Patent Description:
Skin sensitization (allergy) may involve not only local symptoms such as blisters and redness in areas exposed to substances, but also severe, life-threatening systemic allergic reactions known as anaphylaxis. In addition, skin sensitization is thought to be one of the important toxicities because, for example, once it develops, care needs to be taken to avoid exposure over a long period of time.

It is important that chemical substances present in products such as medicines, agricultural chemicals, and cosmetics be substances that do not cause allergic reactions. In product development, the chemical substances used need to be inspected for skin sensitization.

Conventionally, test methods using guinea pigs are commonly known as methods for evaluating chemical substances for skin sensitization, and test methods such as the Guinea Pig Maximisation Test (GPMT), which uses adjuvants, and the Buehler Test, which is a non-adjuvant test, have been widely used for a long time. On the other hand, recently, research and development have been made in alternatives to animal testing to meet ethical and social requirements such as animal welfare. In particular, there is an urgent need for the development of alternatives to skin sensitization tests that show severe symptoms, and in vitro tests using cultured cells and in chemico tests based on chemical reactions have been developed.

Examples of known in vitro tests include the ARE-Nrf2 luciferase KeratinoSens™ test method (KeratinoSens is a registered trademark), the ARE-NrF2 lusiferase LuSens test method (LuSens), the human Cell Line Activation Test (h-CLAT), the Myeloid U937 Skin Sensitization Test (U-SENS), and IL-<NUM> Luc assay.

In chemico tests based on chemical reactions, which do not use cultured cells, have many advantages such as requiring no special technology, knowledge, or equipment. For example, Non Patent Document <NUM> and Non Patent Document <NUM> describe methods using two peptides (cysteine and lysine peptides) as nucleophilic reagents (Direct Peptide Reactivity Assay (DPRA)). In addition, Patent Document <NUM> and Patent Document <NUM> describe skin sensitization measuring reagents and methods for measuring skin sensitization using a cysteine derivative having an aryl ring introduced therein and a lysine derivative having an aryl ring introduced therein as nucleophilic reagents.

The methods described in Non-Patent Documents <NUM> and <NUM> and Patent Documents <NUM> and <NUM> take time for evaluation because two reagents including cysteine and lysine are separately chemically reacted with a test substance and are separately subjected to measurement and quantification for calculation of depletion. Accordingly, a test method in which a skin sensitizer is detected and evaluated using a peptide including both cysteine and lysine has also been reported. However, this evaluation method, in which the reagent used is the heptapeptide Cor1C-<NUM> (Ac-Asn-Lys-Lys-Cys-Asp-Leu-Phe) (derived from the sequence around cysteine at residue <NUM> from the N-terminus of the human Coronin-<NUM> protein, which is a site with very high reactivity toward electrophilic reagents (Non-Patent Document <NUM>)), is a test method in which mass spectrometry is used for measurement; therefore, optical detection using light such as UV or visible light cannot be performed. In addition, this method is not intended to shorten the measurement time by including cysteine and lysine.

Non-Patent Document <NUM> describes five features: (<NUM>) a peptide-test substance adduct (covalent adduct) can be distinguished from peptide oxidation; (<NUM>) the problem of the sedimentation of a test substance does not occur because the concentration of the test substance in the reaction solution can be reduced; (<NUM>) the problem of the solubility of a test substance is alleviated because the preparation concentration of the test substance can be reduced; (<NUM>) the problem of co-elution does not occur because LC-MS measurement is used; and (<NUM>) a test substance with high reactivity can be more accurately evaluated by kinetic measurement. In addition, Non-Patent Document <NUM> states that the evaluation of three peptides, namely, the two peptides used in DPRA and the heptapeptide mentioned above, allows for high prediction accuracy. Furthermore, Patent Document <NUM> describes a skin sensitization detection reagent having a fluorescent dye attached to a terminus of a peptide.

In the skin sensitization measurement methods described in Non Patent Documents <NUM> and <NUM>, two reagents, namely, cysteine and lysine peptides, are used and separately reacted with a test substance. These reaction solutions are separately subjected to measurement by high-performance liquid chromatography (HPLC). The depletion is calculated from the amount of unreacted peptide in each solution, and the mean thereof (mean depletion) is calculated. It is predicted that the test substance is a skin sensitizer if the mean (mean depletion) exceeds a criterion, whereas it is predicted that the test substance is a non-sensitizer if the mean (mean depletion) does not exceed the criterion. This test method takes time because the two reagents are separately reacted and are separately subjected to measurement and quantification. In Non Patent Documents <NUM> and <NUM>, there is no mention of increasing the efficiency of measurement using a peptide including cysteine and lysine for efficient measurement. In addition, although a thiol group derived from cysteine is present in the peptide used as the skin sensitization detection reagent described in Patent Document <NUM>, the amino group used is an α-amino group of an amino acid. The great majority of amino groups in proteins in a living body are those derived from lysine, which differ in reactivity from α-amino groups.

An object of the present invention is to provide a skin sensitization measuring reagent, a method for measuring skin sensitization, and a compound that allow the sensitization of a test substance to be measured using a single reagent.

After conducting intensive research in order to achieve the foregoing object, the inventors have found that an organic compound having at least one thiol group derived from an amino acid and at least one amino group derived from a side chain of an amino acid can be used as a skin sensitization measuring reagent, which has led to the completion of the present invention. According to the present invention, the following inventions are provided.

In a first aspect the present invention relates to a skin sensitization measuring reagent including, as a main measuring agent, an organic compound having at least one thiol group derived from an amino acid and at least one amino group derived from a side chain of an amino acid, the organic compound having an absorption spectrum in an ultraviolet, visible, or near-infrared region and having a molar absorption coefficient of <NUM>/mol·cm or more and <NUM>,<NUM>/mol·cm or less at a maximal absorption wavelength;
wherein the organic compound is represented by the following formula (<NUM>):.

The reagent of the present invention is defined in claim <NUM>.

In general the organic compound has emission at <NUM> to <NUM>.

Also described is a skin sensitization measuring reagent wherein the amino group is an amino group derived from a side chain of lysine.

In typical embodiments W is a q-valent group having a naphthalene ring structure.

Preferably the organic compound has a partial structure represented by any of the following formulas (A) to (F):
<CHM>
<CHM>
<CHM>
wherein R<NUM> represents a hydrogen atom or an alkyl group having <NUM> to <NUM> carbon atoms, n represents an integer of <NUM> to <NUM>, and * represents a linking site.

Typical examples of the organic compound are represented by the following formula (<NUM>), (<NUM>), (<NUM>), (<NUM>), or (<NUM>):
<CHM>
wherein.

<<NUM>> The skin sensitization measuring reagent according to <<NUM>>, wherein
A<NUM> in formula (<NUM>) and A<NUM> in formula (<NUM>) are any of:
<CHM>.

