left side of FIG. 4).

(Example 3) Preparation of peptide-immobilized carrier Gel 7 (negative control, see Fig. 2) or gel 9 having only a spacer compound and no bioorthogonal reaction point was added to the peptide for immobilization prepared in Example 2. 6 μL was added and incubated at room temperature with stirring for 30 minutes (FIG. 4). After the reaction, the gel and the supernatant were separated. The supernatant was diluted with water and excited for fluorescence measurement at a wavelength of 320 nm. The gel was washed and then excited at a wavelength of 365 nm for fluorescence imaging.

  The results are shown in FIG. As a result of the fluorescence measurement, 98% of the fluorescent peptide could be immobilized on the gel 9 in an N-terminal specific manner.

(Example 4) Production of protein immobilization carrier (1) Production of protein to be immobilized Protein expression yeast (JD55; Ubr1 gene deficient type; A. Varshavsky et al., JBC, 274, 18135 (1999)), and Plasmid genes for expressing β-galactosidase with R (arginine), M (methionine), and L (leucine) at the amino terminus in the yeast (pUB23-R-betaGal, pUB23-M-betaGal, pUB23-, respectively) L-betaGal; A. Varshavsky et al., Science, 234, 179 (1986)) was obtained from A. Varshavsky, California Institute of Technology. After transforming each of these three genes into JD55 yeast, the addition of galactose allows β-galactosidase with R (arginine), M (methionine), and L (leucine) at the amino terminus in the yeast. Expressed. After the cell culture was crushed with beads, the supernatant was recovered as a cell extract. Enzyme activity was measured using a substrate X-gal according to a conventional method as to whether or not active β-galactosidase was contained in the cell extract.

  The results are shown in the upper part of FIG. From left to right, a cell extract of yeast transformed with a plasmid containing a β-galactosidase gene having R (arginine), M (methionine), and L (leucine) at the amino terminus, respectively. Since all cell extracts were colored blue, it was confirmed that the cell extract contained β-galactosidase having activity.

(2) Preparation of protein for immobilization (Nexta method)
350 pmol / μl Instead of the fluorescent peptide Lys-Ala-AMC 6 μl (2.1 nmol), the cell extract obtained in Example 4 (1) (cultured and centrifuged at 0.8 mL scale yeast suspension) Was prepared by crushing, and a total of about 18 μL) was reacted in the presence of an unnatural amino acid (azidophenylalanine; 60 nmol, 2 mM) at 37 ° C. for 1 hour (30 μL scale).

(3) Preparation of protein-immobilized carrier To the reaction mixture obtained in Example 4 (2), 6.9 μL of 1.1 M cysteine was added, and then mixed with 7.5 μL of the gel 9 obtained in Example 1. . After mixing for 30 minutes at room temperature for a bioorthogonal reaction, the supernatant was discarded and the gel was washed.
Whether or not β-galactosidase having activity was contained on the gel was measured by the same method as in Example 4 (1) using the substrate X-gal according to a conventional method.

  The results are shown in the lower part of FIG. It was confirmed that only β-galactosidase having R (arginine) at the amino terminus was colored blue. This is because only β-galactosidase with R (arginine) at the amino terminus, after azidophenylalanine is transferred, the amino-terminal azidophenylalanine and the cyclic structure of the gel form a covalent bond to form a covalent bond. This is probably because it was immobilized on the gel while keeping it. On the other hand, β-galactosidase with methionine or leucine at the amino terminus remained white. This is considered to be because β-galactosidase having methionine or leucine at the amino terminus could not transfer azidophenylalanine to the N terminus. As described above, it was shown that only the protein to be immobilized having arginine at the amino terminus can be specifically immobilized on the gel while maintaining the activity from the yeast cell extract containing a large number of proteins.

