JP6889432B2 - Resin-fixed peptide - Google Patents

Description

The present invention relates to novel resin-fixed peptides.

Skin sensitizers induce serious allergic reactions depending on the sensitivity of the body. Therefore, in the development of pharmaceuticals, pesticides, foodstuffs, cosmetics, detergents, etc., it is required that the raw materials, additives, final products, etc. do not contain skin sensitizing substances, and this should be confirmed. Various skin sensitization tests have been reported as methods.

Examples of these skin sensitization tests include a Maximization test, a Buehler test, and a Local Lymph Node Assay, which are tests in which a test substance is applied to an experimental animal. However, in such a test, not only is it necessary to breed and manage experimental animals, but there are also problems such as complicated work such as skin observation. It was also required to find an alternative method from the viewpoint of animal protection.

As a method for solving such a problem, an in vitro test system using peptide bondability as an index such as DPRA (Direct Peptide Reactivity Assay) has been developed (Non-Patent Documents 1 to 3). In this test method, a peptide having a binding property to a skin sensitizing substance is used as a reagent, mixed with the test substance, and then the peptide reduction rate before and after the mixing is calculated by HPLC chromatography to obtain the test substance. This is to evaluate the presence or absence of a skin sensitizing substance contained.

G.F. Gerberick et al., Development of a Peptide Reactivity Assay for Screening. Toxicol. Sci., 81, 332-343: 2004. G.F. Gerberick et al., Quantification of Chemical Peptide Reaction for Screening Contact Allergens: A Classification Tree Model Approach. Toxicol. Sci., 97 (2), 417-427: 2007. EURL ECVAM Recommendation on the Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing. European Commission, Joint Research Center, Institute for Health and Consumer Protection (IHCP), EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), November 2013.

However, in these test systems, there is a problem that the test itself cannot be performed on a test substance having low solubility in water. In addition, some test reagents (peptides) contain cysteine residues, and the formation of disulfide bonds between the peptides eliminates the binding property with the skin sensitizing substance, so that the accuracy of the skin sensitization test is improved. There was a problem that it decreased. Furthermore, when calculating the peptide reduction rate before and after mixing using HPLC chromatography, there is a problem that (a part of) the test substance may overlap with the peptide and the dissolution time cannot be calculated accurately. was there.

Therefore, an object of the present invention is a resin-fixed peptide that is applicable to various test substances and can be used as a method for testing the skin sensitization of a test substance easily, quickly, and with higher accuracy. It is to provide a reagent containing this. The present invention also provides a skin sensitizing substance removing agent containing a resin-fixing peptide.

As a result of diligent studies to solve the above problems, the present inventor, when the skin sensitization of the test substance is tested using a specific resin-fixing peptide, the test substance can be easily and quickly and with higher accuracy. We have found that skin sensitization can be tested and completed the present invention.

That is, the present invention provides a resin-fixing peptide in which a peptide having a binding ability to a skin sensitizing substance and a resin are bound via a cleavage linker.

The peptide having the ability to bind to the skin sensitizing substance is preferably a peptide containing one or more lysine residues or a peptide containing one or more cysteine residues.

The cleavage linker is preferably a light or alkali cleavage linker.

The cleavage linker preferably binds to the resin via the side chain of the C-terminal amino acid of the peptide having the ability to bind to the skin sensitizing substance.

（式中、Ｐはリジン残基を１個以上含むペプチド又はシステイン残基を１個以上含むペプチドを示す。Ｌ’は２価の炭化水素基、エーテル結合、チオエーテル結合、エステル結合、アミド結合、カルボニル基、又はこれらの２以上が結合した２価の基を示す。Ｒは樹脂を示す。） Further, the present invention provides a resin-fixed peptide represented by the following formula (2).

(In the formula, P represents a peptide containing one or more lysine residues or a peptide containing one or more cysteine residues. L'is a divalent hydrocarbon group, ether bond, thioether bond, ester bond, amide bond, Indicates a carbonyl group or a divalent group in which two or more of these are bonded. R indicates a resin.)

（式中、Ｒは樹脂を示す。） Further, the present invention provides a resin-fixing peptide represented by the following formula (I) or (II).

(In the formula, R indicates resin.)

The present invention also provides a test reagent for skin sensitization of a test substance containing the above-mentioned resin-fixing peptide.

The present invention also provides a skin sensitizing substance removing agent containing the above-mentioned resin-fixing peptide.