A<NUM> in formula (<NUM>) and A<NUM> in formula (<NUM>) are any of:
<CHM>.

A<NUM> in formula (<NUM>) is:
<CHM>
wherein R<NUM> represents a hydrogen atom or an alkyl group having <NUM> to <NUM> carbon atoms, n represents an integer of <NUM> to <NUM>, and * represents a linking site.

In a second aspect the present invention relates to a method for measuring skin sensitization, including:.

Typically the test substance is at least one of a perfume, an essential oil, a polymer compound, a medicine, an agricultural chemical, food, a chemical product, or a plant extract made of a naturally derived component.

In one embodiment the method for measuring skin sensitization includes chromatographing the reaction product obtained by the step of reacting the skin sensitization measuring reagent with the test substance.

Preferably the method for measuring skin sensitization is a measurement using a fluorescence detector at an excitation wavelength of <NUM> to <NUM> and a fluorescence wavelength of <NUM> to <NUM>.

In another aspect the present invention relates to a compound represented by the following formula (<NUM>), (<NUM>), (<NUM>), (<NUM>), or (<NUM>):
<CHM>
wherein.

The compound of the present invention is defined in claim <NUM>.

Preferably the group having an absorption spectrum in the ultraviolet, visible, or near-infrared region is a group having a naphthalene ring structure.

Typically A<NUM> in formula (<NUM>) and A<NUM> in formula (<NUM>) are any of:
<CHM>.

According to the present invention, the skin sensitization of chemical substances can be quickly measured in a simple manner.

In the present specification, "to" is meant to include the values recited before and after "to" as the lower and upper limits.

In the present specification, "measurement of skin sensitization" is meant to include assay of skin sensitization and is also meant to include determination of the presence or absence of skin sensitization based on a certain criterion and quantitative measurement of skin sensitization.

Skin sensitization develops through a complicated process composed of a large number of stages. The first event is that a test substance permeates through the skin and covalently bonds to a protein in the skin. Thus, it is thought that, by evaluating this covalent bonding, it is possible to predict whether the target test substance is a skin sensitizer. The reaction of the test substance with the protein in the skin is known to be roughly due to five organic chemical reactions. It is known that the amino acids involved in these five reactions are the SH group of cysteine and the NH<NUM> group of lysine. Accordingly, in the measurement of skin sensitization described in Patent Documents <NUM> and <NUM>, two nucleophilic reagents having a naphthalene ring, which has a high molar absorption coefficient in the UV region, introduced into the N-termini of cysteine and lysine are each chemically synthesized, and these two nucleophilic reagents are reacted with a test substance. By quantifying the unreacted nucleophilic reagent, the reactivity with the test substance is calculated, and skin sensitization is predicted.

In the present invention, a nucleophilic reagent has been synthesized that includes a thiol group derived from an amino acid and an amino group derived from a side chain of an amino acid in the same molecule and that has introduced into the N-terminus or C-terminus thereof a structure having a high molar absorption coefficient in the UV region and having fluorescence (e.g., a naphthalene ring). It has been demonstrated that skin sensitization can be predicted by evaluating and quantifying the reaction of this nucleophilic reagent with a test substance.

A skin sensitization measuring reagent according to the present invention has a structure having UV absorption and fluorescence (e.g., a naphthalene ring) at a terminus of a peptide and is designed to allow high-sensitivity detection. Thus, the skin sensitization measuring reagent allows evaluation with a UV detector or fluorescence detector, which is inexpensive and easily available but is difficult to use in conventional evaluation methods using peptides including cysteine and lysine. According to the present invention, the amount of reagent and the work time can also be reduced as compared to conventional methods, thus leading to a significant efficiency improvement and cost reduction.

In addition, conventional methods using a peptide including a thiol group and an amino group in one molecule have low peptide detection sensitivity and do not allow optical detection; therefore, a detection method using an LC-MS, which is an expensive apparatus, as detection means is employed. In contrast, according to the present invention, evaluation can be performed only using an inexpensive detector using UV or fluorescence.

Furthermore, in test methods using a peptide combined with a chromophore such as one that emits fluorescence, the reactivity of an amino group of a side chain with a test substance is evaluated. However, artificial peptides in which an amino group of a side chain is bonded to the main chain has a problem in that, for example, biological reactions cannot be correctly evaluated because such peptides differ in the form of bonding from peptides present in a living body. On the other hand, the method for measuring skin sensitization according to the present invention uses a peptide that correctly reflects a peptide bond present in a living body and is therefore an evaluation method that more accurately reflects a chemical reaction (biological reaction) that occurs in a living body and thus allows skin sensitization to be correctly predicted.

The skin sensitization measuring reagent according to the present invention includes, as a main measuring agent, an organic compound having at least one thiol group derived from an amino acid and at least one amino group derived from a side chain of an amino acid. The organic compound has an absorption spectrum in the ultraviolet, visible, or near-infrared region and has a molar absorption coefficient of <NUM>/mol·cm or more and <NUM>,<NUM>/mol·cm or less at the maximal absorption wavelength.

The organic compound used in the present invention is a compound that has an absorption spectrum in the ultraviolet, visible, or near-infrared region and that exhibits absorption as-is or in solution form, preferably in a wavelength range of <NUM> to <NUM>,<NUM>, more preferably a compound that exhibits absorption in a wavelength range of <NUM> to <NUM>. Even more preferred is a compound having maximal absorption in the above wavelength range.

The organic compound used in the present invention is a compound that has a molar absorption coefficient of <NUM>/mol·cm or more and <NUM>,<NUM>/mol·cm or less at the maximal absorption wavelength, preferably a compound that has a molar absorption coefficient (L/mol·cm) of <NUM>/mol·cm or more and <NUM>,<NUM>/mol·cm or less at the maximal absorption wavelength. Particularly preferred is a compound that has maximal absorption in a wavelength range of <NUM> to <NUM> and that has a molar absorption coefficient of <NUM>/mol·cm or more and <NUM>,<NUM>/mol·cm or less at the maximal absorption wavelength.

The molar absorption coefficient (ε) is given by the following equation:<MAT> where D represents the absorbance of the solution, c represents the molar concentration (mol/L) of the solute, and d represents the thickness (cm) of the solution layer (optical path length). The molar absorption coefficient can be determined by measuring the absorption spectrum or absorbance using a commercially available spectrophotometer.

The organic compound used in the present invention is preferably a compound having emission at <NUM> to <NUM>, more preferably a compound having emission at <NUM> to <NUM>, even more preferably a compound having emission at <NUM> to <NUM>.

The organic compound used in the present invention has at least one thiol group derived from an amino acid and at least one amino group derived from a side chain of an amino acid. Preferably, the organic compound used in the present invention includes at least an amino acid residue having a thiol group and an amino acid residue having an amino group in a side chain thereof. The amino acid residue having a thiol group may be, for example, a cysteine residue. The amino acid residue having an amino group in a side chain thereof may be, for example, a lysine residue. That is, the thiol group derived from an amino acid is preferably a thiol group derived from cysteine, and the amino group derived from a side chain of an amino acid is preferably an amino group derived from a side chain of lysine.