(Example 5) Production of low molecular weight compound immobilization carrier (1) Production of immobilization carrier Commercially available hydrogel slide for protein chip (Nexterion (registered trademark) Slide H, manufactured by SHOTT, # 1070936) was used as a starting material. . 0.3 μL of 40 mM DMSO solution containing H ₂ N- (CH ₂ CH ₂ O) ₄₄ —CH ₂ CH ₂ —NH ₂ (Fluka, # 14501) was spotted on the hydrogel slide and room temperature in the chamber. Incubated overnight. The next day, the slide was washed with DMF, and the surface was lightly dried. To crush the unreacted reaction sites (OSu residues) on the slide, apply a 40 mM DMSO solution in which diglycolamine (Wako, # 019-10922) is dissolved on the entire surface of the slide. Incubated. The next day, the slide was washed with DMF, and the surface was lightly dried to obtain slide 12. Compound 6 (23 mg; 60 μmol) and dimethylformamide (DMF) 1 mL triethylamine (4.5 μL; 45 μmol) were mixed, spread on slide 12, and incubated in the chamber at room temperature overnight. The next day, the supernatant was removed and the slides were washed 5 times with DMF and then twice with water. Thereby, a slide 13 having a strained triple bond as a bioorthogonal reaction point was produced (FIG. 7).

(2) Preparation of low molecular weight compounds for immobilization
Using TAMRA-OSu (Sigma; official name: (5 (6) -carboxytetramethylrhodamine N-hydroxysuccinimide ester) and 11-Azido-3,6,9-trioxaundecan-1-amine (Fluka), Example 1 ( A low molecular compound 11 having an azide group was synthesized by the same method as in 4) (Scheme 2).

(3) Preparation of low-molecular-weight compound-immobilized carrier Compound 11 dissolved in water is spotted at 1 μL / spot on each spot on the slide 13, and incubated in a humid chamber at room temperature for 3 hours. Was made. As a negative control experiment, a slide 12 having no bioorthogonal reaction points and a compound 11 dissolved in water were mixed and incubated in the same manner. After the reaction, the slide was washed with TBST buffer solution three times for 20 minutes, excited with 488 nm light, and a fluorescent image of the spot on the slide was taken with a fluorescence imager.

The results are shown in FIG. The upper row shows the result of producing the slide 14 using the slide 13. The lower row shows the result of using slide 12 (negative control). Since no fluorescence was observed in the lower stage, it was found that no compound 11 having a fluorescent group was bound on the slide. From these results, it was considered that a reaction specific to the bioorthogonal reaction point occurred and Compound 11 was immobilized on the slide 13.
In FIG. 8, the amount of compound 11 contained in 1 μL of the spotted solution is 1 nmol, 100 pmol, 10 pmol, 1 pmol in order from the left, and the display width (lateral width of the slide) is about 1.5 cm. The detection limit of Compound 11 under the conditions of this example was found to be 10 pmol. In addition, since the fluorescence intensity of the 1 nmol (leftmost) spot is equal to the fluorescence intensity of 100 pmol (second from the left), the number of moles of bioorthogonal reaction points contained in each spot on the slide is 100 pmol. Calculated as / spot.

(Example 6) Preparation of biotinylated peptide for immobilization In order to bind azidophenylalanine to compound 15 (fluorescent peptide Lys-Ala-AMC), the following reaction was performed (FIG. 9).
0.025 OD / μl E. coli tRNA ^Phe 24μl (0.6OD; 920 pmol), 5 × aat buffer (50 mM MgCl ₂ , 5 mM Spermine, 250 mM Hepes buffer (pH7.) Containing ATP (Na) -KOH (pH 7.0) 6), 100 mM KCl) 24 μl, 350 pmol / μl fluorescent peptide Lys-Ala-AMC (Compound 15) 12 μl (4.2 nmol), azidophenylalanine 96 nmol, 20 μM E. coli PheRS (Ala294 → Gly) 6 μl (120 pmol), 5.7 A mixed solution (adjusted to a total amount of 120 μl with ultrapure water) was prepared by mixing 27 μl (160 pmol) of μM L / F transferase (wild type). The mixed solution was incubated at 37 ° C. for 120 minutes. The mixture after the reaction was filtered using a YM10 filter to remove an enzyme having a molecular weight of 10,000 or more.
30 μL of the filtered solution was analyzed by HPLC. HPLC conditions are as follows: Column: GRACE Vydac 218TP, Detection: Fluorescence (Ex: 324 nm, Em: 320 nm), A solvent: 0.1% TFA aqueous solution, B solvent: acetonitrile, HPLC profile (ratio of B solvent): 0-5min 0%, 5-7min 0-10%, 7-27min 10-50%, 27-37min 50-100%.