INDUSTRIAL APPLICABILITY The present invention provides a resin-fixed peptide that is adaptable to various test substances and is used in a method for testing the skin sensitization of a test substance easily, quickly, and with higher accuracy, and a reagent containing the same. can do. Further, according to the present invention, it is possible to provide a skin sensitizing substance removing agent containing a resin-fixing peptide.

In the present invention, various methods are used by using a resin-fixing peptide (hereinafter, may be referred to as "resin-fixing peptide") in which a peptide having a binding ability to a skin-sensitizing substance and a resin are bound via a cleavage linker. It is applicable to the test substance of the above, and the skin sensitivities of the test substance can be tested easily, quickly, and with higher accuracy. The resin-fixed peptide is a reagent in the skin sensitization test method of the test substance of the present invention (hereinafter, may be referred to as "test method of the present invention") (hereinafter, referred to as "test reagent of the present invention"). There is). That is, in the present invention, the skin sensitization property of the test substance can be tested using a reagent containing a resin-fixing peptide.

<Testing method for skin sensitization of test substance>
The assay method of the present invention is characterized in that after the test substance is brought into contact with the resin-fixed peptide, the presence or absence of binding between the peptide and the test substance is measured.

When the test substance contains a skin-sensitizing substance, by contacting the resin-fixing peptide with the test substance, the skin-sensitizing substance contained in the test substance reacts and binds to the peptide site of the resin-fixing peptide. A conjugate is formed. On the other hand, when the test substance does not contain a skin sensitizing substance, the above-mentioned conjugate does not occur. The present invention determines whether or not a skin sensitizing substance is contained in a test substance by measuring the presence or absence of such a bond (presence or absence of a binder).

After contacting the test substance with the resin-fixed peptide, the linker portion of the resin-fixed peptide was cleaved, and the presence or absence of binding between the peptide and the test substance was measured for the compound containing the generated peptide site. May be good. Further, the presence or absence of binding between the peptide and the test substance may be measured by using a chromatography method.

In the assay method of the present invention, for example, after contacting the resin-fixed peptide with the test substance, a resin-fixed peptide and / or a conjugate of the resin-fixed peptide and the test substance (skin-sensitizing substance) is obtained from the mixture thereof. After separation, the linker portion of the obtained resin-fixed peptide (including the one bound to the test substance) was cleaved, and the compound containing the peptide site generated by the cleavage (including the one bound to the test substance) was separated. It may be characterized by measuring the presence or absence of binding between the peptide and the test substance.

That is, the verification method of the present invention may include the following steps A to E.
Step A: Contacting the resin-fixed peptide with the test substance Step B: Separation of the resin-fixed peptide (including those bound to the test substance) from the mixture obtained in Step A Step C: Separation in Step B Step D: Step D: Separation of the compound containing the peptide moiety generated in Step C (including those bound to the test substance). E: A step of measuring the presence or absence of binding between a peptide and a test substance for a compound containing a peptide moiety separated in step D (including one bound to the test substance).

[Step A]
Step A in the assay method of the present invention is a step of bringing the resin-fixed peptide into contact with the test substance. In this step, when the test substance contains a skin sensitizing substance, the peptide site of the resin-fixing peptide and the skin sensitizing substance are bound to each other by contacting the resin-fixing peptide with the test substance. .. Examples of the contacting method include a method of mixing the resin-fixed peptide and the test substance in a solvent. Further, it may have a step of allowing it to stand after mixing.

The contact time in the step A is not particularly limited, but is preferably about 1 minute to 120 hours, more preferably about 5 minutes to 48 hours, and even more preferably about 10 minutes to 36 hours. The temperature (reaction temperature) in the step A is preferably about 0 to 100 ° C, more preferably about 10 to 60 ° C, and even more preferably about 20 to 40 ° C.

The solvent used in the step A is not particularly limited as long as it can dissolve the test substance, and is, for example, water, an aqueous buffer solution such as sodium phosphate or ammonium acetate, an organic solvent, or a solvent thereof. A mixed solvent can be used. Examples of the organic solvent include dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMAc), N-methyl-2-pyrrolidone (NMP), 2-pyrrolidone, acetonitrile, acetone, and the like. Examples thereof include vegetable oils such as chloroform, hexane, cyclohexane and olive oil, and monovalent alcohols having 1 to 4 carbon atoms. Examples of monohydric alcohols having 1 to 4 carbon atoms include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol and the like. These solvents may be used alone or in combination of two or more.