As described above, the organic compound used in the present invention is a compound including an amino acid residue, and is a compound represented by the following formula (<NUM>):.

W represents a q-valent group having an absorption spectrum in the ultraviolet, visible, or near-infrared region.

Preferably, W is a q-valent group derived from an organic compound having an absorption spectrum in the ultraviolet, visible, or near-infrared region.

The organic compound having an absorption spectrum in the ultraviolet, visible, or near-infrared region is a compound having absorption in the range from <NUM> to <NUM>,<NUM>. Examples of such compounds include naphthalene derivatives, anthracene derivatives, phenanthrene derivatives, tetracene derivatives, pentacene derivatives, benzopyrene derivatives, chrysene derivatives, pyrene derivatives, triphenylene derivatives, corannulene derivatives, coronene derivatives, ovalene derivatives, acridine derivatives, luciferin derivatives, pyranine derivatives, stilbene derivatives, benzofuran derivatives, dihydroquinoxalinone derivatives, phthalimidinyl derivatives, dansyl derivatives, merocyanine derivatives, perylene derivatives, rhodamine derivatives, coumarin derivatives, <NUM>-(dicyanomethylene)-<NUM>-methyl-<NUM>-(<NUM>-dimethylaminostyryl)-<NUM>-pyran (DCM) derivatives, pyrromethene derivatives, fluorescein derivatives, umbelliferone derivatives, benzothiazole derivatives, benzoxadiazole derivatives, shikonin derivatives, fluoranthene derivatives, carbazole derivatives, tetraphene derivatives, acenaphthene derivatives, and fluorene derivatives. Specific examples include compounds derived from <NUM>-naphthylacetyl chloride, <NUM>-(<NUM>,<NUM>-dimethoxy-N-phthalimidinyl)benzenesulfonic acid chloride (DPS-CL), <NUM>-chloro-<NUM>-nitro-<NUM>,<NUM>,<NUM>-benzoxadiazole (NBD-CL), fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), <NUM>-fluoro-<NUM>-nitro-<NUM>,<NUM>,<NUM>-benzoxadiazole (NDB-F), <NUM>-(N,N-dimethylaminosulfonyl)-<NUM>-fluoro-<NUM>,<NUM>,<NUM>-benzoxadiazole (DBD-F), <NUM>-(N-phthalimidinyl)benzenesulfonic acid chloride (PHISYL-CL), <NUM>-aminosulfonyl-<NUM>-fluoro-<NUM>,<NUM>,<NUM>-benzoxadiazole (ABD-F), N-[<NUM>-(<NUM>-dimethylamino-<NUM>-benzofuranyl)phenyl]maleimide (DBPM), <NUM>-(<NUM>-maleimidophenyl)-<NUM>-methylbenzothiazole (MBPM), N-(<NUM>-acridinyl)maleimide (NAM), <NUM>-chloro-<NUM>-sulfobenzofurazane ammonium salt (SBD-CL), <NUM>-fluorobenzofurazane-<NUM>-sulfonic acid ammonium salt (SBD-F), <NUM>,<NUM>-diamino-<NUM>,<NUM>-dimethoxybenzene (DDB), <NUM>-(N,N-dimethylaminosulfonyl)-<NUM>-hydrazino-<NUM>,<NUM>,<NUM>-benzoxadiazole (DBD-H), <NUM>-hydrazino-<NUM>-nitro-<NUM>,<NUM>,<NUM>-benzoxadiazole hydrazine (DBD-H), <NUM>,<NUM>'-dithiodi(<NUM>-naphthylamine) (DTAN), <NUM>-amino-<NUM>-penten-<NUM>-one (FLUORAL-P), <NUM>,<NUM>-amino-<NUM>,<NUM>-methylenedioxybenzene (MDB), <NUM>-(<NUM>,<NUM>-dimethoxybenzothiazol-<NUM>-yl)benzoic acid hydrazide (BHBT), <NUM>-(N,N-dimethylaminosulfonyl)-<NUM>-(N-hydrazinocarbonylmethyl-N-methyl)amino-<NUM>,<NUM>,<NUM>-benzoxadiazole (DBD-CO-HZ), <NUM>-(N-hydrazinocarbonylmethyl-N-methylamino)-<NUM>-nitro-<NUM>,<NUM>,<NUM>-benzoxadiazole (NBD-CO-HZ), <NUM>-bromomethyl-<NUM>,<NUM>-dimethoxy-<NUM>-methyl-<NUM>,<NUM>-dihydroquinoxalin-<NUM>-one (BR-DMEQ), <NUM>-bromomethyl-<NUM>-methoxycoumarin (BR-MMC), <NUM>-(N,N-dimethylaminosulfonyl)-<NUM>-piperazino-<NUM>,<NUM>,<NUM>-benzoxadiazole (DBD-PZ), <NUM>-nitro-<NUM>-piperazino-<NUM>,<NUM>,<NUM>-benzoxadiazole (NBD-PZ), <NUM>-(N,N-dimethylaminosulfonyl)-<NUM>-(<NUM>-aminoethylamino)-<NUM>,<NUM>,<NUM>-benzoxadiazole (DBD-ED), <NUM>-chlorocarbonyl-<NUM>,<NUM>-dimethoxy-<NUM>-methyl-<NUM>(<NUM>)-quinoxalinone (DMEQ-COCL), and <NUM>-(<NUM>-chlorocarbonyl-<NUM>-oxazolyl)-<NUM>,<NUM>-methylenedioxybenzofuran (OMB-COCL).

Of these, W is preferably a q-valent group having a naphthalene ring structure.

If W is a q-valent group having a naphthalene ring structure, the organic compound preferably has a partial structure represented by any of the following formulas (A) to (F):
<CHM>
<CHM>
<CHM>
where R<NUM> represents a hydrogen atom or an alkyl group having <NUM> to <NUM> carbon atoms, n represents an integer of <NUM> to <NUM>, and * represents a linking site.

L<NUM> and L<NUM> each independently represent a single bond or a linking group.

The linking group represented by L<NUM> and L<NUM> may be an alkylene group having <NUM> to <NUM> carbon atoms, -NH-, -CO-, -O-, -COO-, -OCO-, or any combination thereof. Particularly preferred examples of linking groups represented by L<NUM> and L<NUM> include -CH<NUM>-NH- and -NH-CH<NUM>-.

Xaa represents an amino acid residue, provided that p × q Xaa moieties include at least one lysine residue and at least one cysteine residue.