  Of the remaining 90 μL solution, 30 μL was used for the next reaction. 5 μL of a methanol solution containing 20 mM of compound 17 (biotin derivative) (synthesized, purified and identified according to Ning et al., Angew. Chem. Int. Ed. Engl., 47, 2253 (2008)) In addition to the reaction solution of phenylalanine and compound 15, the total was 35 μL, and the reaction was performed at 37 ° C. for 60 minutes. The reaction mixture was filtered using a DISMIC 03 filter and the whole was analyzed by HPLC.

  The results are shown in FIG. The HPLC chromatograms of Compounds 15, 16, and 18 are shown from top to bottom, respectively. It was confirmed by MALDI-TOF-MS spectrum that the mass of the compound present in these peaks was the same as the theoretical value. From the HPLC results, the conversion efficiency from compound 15 to compound 16 and the conversion efficiency from compound 16 to compound 18 were determined. As a result, Compound 18 could be synthesized almost quantitatively (100%). Moreover, the compound 18 was also identified by the MALDI-TOF-MS spectrum. In the chromatography of compound 18, three (or more) peaks are thought to be due to the presence of four isomers of compound 18.

  According to the immobilization carrier, the immobilization substance, and the method for producing the substance immobilization carrier of the present invention, it is possible to immobilize the substance on the support specifically and in a uniform orientation, and the substance is densely distributed. An immobilized substance-immobilized carrier can be obtained. In addition, any substance that can introduce a bioorthogonal reactive site can be immobilized on a carrier, and the immobilization operation can be performed in a state in which various contaminating proteins such as cell extracts are mixed. It can be carried out. Such a substance-immobilized carrier of the present invention can appropriately exhibit the characteristics of the immobilized substance to be immobilized, and not only an array for analysis but also a chromatography gel, a micro It can also be used as an immobilized catalyst for reactors and is extremely useful.

It is a figure which shows the concept of this invention. It is a figure which shows the preparation process of the gel for fixation. (Example 1 (1) (2)) It is the photograph which shows that the bioorthogonal reaction point was introduce | transduced on the gel. (Example 1 (3)) It is a figure which shows the preparation process of a peptide fixed gel or a protein fixed gel. (Examples 2 to 4) It is the figure which confirmed the immobilization rate of the peptide on a gel. Example 3 It is the photograph which confirmed the fixation | immobilization of the protein on a gel. (Example 4) It is a figure which shows the preparation processes of a low molecular compound fixed support | carrier. (Example 5) It is the photograph which confirmed the fixation | immobilization of the fluorescent low molecular weight compound on a slide. (Example 5 (3)) It is a figure which shows the preparation process of a biotinylated peptide. (Example 6) It is a figure which shows the analysis result regarding a biotinylated peptide. (Example 6)

Claims (15)