[Step B]
Step B in the assay method of the present invention is a step of separating the resin-fixed peptide (including those bound to the test substance) from the mixture obtained in step A. Examples of the separation method in step B include centrifugation, separation by filter filtration, separation using magnetic beads, and the like. The solvent used in step B is not particularly limited, and for example, the solvent exemplified in step A can be used.

Centrifugation involves centrifuging the mixture obtained through step A to obtain a resin-fixed peptide (including one bound to the test substance) in the mixture and a test substance not bound to the resin-fixed peptide. Is a method of separating. For example, the resin-fixed peptide can be obtained with high purity by removing the supernatant after subjecting the mixture to centrifugation.

In the separation by filter filtration, the mixture obtained through step A is subjected to filter filtration to separate the resin-fixed peptide (including the one bound to the test substance) and the test substance not bound to the resin-fixed peptide. It is a method of separating according to the size, and is particularly effective when the difference between these sizes is large. For example, by subjecting the mixture to filter filtration, the resin-fixed peptide can be obtained with high purity.

Separation using magnetic beads is an effective separation method when magnetic beads are used for the resin portion of the resin-fixed peptide (including those bound to the test substance), and is a separation method using the magnetic force of the magnetic beads and the magnet. Is. Specifically, by bringing the magnet into contact with the mixture obtained in step A, a resin-fixed peptide (including one bound to the test substance) is attached to the magnet, and the test is not bound to the resin-fixed peptide. It is a method of separating from a substance. Examples of the method of dissociating the resin-fixed peptide from the magnet include a method of losing the magnetic force of the magnet (for example, a method of using an electromagnet as the magnet). Further, there is also a method of obtaining a peptide containing a part of a cleavage linker (including one bound to a test substance) by performing steps C and D described later in a state where the resin-fixed peptide is attached to the magnet. Thereby, a resin-fixed peptide (including one bound to the test substance) and a peptide containing a part of the cleavage linker (including one bound to the test substance) can be obtained with high purity.

When the assay method of the present invention includes step B, a test substance having no binding ability to the resin-fixing peptide can be removed from the mixture obtained through step A, so that the resin-fixing peptide and the test substance (skin-sensitizing substance) can be removed. Only the resin-fixed peptide bound to and is obtained, and as a result, the test accuracy of skin sensitization is improved.

[Step C]
Step C in the assay method of the present invention is a step of cleaving the linker portion of the resin-fixed peptide (including those bound to the test substance) separated in step B. As a result, a peptide containing a part of the cleavage linker (including one bound to the test substance) and a resin containing a part of the cleavage linker can be obtained. A peptide containing a part of a cleavage linker may be referred to as a "compound containing a peptide moiety". The solvent used in step C is not particularly limited, and for example, the solvent exemplified in step A can be used.

When cutting with light in step C, the cutting method is not particularly limited, and examples thereof include a method of irradiating a resin-fixed peptide (including one bound to a test substance) with light. The type of light to be irradiated is not particularly limited, and examples thereof include ultraviolet rays (wavelength 315 nm to 380 nm). The light source is not particularly limited, but a low-pressure mercury lamp, a medium-pressure mercury lamp, a high-pressure mercury lamp, an ultra-high-pressure mercury lamp, a chemical lamp, a black light lamp, a microwave-excited mercury lamp, a metal halide lamp, or the like is preferably used.

When cleaving with an alkali in step C, the cleaving method is not particularly limited, and examples thereof include mixing an alkaline solution with a solution containing a resin-fixing peptide (including one bound to a test substance). The alkali used is not particularly limited, and for example, ammonia, hydrazine, triethylamine, sodium borohydride, and sodium hydroxide can be used. Alkaline solutions can be prepared by dissolving these alkalis in a solvent (eg, water, an organic solvent, or a mixed solvent thereof, etc.).

Examples of the cutting method other than light or alkali in step C include heating, acid, and enzymatic reaction.

When the assay method of the present invention includes step C, a compound containing a peptide moiety (including one bound to a test substance) and a resin containing a part of a cleavage linker can be obtained. Since these have different physical characteristics (for example, molecular weight, size, etc.), they can be separated by various separation methods (for example, the separation method in step D).