The amino acid of the amino acid residue represented by Xaa is not particularly limited as long as the amino acid is a compound having an amino group and a carboxyl group. The amino acid may be an α-amino acid, a β-amino acid, or a γ-amino acid. The α-amino acid may be, for example, a naturally occurring amino acid that forms a protein, a naturally occurring amino acid that does not form a protein, or a non-naturally-occurring amino acid. That is, the amino acid residue represented by Xaa may be an α-amino acid residue, a β-amino acid residue, or a γ-amino acid residue.

The α-amino acid is preferably an amino acid that forms a protein. Specific examples include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine.

The β-amino acid may be, for example, β-alanine.

The γ-amino acid may be, for example, γ-aminobutyric acid.

Z represents a hydrogen atom or an alkyl group having <NUM> to <NUM> carbon atoms. The alkyl group having <NUM> to <NUM> carbon atoms is optionally substituted with a substituent selected from the group consisting of a carboxyl group, an amino group, a hydroxy group, a guanidyl group, a pyridine ring, and an imidazole ring.

p represents an integer of <NUM> to <NUM>. Preferably, p represents an integer of <NUM> to <NUM>.

q represents an integer of <NUM> to <NUM>. Preferably, q represents <NUM> or <NUM>.

However, p and q are not simultaneously <NUM>.

If p is <NUM> or more, L<NUM>-Xaa-L <NUM> moieties may be the same or different. If q is <NUM> or more, [(L<NUM>-Xaa-L<NUM>)p-Z] moieties may be the same or different.

Particularly preferably, the organic compound used in the present invention is a compound represented by the following formula (<NUM>), (<NUM>), (<NUM>), (<NUM>), or (<NUM>). According to the present invention, there is provided a compound represented by the following formula (<NUM>), (<NUM>), (<NUM>), (<NUM>), or (<NUM>):
<CHM>
where.

The groups having an absorption spectrum in the ultraviolet, visible, or near-infrared region in formulas (<NUM>) to (<NUM>) are preferably groups having a naphthalene ring structure.

A<NUM> in formula (<NUM>) and A<NUM> in formula (<NUM>) are preferably any of:
<CHM>.

A<NUM> in formula (<NUM>) is preferably:
<CHM>.

In the above formulas, R<NUM> represents a hydrogen atom or an alkyl group having <NUM> to <NUM> carbon atoms, n represents an integer of <NUM> to <NUM>, and * represents a linking site.

In formula (<NUM>), R<NUM> to R<NUM> represent a hydrogen atom or a substituent.

In formula (<NUM>), R<NUM> to R<NUM> represent a hydrogen atom or a substituent,.

The substituents represented by R<NUM> to R<NUM>, R<NUM> to R<NUM>, R<NUM> to R<NUM>, R<NUM> to R<NUM>, and R<NUM> to R<NUM> may be, but is not limited to, an alkyl group having <NUM> to <NUM> carbon atoms, an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with a hydroxy group, an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with a carboxyl group, an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with -CONH<NUM>, an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with an amino group (-NH<NUM>), an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with a thiol group (-SH), an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with -S-CH<NUM>, or an alkyl group having <NUM> to <NUM> carbon atoms that is substituted with any of:
<CHM>.

If any of: R<NUM>, R<NUM>, R<NUM>, R<NUM> R<NUM> R<NUM>, R<NUM>, R<NUM>, R<NUM> R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM> R<NUM> R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM>, R<NUM>, and R<NUM> represents an alkyl group having <NUM> to <NUM> carbon atoms, the alkyl group may be attached to the adjacent N atom to form a ring.

Preferably, R<NUM> and R<NUM> are hydrogen atoms.

Preferably, one of R<NUM> and R<NUM> is a hydrogen atom, and the other is the substituent described above.

Preferably, R<NUM>, R<NUM>, and R<NUM> are hydrogen atoms.

The α-amino acid residues, the β-amino acid residues, and the γ-amino acid residues in formulas (<NUM>) to (<NUM>) are as described above for the amino acid residue represented by Xaa.

The peptide residues in formulas (<NUM>) to (<NUM>) may be those composed of two or more amino residues (where the amino acid residues may be an α-amino acid residue, a β-amino acid residue, or a γ-amino acid residue).

In the present specification, examples of alkyl groups having <NUM> to <NUM> carbon atoms include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, and cyclopropyl groups.

The organic compound used in the present invention can be manufactured by a known peptide synthesis method. Specifically, the organic compound can be manufactured in accordance with the method described later in the Examples section. That is, the organic compound used in the present invention can be synthesized by solid-phase peptide synthesis using a commercially available automated peptide synthesizer.

Synthesis can be performed by setting to the synthesizer a resin for solid-phase synthesis, N-methyl-<NUM>-pyrrolidone (NMP) solutions of Fmoc-amino acids, an NMP solution of ethyl cyano-hydroxyimino-acetate, an NMP solution of diisopropylethylamine, an NMP solution of diisopropylcarbodiimide, an NMP solution of piperidine, and an NMP solution of acetic anhydride. One cycle includes Fmoc deprotection, washing with NMP, Fmoc-amino acid condensation, and washing with NMP. By repeating this cycle, a peptide chain can be extended.

The skin sensitization measuring reagent according to the present invention may be composed only of the organic compound or may include one or more additives in addition to the organic compound serving as the main measuring agent. Examples of additives include pH adjusters and stabilizers. The skin sensitization measuring reagent according to the present invention may be prepared by dissolving the main measuring agent and optionally the additives in water, an aqueous buffer solution, an organic solvent, or a mixture thereof.

The skin sensitization measuring reagent according to the present invention may be provided in solution form, in liquid form, or in solid form (e.g., powders, granules, freeze-dried products, or tablets).

A method for measuring skin sensitization according to the present invention includes:.

For example, the skin sensitization measuring reagent according to the present invention may be used in a form in which the skin sensitization measuring reagent is dissolved in an aqueous buffer solution such as a phosphate buffer solution or an organic solvent such as dimethyl sulfoxide (DMSO) and is optionally further diluted with an aqueous buffer solution such as a phosphate buffer solution or another organic solvent, for example, at an organic compound concentration of about <NUM>µmol/L to about <NUM> mol/L, typically about <NUM>µmol/L to about <NUM>µmol/L.

Although the type of test substance is not particularly limited, the test substance is, for example, at least one of a perfume, an essential oil, a polymer compound, a medicine, an agricultural chemical, food, a chemical product, or a plant extract made of a naturally derived component. For example, the test substance may be dissolved in an organic solvent such as methanol, ethanol, acetonitrile, acetone, or a mixture thereof, for example, at a concentration of about <NUM>µmol/L to about <NUM> mol/L, typically about <NUM> mmol/L to about <NUM> mmol/L.

The organic compound serving as the main measuring agent of the skin sensitization measuring reagent according to the present invention may be mixed and reacted with a test substance solution such that the ratio of the molar concentration of the organic compound to the molar concentration of the test substance is, for example, <NUM>:<NUM> to <NUM>:<NUM>. The reaction can be performed by stirring a solution including the organic compound and the test substance or allowing the solution to stand, typically for about <NUM> minute to about <NUM> days, while maintaining the solution in the temperature range of, for example, about <NUM> to about <NUM>.