A substance-immobilized carrier comprising at least one binding structure with bioorthogonal reaction points and represented by the general formula XSR, wherein X is an arbitrary carrier, S is a spacer, and R is an immobilizing substance. A substance-immobilized carrier. The substance immobilization according to claim 1, wherein the binding structure by the bioorthogonal reaction point is present in (i) at least a binding portion between the immobilizing substance and the spacer, or (ii) included in at least the immobilizing substance. Carrier. The substance immobilization carrier according to claim 1 or 2, wherein the substance to be immobilized in the substance for immobilization is selected from the group consisting of proteins, peptides and low molecular weight compounds. The bond structure by the bioorthogonal reaction point is a combination of a functional group having a carbon-carbon triple bond and an azide group, and a carbon-carbon double bond represented by the following chemical formula I and three The substance-immobilized carrier according to any one of claims 1 to 3, which has a nitrogen-containing five-membered ring structure composed of nitrogen atoms.
Bond structure by bioorthogonal reaction point
(A) When the substance to be immobilized is a protein or peptide, a functional group represented by the following chemical formula XV is bonded to an azide group attached to the protein or peptide; or
(A) When the substance to be immobilized is a low molecular compound, a functional group represented by the following chemical formula XV is bonded to an azide group in the low molecular compound;
The substance-immobilized carrier according to any one of claims 1 to 4, wherein the binding structure based on the bioorthogonal reaction point is a structure represented by the following chemical formula XVI.
In compounds XV and XVI, R ₁ , R ₂ , R ₃ and R ₄ are each an arbitrary substituent, or R ₁ and R ₂ form a cyclic structure, and R ₃ and R ₄ are each an arbitrary substitution. R ₃ and R ₄ form a cyclic structure, and R ₁ and R ₂ are optional substituents, respectively, or R ₁ and R ₂ form a cyclic structure and R ₃ and R ₄ Form a ring structure. Bond structure by bioorthogonal reaction point
(C) When the substance to be immobilized is a protein or peptide, a functional group represented by the following chemical formula XIII is bonded to an azide group attached to the protein or peptide; or
(D) When the substance to be immobilized is a low molecular compound, a functional group represented by the following chemical formula XIII is bonded to an azide group in the low molecular compound;
The substance-immobilized carrier according to any one of claims 1 to 5, wherein a binding structure by a bioorthogonal reaction point is a structure represented by the following chemical formula XIV.
The immobilization target substance is a solidification target protein or peptide, the amino terminus of the solidification target protein or peptide is a basic amino acid, and an amino acid having a bioorthogonal reaction point is bound to the basic amino acid. The substance-immobilized carrier according to any one of claims 1 to 6. A carrier for immobilization represented by the general formula XS, wherein X is an arbitrary carrier, S is a spacer having a bioorthogonal reaction point,
A carrier for immobilization for immobilizing a substance for immobilization having a bioorthogonal reaction point that reacts with a bioorthogonal reaction point present in a spacer. An immobilization protein or peptide immobilized on an immobilization carrier,
The carrier for immobilization is represented by the general formula XS, X is an arbitrary carrier, S is a spacer,
A protein or peptide for immobilization, wherein the protein or peptide for immobilization has a binding structure by bioorthogonal reaction points or a bioorthogonal reaction point. A method for producing the substance-immobilized carrier according to claim 1, comprising the following steps:
(A) preparing an immobilization carrier represented by the general formula XS;
(B) providing a bioorthogonal reaction point to the immobilization target substance to produce an immobilization substance;
(C) A step of producing a substance-immobilized carrier by bringing the immobilizing carrier into contact with the immobilizing substance. The method according to claim 10, wherein a bio-orthogonal reaction is included in step (b) or step (c), and the bio-orthogonal reaction does not use a catalyst. The substance to be immobilized is a protein or peptide, and the step (b) comprises the step of immobilizing the protein or peptide to be immobilized, a non-natural amino acid having a bioorthogonal reactive site, tRNA, aminoacyl tRNA synthetase, and aminoacyl tRNA protein transferase. The method of Claim 10 or 11 which uses the mixed solution containing. The array which consists of a substance fixed support | carrier of any one of Claims 1-7 containing 1 or more substance to be fix | immobilized. A chromatography gel comprising the substance-immobilized carrier according to claim 1, comprising one or more substances to be immobilized. An immobilization catalyst comprising the substance-immobilized carrier according to claim 1, comprising at least one substance to be immobilized.