[Step D]
Step D in the assay method of the present invention is a step of separating a compound containing a peptide moiety generated in step C (including one bound to a test substance). More specifically, a compound containing a peptide moiety generated in step C (combined with a test substance) produced in step C by centrifugation, separation by filter filtration, separation using magnetic beads, etc., which is exemplified as the separation method in step B, is used. This is a step of separating (including) and removing the resin containing a part of the cleavage linker. The solvent used in step D is not particularly limited, and for example, the solvent exemplified in step A can be used.

When the assay method of the present invention includes step D, a compound containing a highly pure peptide moiety (including one bound to a test substance) can be obtained, so that the accuracy of skin sensitization assay is further improved.

[Step E]
Step E in the assay method of the present invention is a step of measuring the presence or absence of binding between the peptide and the test substance in the compound containing the peptide site separated in step D (including those bound to the test substance). The specific measurement method is not particularly limited, but for example, a chromatography method such as ion exchange chromatography, gel filtration chromatography, normal phase chromatography, reverse phase chromatography, or thin layer chromatography (TLC) was used. The method can be mentioned. Specifically, for example, a sample (solution) obtained by contacting the resin-fixed peptide with the test substance and a reference (solution) containing only the resin-fixed peptide are prepared, and if necessary, the above steps B to B to After performing D, each is measured by a chromatography method, and it is determined from the difference in the obtained amount ratio (for example, the area of the peak obtained by HPLC) whether or not a bond between the peptide and the test substance is formed. There is a way to do it.

Measurement methods other than the chromatography method in the assay method of the present invention include, for example, nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser ionization (MALDI) method, electrospray ionization (ESI) method, and atmospheric chemical ionization (ESI). It is possible to use known or commonly used methods in this field such as mass spectrometry using APCI) method, electron shock ionization (EI) method, fast atom bombardment (FAB) method, infrared spectroscopy (IR method), etc. it can. It should be noted that these measurement methods can also be used in combination of two or more.

<Resin-fixed peptide>
The resin-fixing peptide of the present invention is a resin-fixing peptide in which a peptide having a binding ability to a skin-sensitizing substance (hereinafter, may be referred to as "peptide") and a resin are bound via a cleavage linker. The resin-fixed peptide of the present invention can be represented by the following formula (1), in which P is a peptide having the ability to bind to a skin sensitizing substance (peptide site) and L is a cleavage linker (linker site). , R indicate a resin (resin portion).
P-L-R (1)

The peptide is not particularly limited as long as it is a peptide having a binding ability to a skin sensitizing substance. In addition, the peptide may have a protecting group. Examples of such peptides include peptides containing one or more lysine residues and peptides containing one or more cysteine residues. These peptides bind to skin sensitizers via lysine and cysteine residues. The degree of polymerization of the amino acid in the peptide having the ability to bind to a skin sensitizing substance is not particularly limited, but is preferably, for example, 3 to 20, more preferably 4 to 15, and further preferably 5 to 10.

The cleavage linker may be bound to any amino acid contained in the peptide, but from the viewpoint of binding ability to the skin sensitizing substance, it may be bound to the C-terminal or N-terminal amino acid of the peptide. preferable. When the cleavage linker is bound to the C-terminal or N-terminal amino acid of the peptide, it may be bound via the side chain of the amino acid at the C-terminal or N-terminal of the peptide, or the C-terminal of the peptide. It may be bonded via the carboxyl group of the above or the amino group at the N-terminal. Among these, from the viewpoint of binding ability to a skin sensitizing substance, it is preferable that the cleavage linker is bound via the side chain of the amino acid at the C-terminal or N-terminal of the peptide.

When the cleaved linker is bound via the side chain of the amino acid of the peptide, the amino acids include lysine (K), arginine (R), aspartic acid (D), glutamic acid (E), and cysteine (C). It is preferably an amino acid having a reactive functional group in the chain.

The peptide containing one or more lysine residues is preferably a peptide containing RFAAKAD or RFAAKAA as a peptide sequence, and more preferably has a protecting group at the N-terminal and / or C-terminal. Good H-RFAKAD-OH and H-RFAKAA-OH. The peptide containing one or more cysteine residues is preferably a peptide containing RFAACAD or RFAACAA as a peptide sequence, and more preferably has a protecting group at the N-terminal and / or C-terminal. It may be H-RFAACAD-OH or H-RFAACAA-OH. In addition, these peptides may have a protecting group. As the protecting group, a protecting group usually used in peptide synthesis can be used, for example, an N-terminal protecting group (for example, Ac (acetyl) group, Boc (t-butyloxycarbonyl) group, Z (benzyloxycarbonyl)). Groups, Fmoc (9-fluorenylmethyloxycarbonyl) groups, C-terminal protecting groups (eg, OBzl (benzyl ester) groups, OtBu (t-butyl ester) groups), and amino acid side-chain protecting groups (Tos) ) Group, Pbf (4-methoxytrityl) group, OccHex (cyclohexyl ester) group, MeBzl (methylbenzyl) group, Trt (trityl) group). Ac-RFAAKAD-OH and the like are exemplified as those having a protecting group, and Ac-RFAACAD-OH and the like are exemplified as a peptide containing one or more of the above-mentioned cysteine residues and having a protecting group. Be done.