The skin sensitization of the test substance can be measured by examining the reactivity of the organic compound with the test substance by the above reaction. To examine the reactivity, the mixture of the skin sensitization measuring reagent solution and the test substance solution may be analyzed for the amount of residual organic compound and/or the amount of the reaction product of the organic compound with the test substance. This analysis is performed over time to determine the reaction rate constant of the organic compound for the test substance. The skin sensitization of the test substance can be evaluated by comparing the reaction rate constants of different test substances or by comparing the reaction rate constant of the test substance with the reaction rate constant determined for a compound that has been examined for the presence or absence or strength of skin sensitization by animal testing.

If the skin sensitization measuring reagent can undergo any change in the reaction solution during the analysis of the amount of residual organic compound, a reaction solution from which only the test substance is excluded (control group) may optionally be separately prepared and analyzed, and correction may be made based on the amount of residual organic compound in this reaction solution.

The method according to the present invention may include chromatographing the reaction product obtained by the step of reacting the skin sensitization measuring reagent with the test substance. That is, although the method for analyzing the compound and the compound produced by the reaction is not particularly limited, the compound produced by the reaction, the organic compound, and the test substance can be separated and analyzed by a technique such as high-performance liquid chromatography (HPLC), gas chromatography (GC), or thin layer chromatography (TLC).

Examples of chromatography techniques that can be used for HPLC, GC, or TLC include reversed-phase chromatography, normal-phase chromatography, and ion-exchange chromatography. Examples of commercially available columns and TLC plate that can be used for such chromatography techniques include LC columns such as CAPCELL-PAK (manufactured by Shiseido Co. ), L-column ODS (manufactured by Chemicals Evaluation and Research Institute, Japan), and Shodex Asahipak (manufactured by Showa Denko K. ); and TLC plates such as Silica Gel <NUM> F254 (manufactured by Merck) and Silica Gel Plate (manufactured by Nacalai Tesque, Inc.

Although the method for detecting the compound produced by the reaction or the residual organic compound is not particularly limited, examples of detectors that can be used for the HPLC analysis include ultraviolet-visible detectors, near-infrared detectors, fluorescence detectors, refractive index detectors, conductivity detectors, and evaporative light scattering detectors. Examples of ultraviolet-visible detectors include single-wavelength ultraviolet-visible detectors, dual-wavelength ultraviolet-visible detectors, and photodiode array detectors. Examples of commercially available detectors that can be used for such detection methods include ultraviolet-visible detectors, refractive index detectors, and conductivity detectors such as those manufactured by Shimadzu Corporation, Hitachi, Ltd. , Waters Corporation, and Shiseido Co. and evaporative light scattering detectors such as those manufactured by Shimadzu Corporation.

In the present invention, the optical measurement is preferably performed using a fluorescence detector, more preferably at an excitation wavelength of <NUM> to <NUM> and a fluorescence wavelength of <NUM> to <NUM>.

The detection in the method for measurement using the skin sensitization measuring reagent according to the present invention is not limited to the above, but may be performed by, for example, detecting ions with a particular mass based on molecular weight in accordance with the method described in <CIT> or <CIT>.

In the method using the skin sensitization measuring reagent according to the present invention, an optical detection method can preferably be used. Preferably, an ultraviolet-visible detector or a near-infrared detector as mentioned above may be used.

The present invention will now be specifically described with reference to the following examples, although these examples are not intended to limit the present invention.

To <NUM> of distilled water (manufactured by FUJIFILM Wako Pure Chemical Corporation), <NUM> of TFA (manufactured by FUJIFILM Wako Pure Chemical Corporation, Special Grade) is added.

To <NUM> of distilled water (manufactured by FUJIFILM Wako Pure Chemical Corporation), <NUM> of TFA is added.

To <NUM> of HPLC-grade acetonitrile (manufactured by FUJIFILM Wako Pure Chemical Corporation, for HPLC), <NUM> of TFA is added.

The same stock solution is used for one test. The stock solution is stored in portions that can be used up for each test. A specific example of preparation is given below:.

One solvent with which a <NUM> mmol/L test substance solution can be prepared is selected according to the following order of priority: water, acetonitrile, acetone, and a <NUM>% solution of DMSO in acetonitrile. If water, acetonitrile, or acetone is selected, a <NUM> mmol/L test substance solution is first prepared. A suitable amount of the test substance is weighed and completely dissolved by adding the solvent to prepare a <NUM> mmol/L solution. A portion of the <NUM> mmol/L solution is then collected and diluted <NUM>-fold with the same solvent to prepare a <NUM> mmol/L test substance solution. If a <NUM>% by mass solution of DMSO in acetonitrile is selected, a <NUM> mmol/L test substance solution is prepared in the same manner as above. A portion of the solution is then collected and diluted <NUM>-fold with acetonitrile to prepare a <NUM> mmol/L test substance solution.

Test substance solutions are prepared on a <NUM>-well plate (U96 PP-<NUM> NATURAL, Thermo (NUNC)), mainly using a <NUM>-channel pipette, and the reagent is added in the following amount:.

The plate is firmly sealed with a plate seal (resistant embossed seal (TORAST™ <NUM>-well Seal E Type (TORAST is a registered trademark)), Shimadzu GLC Ltd. ) and is shaken on a plate shaker (Titramax <NUM>, Heidolph Instruments). After being spun down in a centrifuge, the reaction solutions are incubated at <NUM> in a light-shielded state for <NUM> hours.

After incubation for <NUM> hours, the plate seal is removed, and <NUM>µL of the reaction stop solution is added to each sample to stop the reaction.

The HPLC measurement conditions for the nucleophilic reagents are given below. As the elution conditions, condition <NUM>, <NUM> or <NUM> was selected depending on the nucleophilic reagent.

The depletion of the nucleophilic reagent is calculated from the mean peak area of the nucleophilic reagent by the following equation: <MAT>.

The reaction solution as prepared (<NUM> hours) and the reaction solution incubated at <NUM> for <NUM> hours (<NUM> hours) are each subjected to measurement by HPLC-UV. Because the nucleophilic reagent and oxidized nucleophilic reagent can be identified on the HPLC, the residual fraction of the nucleophilic reagent is calculated based on the following equation: <MAT>.

In fluorescence detection, the peak area detected with excitation light (<NUM>) and fluorescence (<NUM>) is determined. This value is compared with those of NAC and NAL described in Patent Documents <NUM> and <NUM>.

Ten substances shown in the following table were used for reactivity evaluation. For primary evaluation, the upper three substances were used. The substances selected for the primary evaluation were a substance that mainly reacts with cysteine, a substance that mainly reacts with lysine, and a substance that does not react. For secondary evaluation, the lower seven substances were used. The substances selected for the secondary evaluation were substances that are difficult to distinguish between sensitizers and non-sensitizers by analysis using NAC and NAL. As above, the substances selected for the secondary evaluation were five substances that mainly react with cysteine and two substances that mainly react with lysine.

The reactivity was compared with the depletions of NAC and NAL or the mean depletion (mean % depletion).

The synthesis route and yield of the nucleophilic reagent according to the present invention are shown.

Solid-phase peptide synthesis was performed using an automated peptide synthesizer (Syro I manufactured by Biotage). Synthesis was performed by setting to the synthesizer a resin for solid-phase synthesis, N-methyl-<NUM>-pyrrolidone (NMP) solutions of Fmoc-amino acids (<NUM> mol/L), an NMP solution of ethyl cyano-hydroxyimino-acetate (<NUM> mol/L), an NMP solution of diisopropylethylamine (<NUM> mol/L), an NMP solution of diisopropylcarbodiimide (<NUM> mol/L), an NMP solution of piperidine (<NUM>% v/v), and an NMP solution of acetic anhydride (<NUM>% v/v).

One cycle included Fmoc deprotection (<NUM> minutes), washing with NMP, Fmoc-amino acid condensation (<NUM> hour), and washing with NMP. By repeating this cycle, a peptide chain was extended.

The resulting crude product was purified by liquid chromatography.

The Fmoc-amino acids were obtained from Watanabe Chemical Industries, Ltd.

N-Methyl-<NUM>-pyrrolidone, diisopropylethylamine, diisopropylcarbodiimide, piperidine, and acetic anhydride were obtained from FUJIFILM Wako Pure Chemical Corporation.

Ethyl cyano-hydroxyimino-acetate was obtained from Tokyo Chemical Industry Co.

Solid-phase peptide synthesis was performed using <NUM>-Fmoc hydrazinebenzoyl AM NovaGel (manufactured by Novabiochem) (<NUM> mmol/g, <NUM>) as a resin for solid-phase synthesis.

(S)-<NUM>-((((<NUM>-Fluoren-<NUM>-yl)methoxy)carbonyl)amino)-N-ε-(tert-butoxycarbonyl)-L-lysine (Fmoc-Lys(Boc)-OH), (S)-<NUM>-((((<NUM>-fluoren-<NUM>-yl)methoxy)carbonyl)amino)-S-trityl-L-cysteine (Fmoc-Cys(Trt)-OH), and acetic anhydride were condensed in that order. After completion of extension, the resin was washed with dichloromethane (manufactured by FUJIFILM Wako Pure Chemical Corporation), and <NUM>µL of dimethylformamide (DMF) (manufactured by FUJIFILM Wako Pure Chemical Corporation), <NUM> of copper(II) acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation), <NUM>µL of pyridine (manufactured by FUJIFILM Wako Pure Chemical Corporation), and <NUM>µL of <NUM>-naphthylmethylamine (manufactured by Alfa Aesar) were added, followed by shaking for <NUM> hours. The resin was filtered out and washed twice with <NUM> of ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation). To the filtrate was added <NUM> of <NUM> mol/L hydrochloric acid (manufactured by FUJIFILM Wako Pure Chemical Corporation). After thorough mixing, the mixture was allowed to stand, and the ethyl acetate layer was recovered. The solvent was then removed by distillation under reduced pressure. A solid was formed by adding <NUM> of trifluoroacetic acid (TFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation):triisopropylsilane (manufactured by Tokyo Chemical Industry Co. ):water (= <NUM>:<NUM>:<NUM>) and adding <NUM> of n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation)):methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM>). After the solid was settled by centrifugation, the supernatant was removed. After the solid was washed with methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation), the solvent was removed by distillation under reduced pressure. After the resulting residue was purified by liquid chromatography, the solvent was removed by distillation under reduced pressure, followed by freeze drying to yield <NUM> of a white solid.

<NUM>-NMR (DMSO-d6) δ: <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (H<NUM>O), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m).

Compounds <NUM> to <NUM> were synthesized in the same manner as Compound <NUM>. The reagents used for solid-phase peptide synthesis were changed as in the following table.

Succinic anhydride and glycolic acid were obtained from FUJIFILM Wako Pure Chemical Corporation.

Solid-phase peptide synthesis was performed using Rink Amide-ChemMatrix (manufactured by Biotage) (<NUM> mmol/g, <NUM>) as a resin for solid-phase synthesis.

(S)-<NUM>-((((<NUM>-Fluoren-<NUM>-yl)methoxy)carbonyl)amino)-N-ε-(tert-butoxycarbonyl)-L-lysine (Fmoc-Lys(Boc)-OH), (S)-<NUM>-((((<NUM>-fluoren-<NUM>-yl)methoxy)carbonyl)amino)-S-trityl-L-cysteine (Fmoc-Cys(Trt)-OH), (S)-<NUM>-((((<NUM>-fluoren-<NUM>-yl)methoxy)carbonyl)amino)-O-tert-butyl-L-tyrosine (Fmoc-Tyr(OtBu)-OH), and <NUM>-naphthylacetic acid (manufactured by FUJIFILM Wako Pure Chemical Corporation) were condensed in that order. After completion of extension, the resin was washed with dichloromethane (manufactured by FUJIFILM Wako Pure Chemical Corporation), and the solvent was then then removed by distillation under reduced pressure. By adding <NUM> of trifluoroacetic acid (TFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation):triisopropylsilane (manufactured by Tokyo Chemical Industry Co. ):water (= <NUM>:<NUM>:<NUM>), the peptide was cleaved from the resin while deprotection was simultaneously performed. After <NUM> hours, the resin was filtered out, and <NUM> of n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM>) was added to the filtrate to form a solid. After the solid was settled by centrifugation, the supernatant was removed. After the solid was washed with methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation), the solvent was removed by distillation under reduced pressure. After the resulting residue was purified by liquid chromatography, the solvent was removed by distillation under reduced pressure, followed by freeze drying to yield <NUM> of a white solid.

<NUM>-NMR (DMSO-d6) δ: <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (H<NUM>O), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m).

Solid-phase peptide synthesis was performed using N-ε-(t-butoxycarbonyl)-L-lysine <NUM>-chlorotrityl resin (manufactured by Watanabe Chemical Industries, Ltd. (<NUM> mmol/g, <NUM>) as a resin for solid-phase synthesis.