When the peptide is a peptide containing RFAAKAD as a peptide sequence, the cleavage linker binds via the carboxyl group of the side chain of aspartic acid (D) from the viewpoint of binding ability to a skin sensitizer. Is even more preferable. The same applies when the peptide is a peptide containing RFAACAD as a peptide sequence.

Examples of the cleavage linker include a photo-cleaving linker (a linker that can be cleaved by light), an alkali-cleaving linker (a linker that can be cleaved by an alkali), and a thermal cleavage linker (a linker that can be cleaved by heat). , An acid-cleaving linker (a linker capable of cleaving with an acid), an enzyme-cleaving linker (a linker capable of cleaving with an enzyme), and a photo-cleaving linker or an alkali-cleaving linker is preferably used. The molecular weight of these linkers is not particularly limited, but is preferably about 60 to 1000, and more preferably 100 to 400.

As the photo-cleaving linker, a known photo-cleaving linker can be appropriately selected and used, and the photo-cleaving linker has a 2-nitrobenzene skeleton, a 5-nitrophenol skeleton, a nitroindole skeleton, or a coumarin skeleton, and is not particularly limited. Among them, a photocleaving linker having a 2-nitrobenzene skeleton and a 5-nitrophenol skeleton is preferable.

The photocleaving linker can be introduced, for example, by a photocleaving linker agent. Examples of such a photo-cleaving linker include Fmoc-Phg (2-NO2-)-(C # CH2) OH (manufactured by Watanabe Chemical Industry Co., Ltd .: a photo-cleaving linker having a 2-nitrobenzene skeleton). Can be done.

As the alkali cleavage linker, a known alkali cleavage linker can be appropriately selected and used, and the alkali cleavage linker is not particularly limited, and examples thereof include an alkali cleavage linker containing 4-hydroxymethylbenzoic acid. The alkali-cleaving linker can be introduced by an alkali-cleaving linker, and examples of the alkali-cleaving linker include a linker containing 4-hydroxymethylbenzoic acid.

The resin is not particularly limited, but is, for example, polyoxyalkylene polyol (for example, polyethylene glycol, polypropylene glycol, etc.), polyvinyl alcohol, polyacrylic acid, polyacrylic acid ester, polyester (for example, polyethylene terephthalate, polyethylene naphthalate, poly). Butylene terephthalate, polybutylene naphthalate, etc.), polyolefin (for example, polyethylene, polypropylene, ethylene-propylene copolymer, etc.), polyvinyl chloride, polystyrene, and resins that combine two or more of these (for example, copolymers thereof). ). Further, a resin that can be used as magnetic beads can be used. When a resin insoluble in the solvent used is used in the assay method of the present invention, separation of the resin-fixed peptide and the test substance (for example, separation by centrifugation or filter filtration) becomes easy. From the viewpoint of introducing a cleavage linker, these resins are preferably resins having a functional group such as an amino group, a carboxyl group, or a hydroxyl group. Examples of such a resin include Fmoc-NH-PEG resin (manufactured by Watanabe Chemical Industry Co., Ltd.), Amino PEGA resin, NovaPEG amino resin, NovaSyn TG amino resin (manufactured by Merck Co., Ltd.) and the like. Further, as the resin, a resin having a cutting linker (light or alkali cutting linker, etc.) can also be used. Examples of such resins include 4- [4- (1-Fmoc-aminoethyl) -2-methoxy-5-nitrophenoxy] butyramidomethyl-polystyrene (manufactured by Sigma-Aldrich Japan Co., Ltd.) and HMBA-PEG resin (Watanabe Chemical Industry Co., Ltd.). Made) and the like.

The resin-fixed peptide of the present invention is stable over a long period of time without forming a disulfide bond between the peptides even when the peptide site is a peptide containing a cysteine residue. It is considered that one of the reasons for this is that by immobilizing the peptide on the resin, the chances of the cysteine residue contained in the peptide reacting are reduced as compared with the case where the peptide itself is used as a test reagent.

The resin-fixing peptide of the present invention is preferably a resin-fixing peptide represented by the following formula (2). In addition, P in the formula (2) indicates a peptide site having an ability to bind to a skin sensitizing substance. In addition, P is preferably a peptide containing one or more lysine residues or a peptide containing one or more cysteine residues. L'indicates a part of the linker, and examples thereof include a divalent hydrocarbon group, an ether bond, a thioether bond, an ester bond, an amide bond, a carbonyl group, and a divalent group in which two or more of these are bonded. Examples of the divalent hydrocarbon group include a divalent aliphatic hydrocarbon group (for example, a divalent linear hydrocarbon group or a divalent branched chain hydrocarbon group) and a divalent hydrocarbon group. Alicyclic hydrocarbon group, a divalent aromatic hydrocarbon group, and a divalent hydrocarbon group in which two or more of these are bonded, and having 1 to 20 carbon atoms (preferably 2 to 10 carbon atoms). Hydrocarbon groups can be mentioned. R indicates a resin.

The resin-fixing peptide is preferably a resin-fixing peptide represented by the following formula (3). Note that P and R in the equation are the same as those in the equation (2).

Furthermore, the resin-fixing peptide is preferably a resin-fixing peptide represented by the following formula (I) or (II). In addition, R in the formula is the same as that in the formula (2).

The method for producing the resin-fixed peptide of the present invention is not particularly limited, but it can be produced, for example, by using a peptide solid-phase synthesis method or the like. Specifically, a resin whose surface is modified with an amino group or the like is used as a solid phase, and after reacting with a light or alkali-cleaving linker agent, the amino acid chains are extended one by one by a dehydration reaction to form a peptide moiety. There is a method of producing. A method of preparing a peptide site by using a resin having a cleavage linker and extending amino acid chains one by one by a dehydration reaction may be used.

The test reagent of the present invention may consist only of the above-mentioned resin-fixed peptide, and may further contain one or more additives. As the additive, for example, a pH adjuster, a stabilizer, a solvent and the like may be contained. The test reagent of the present invention may be provided in either a solid form (powder form, granule form, tablet form, etc.) or a liquid form (slurry form).

The resin-fixing peptide of the present invention can also be used as a skin sensitizing substance removing agent. That is, in the development of pharmaceuticals, pesticides, foodstuffs, cosmetics, detergents, etc., the resin-fixing peptide of the present invention is added to the raw materials, additives, final products, etc., and then appropriately removed to be included in these. It is also possible to remove skin sensitizers. The skin sensitizing substance removing agent may consist only of the resin-fixing peptide, or may further contain one or more additives. As the additive, for example, a pH adjuster, a stabilizer, a solvent and the like may be contained. The skin sensitizing substance removing agent may be provided in either a solid form (powder form, granule form, tablet form, etc.) or a liquid form (slurry form).

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

[Example 1: Preparation of resin-fixed peptide (1)]
The amino group on the NH _2- PEG resin (functional group introduction rate 0.2 mmol / g) was coupled with Fmoc-Phg (2-NO2-)-(C # CH2) OH. Next, the Fmoc group of the obtained reaction product was deprotected with piperidine, and the deprotected amino group and Fmoc-βAla-OH were coupled. Hereinafter, using the same method, Fmoc-Asp-Otbu, Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc- Coupling was performed in the order of Arg (Pbf) -OH, and after reacting with anhydrous acetic acid, it was used in a deprotecting solution (water: triisopropylsilane: TFA (volume ratio) = 0.02: 0.02: 1) for 2 hours. By reacting, washing and drying, a resin-fixed peptide (1) having the following model structure was prepared.

[Example 2: Preparation of resin-fixed peptide (2)]
The amino group on the NH _2- PEG resin (functional group introduction rate 0.2 mmol / g) was coupled with Fmoc-Phg (2-NO2-)-(C # CH2) OH. Next, the Fmoc group of the obtained reaction product was deprotected with piperidine, and the deprotected amino group and Fmoc-βAla-OH were coupled. Hereinafter, using the same method, Fmoc-Asp-Otbu, Fmoc-Ala-OH, Fmoc-Cys (Trt) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc- Coupling was performed in the order of Arg (Pbf) -OH, and after reacting with anhydrous acetic acid, it was used in a deprotecting solution (water: triisopropylsilane: TFA (volume ratio) = 0.02: 0.02: 1) for 2 hours. By reacting, washing and drying, a resin-fixed peptide (2) having the following model structure was prepared.