(S)-<NUM>-((((<NUM>-Fluoren-<NUM>-yl)methoxy)carbonyl)amino)-S-trityl-L-cysteine (Fmoc-Cys(Trt)-OH), (S)-<NUM>-((((<NUM>-fluoren-<NUM>-yl)methoxy)carbonyl)amino)-O-tert-butyl-L-tyrosine (Fmoc-Tyr(OtBu)-OH), and <NUM>-naphthylacetic acid (manufactured by FUJIFILM Wako Pure Chemical Corporation) were condensed in that order. After completion of extension, the resin was washed with dichloromethane (manufactured by FUJIFILM Wako Pure Chemical Corporation), and the solvent was then removed by distillation under reduced pressure. By adding <NUM> of trifluoroacetic acid (TFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation):triisopropylsilane (manufactured by Tokyo Chemical Industry Co. ):water (= <NUM>:<NUM>:<NUM>), the peptide was cleaved from the resin while deprotection was simultaneously performed. After <NUM> hours, the resin was filtered out, and <NUM> of n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM>) was added to the filtrate to form a solid. After the solid was settled by centrifugation, the supernatant was removed. After the solid was washed with methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation), the solvent was removed by distillation under reduced pressure. After the resulting residue was purified by liquid chromatography, the solvent was removed by distillation under reduced pressure, followed by freeze drying to yield <NUM> of a white solid.

<NUM>-NMR (DMSO-d6) δ: <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM> (<NUM>, d, J = <NUM>), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (H<NUM>O), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m).

Solid-phase peptide synthesis was performed using S-Trityl-L-cysteine <NUM>-chlorotrityl resin (manufactured by Watanabe Chemical Industries, Ltd. (<NUM> mmol/g, <NUM>) as a resin for solid-phase synthesis.

(S)-<NUM>-((((<NUM>-Fluoren-<NUM>-yl)methoxy)carbonyl)amino)-N-ε-(tert-butoxycarbonyl)-L-lysine (Fmoc-Lys(Boc)-OH), (S)-<NUM>-((((<NUM>-fluoren-<NUM>-yl)methoxy)carbonyl)amino)-O-tert-butyl-L-tyrosine (Fmoc-Tyr(OtBu)-OH), and <NUM>-naphthylacetic acid (manufactured by FUJIFILM Wako Pure Chemical Corporation) were condensed in that order. After completion of extension, the resin was washed with dichloromethane (manufactured by FUJIFILM Wako Pure Chemical Corporation), and the solvent was then removed by distillation under reduced pressure. By adding <NUM> of trifluoroacetic acid (TFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation):triisopropylsilane (manufactured by Tokyo Chemical Industry Co. ):water (= <NUM>:<NUM>:<NUM>), the peptide was cleaved from the resin while deprotection was simultaneously performed. After <NUM> hours, the resin was filtered out, and <NUM> of n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM>) was added to the filtrate to form a solid. After the solid was settled by centrifugation, the supernatant was removed. After the solid was washed with methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation), the solvent was removed by distillation under reduced pressure. After the resulting residue was purified by liquid chromatography, the solvent was removed by distillation under reduced pressure, followed by freeze drying to yield <NUM> of a white solid.

<NUM>-NMR (DMSO-d6) δ: <NUM>-<NUM> (<NUM>, m), <NUM> (<NUM>, t), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM> (<NUM>, s), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (H<NUM>O), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m).

Into a recovery flask were placed <NUM> of <NUM>,<NUM>-naphthalenediacetic acid (manufactured by A1 Biochem Lab), <NUM> of N-hydroxysuccinimide (manufactured by Tokyo Chemical Industry Co. ), and <NUM> of dimethylformamide (manufactured by FUJIFILM Wako Pure Chemical Corporation). Next, <NUM> of <NUM>-(<NUM>-dimethylaminopropyl)-<NUM>-ethylcarbodiimide hydrochloride (manufactured by Dojindo Laboratories) was added, followed by stirring for <NUM> hours. After completion of the reaction, water was added to the reaction solution, and extraction was performed with ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation). After the organic layer was washed with water and saturated saline solution, the organic layer was dried over anhydrous sodium sulfate (manufactured by FUJIFILM Wako Pure Chemical Corporation). The anhydrous sodium sulfate was filtered out, and the filtrate was subjected to vacuum distillation. The residue was then purified by silica gel chromatography (n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM> to <NUM>:<NUM>)) to yield <NUM> of bis(<NUM>,<NUM>-dioxopyrrolidin-<NUM>-yl)<NUM>,<NUM>'-(naphthalene-<NUM>,<NUM>-diyl) diacetate as a white solid.

Next, <NUM> of bis(<NUM>,<NUM>-dioxopyrrolidin-<NUM>-yl)<NUM>,<NUM>'-(naphthalene-<NUM>,<NUM>-diyl) diacetate, <NUM> of S-trityl-L-cysteine (manufactured by Tokyo Chemical Industry Co. ), and <NUM> of dimethylformamide (manufactured by FUJIFILM Wako Pure Chemical Corporation) were placed into a recovery flask, followed by stirring in an oil bath at <NUM> for <NUM> hours. The reaction solution was then cooled to room temperature, and <NUM> of t-butyl-(2R)-<NUM>-amino-<NUM>(t-butoxycarbonylamino)hexanoate (manufactured by Combi-Blocks, Inc. ) was added, followed by stirring for <NUM> hour. After completion of the reaction, water was added to the reaction solution, and extraction was performed with ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation). After the organic layer was washed with water and saturated saline solution, the organic layer was dried over anhydrous sodium sulfate (manufactured by FUJIFILM Wako Pure Chemical Corporation). The anhydrous sodium sulfate was filtered out, and the filtrate was subjected to vacuum distillation. Next, <NUM> of trifluoroacetic acid (TFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation):triisopropylsilane (manufactured by Tokyo Chemical Industry Co. ):water (= <NUM>:<NUM>:<NUM>) was added. After <NUM> hours, <NUM> of n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM>) was added to form a solid. After the solid was settled by centrifugation, the supernatant was removed. After the solid was washed with methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation), the solvent was removed by distillation under reduced pressure. After the resulting residue was purified by liquid chromatography, the solvent was removed by distillation under reduced pressure, followed by freeze drying to yield <NUM> of a white solid.

Into a recovery flask were placed <NUM> of <NUM>,<NUM>-naphthalendiacetic acid synthesized by a technique described in literature (Rikagaku Kenkyusho Iho, <NUM>, vol. <NUM>, <NUM>), <NUM> of N-hydroxysuccinimide (manufactured by Tokyo Chemical Industry Co. ), and <NUM> of dimethylformamide (manufactured by FUJIFILM Wako Pure Chemical Corporation). Next, <NUM> of <NUM>-(<NUM>-dimethylaminopropyl)-<NUM>-ethylcarbodiimide hydrochloride (manufactured by Dojindo Laboratories) was added, followed by stirring for <NUM> hours. After completion of the reaction, water was added to the reaction solution, and extraction was performed with ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation). After the organic layer was washed with water and saturated saline solution, the organic layer was dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered out, and the filtrate was subjected to vacuum distillation. The residue was then purified by silica gel chromatography (n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM> to <NUM>:<NUM>)) to yield <NUM> of a white solid.