_{The NH 2-} PEG resin used in the preparation of the resin-fixing peptides (1) and (2), Fmoc-Phg (2-NO2-)-(C # CH2) OH, Fmoc-βAla-OH, Fmoc-Asp- Otbu, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Lys (Boc) -OH are all products of Watanabe Chemical Industry Co., Ltd. is there.

[Example 3: Test of skin sensitization of ethyl acrylate 1]
The skin sensitization of ethyl acrylate was tested by the following procedure.
1 mg of the resin-fixed peptide (1) was placed in a 5 ml tube (manufactured by Hypep Laboratory Co., Ltd.), washed 5 times with 500 μl of DMF, and then 500 μl of A solvent (water: TFA (volume ratio) = 100: 0). .1) was washed 5 times. Next, the resin-fixed peptide (1) was added to DMF containing 1 ml of 0.1 wt% triethylamine so that the concentration of ethyl acrylate was 100 mM, and the mixture was reacted for 24 hours. Next, it was washed 5 times with 500 μl DMF and then 5 times with 500 μl A solvent. Next, 500 μl of solvent A was added and stirred, and then collected in an Eppendorf tube using a pipette. Next, the mixture was centrifuged under the condition of 15,000 rpm × 1 min, the supernatant was discarded, and then the operation of adding 500 μl of solvent A again and stirring was repeated 4 times. Next, 400 μl of A solvent was added, and using an ultraviolet-visible fiber light source (Hamamatsu Photonics, LED light source controller (C11924)), light (wavelength: wavelength:) was used for 20 minutes under the conditions of 80% light intensity and 5 cm irradiation distance. Approximately 365 nm) was irradiated. Next, the sample after light irradiation is centrifuged (15000 rpm × 1 min), and the supernatant is transferred to a filter (Millipore 0.45 μm 100 / pk: manufactured by Merck) and centrifuged (15000 rpm × 1 min) to obtain 100 μl. HPLC measurement (column; SB-C18 (2.1 × 100 mm) manufactured by zorbax, solvent A; water: TFA (volume ratio) = 100: 0.1, solvent B; acetonitrile: TFA = 100: 0. 08, gradient; 0 min / B solvent 10%, 11 min / B solvent 90%; flow velocity: 0.35 ml / min). The model of the cut product obtained by irradiating the resin-fixed peptide (1) with light is shown below.

[Example 4: Test of skin sensitization of cyclamen aldehyde 1]
HPLC measurement was performed in the same manner as in Example 3 except that cyclamen aldehyde was used instead of ethyl acrylate.

(Reference of Examples 3 and 4)
HPLC measurement was carried out in the same manner as in Example 3 except that a skin sensitizing substance (ethyl acrylate) was not used.

[Example 5: Test of skin sensitization of ethyl acrylate 2]
HPLC measurement was performed in the same manner as in Example 3 except that the resin-fixed peptide (2) was used instead of the resin-fixed peptide (1).

[Example 6: Test of skin sensitization of cyclamen aldehyde 2]
HPLC measurement was performed in the same manner as in Example 4 except that the resin-fixed peptide (2) was used instead of the resin-fixed peptide (1).

(Reference of Examples 5 and 6)
HPLC measurement was performed in the same manner as in Example 5 except that no skin sensitizing substance was used.

<Evaluation>
By comparing the HPLC chart obtained in Example 3 with the HPLC chart obtained by the reference, it was confirmed that the elution peak area derived from the peptide site of the resin-fixed peptide was reduced. This suggests that in Example 3, ethyl acrylate was bound to the peptide site of the resin-fixed peptide. Furthermore, an elution peak considered to be derived from the conjugate of the peptide moiety cleaved from the resin-fixed peptide (1) and ethyl acrylate was observed. Further, the same results were obtained in Examples 4 to 6.

Claims (3)

Formula (I) or (II) in represented ∎ it can butter anchoring peptide.

(In the formula, R indicates a resin containing a polyoxyalkylene polyol.) A reagent for determining the skin sensitization of a test substance containing the resin-fixing peptide according to claim 1. A skin sensitizing substance removing agent containing the resin-fixing peptide according to claim 1.