Next, <NUM> of bis(<NUM>,<NUM>-dioxopyrrolidin-<NUM>-yl)<NUM>,<NUM>'-(naphthalene-<NUM>,<NUM>-diyl) diacetate, <NUM> of S-trityl-L-cysteine (manufactured by Tokyo Chemical Industry Co. ), and <NUM> of dimethylformamide (manufactured by FUJIFILM Wako Pure Chemical Corporation) were placed into a recovery flask, followed by stirring in an oil bath at <NUM> for <NUM> hours. The reaction solution was then cooled to room temperature, and <NUM> of t-butyl-(2R)-<NUM>-amino-<NUM>(t-butoxycarbonylamino)-hexanoate (manufactured by Combi-Blocks, Inc. ) was added, followed by stirring for <NUM> hour. After completion of the reaction, water was added to the reaction solution, and extraction was performed with ethyl acetate (manufactured by FUJIFILM Wako Pure Chemical Corporation). After the organic layer was washed with water and saturated saline solution, the organic layer was dried over anhydrous sodium sulfate (manufactured by FUJIFILM Wako Pure Chemical Corporation). The anhydrous sodium sulfate was filtered out, and the filtrate was subjected to vacuum distillation. Next, <NUM> of trifluoroacetic acid (TFA) (manufactured by FUJIFILM Wako Pure Chemical Corporation):triisopropylsilane (manufactured by Tokyo Chemical Industry Co. ):water (= <NUM>:<NUM>:<NUM>) was added. After <NUM> hours, <NUM> of n-hexane (manufactured by FUJIFILM Wako Pure Chemical Corporation):methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation) (= <NUM>:<NUM>) was added to form a solid. After the solid was settled by centrifugation, the supernatant was removed. After the solid was washed with methyl t-butyl ether (manufactured by FUJIFILM Wako Pure Chemical Corporation), the solvent was removed by distillation under reduced pressure. After the resulting residue was purified by liquid chromatography, the solvent was removed by distillation under reduced pressure, followed by freeze drying to yield <NUM> of a white solid.

<NUM>-NMR (DMSO-d6) δ: <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, br), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (H<NUM>O), <NUM>-<NUM> (<NUM>, m), <NUM>-<NUM> (<NUM>, m).

The structures of Compounds <NUM> to <NUM> are shown below. <CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

The mass spectrum (MS) was measured using an ACQUITY SQD LC/MS System (Waters Corporation, method for ionization: electrospray ionization (ESI)).

The retention time (RT) was measured using the ACQUITY SQD LC/MS System (Waters Corporation) and was expressed in minutes (min).

Fourteen nucleophilic reagents with a naphthylmethyl group at the N-terminus thereof (Compounds <NUM> to <NUM>) were evaluated.

Using the sensitizers shown in "(<NUM>-<NUM>) Evaluation of Reactivity with Sensitizers" above, <NUM> mmol/L solutions were prepared and used for testing.

The nucleophilic reagent depletion (%) was determined under the HPLC measurement conditions shown in "(<NUM>) HPLC Measurement" above.

The residual fractions of the <NUM> nucleophilic reagents as prepared (<NUM> hours) and after <NUM> hours were calculated. The results are shown in <FIG>. The residual fractions of all <NUM> nucleophilic reagents were <NUM>% or more after <NUM> hours. In particular, the residual fractions of all nucleophilic reagents except Compounds <NUM> and <NUM> were <NUM>% or more after <NUM> hours. There was no nucleophilic reagent whose residual fraction decreased considerably after <NUM> hours.

The reactivity of the <NUM> nucleophilic reagents with three evaluation substances was calculated. The results are shown in <FIG>. Trimellitic anhydride, which is known to mainly react with lysine, exhibited a reactivity of almost <NUM>% for all nucleophilic reagents. This is comparable to the reactivity of NAL alone. In addition, <NUM>-methyl-<NUM>-isothiazol-<NUM>-one, which is known to mainly react with cysteine, exhibited a reactivity of about <NUM>% to about <NUM>%. In particular, Compounds <NUM>, <NUM> and <NUM> exhibited a reactivity as high as almost <NUM>%. This value exceeds the reactivity of NAC alone. On the other hand, salicylic acid, which is a non-sensitizer, was almost completely unreactive with any nucleophilic reagent. These results show that all nucleophilic reagents exhibit a reactivity higher than or equal to that of NAC or NAL alone with sensitizers but are completely unreactive with non-sensitizers and can thus be used as detection reagents for sensitizers.

Three nucleophilic reagents that were thought to have high performance in the above results, namely, Compounds <NUM>, <NUM>, and <NUM>, were subjected to secondary evaluation. The results are shown in <FIG>. The evaluation was performed for seven sensitizers including five sensitizers that are difficult to evaluate in conventional methods for measuring skin sensitization (cyclamen aldehyde, imidazolidinyl urea, <NUM>-methyl-<NUM>,<NUM>-hexanedione, ethylene glycol dimethacrylate, and hydroxycitronellal). The results show that all six substances except <NUM>-methyl-<NUM>,<NUM>-hexanedione exhibited a reactivity higher than that of NAC or NAL alone and the mean depletion. This demonstrates that there is a possibility that sensitizers can be predicted with a higher sensitivity than conventional methods for measuring skin sensitization and that there is a high possibility that sensitizers that are difficult to evaluate by conventional methods for measuring skin sensitization can also be correctly predicted. It is believed that <NUM>-methyl-<NUM>,<NUM>-hexanedione can also be correctly predicted as a sensitizer because the depletions were similar to the mean depletion of NAC and NAL.

Twenty-two nucleophilic reagents with a naphthylmethyl group at the C-terminus thereof (Compounds <NUM> to <NUM>)) were evaluated.

The residual fractions of the <NUM> nucleophilic reagents as prepared (<NUM> hours) and after <NUM> hours were calculated. The results are shown in <FIG>. The results show that the residual fractions of all nucleophilic reagents were <NUM>% or more after <NUM> hours and that there was no nucleophilic reagent whose residual fraction decreased considerably after <NUM> hours.

The reactivity of the <NUM> nucleophilic reagents with three evaluation substances was calculated. The results are shown in <FIG>. Trimellitic anhydride, which is known to mainly react with lysine, exhibited a reactivity of almost <NUM>% for all nucleophilic reagents. This is comparable to the reactivity of NAL alone. In addition, <NUM>-methyl-<NUM>-isothiazol-<NUM>-one, which is known to mainly react with cysteine, exhibited a reactivity of about <NUM>% to about <NUM>%. In particular, Compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> exhibited a reactivity as high as almost <NUM>%. This value exceeds the reactivity of NAC alone. On the other hand, salicylic acid, which is a non-sensitizer, was almost completely unreactive with any nucleophilic reagent. These results show that all nucleophilic reagents exhibit a reactivity higher than or equal to that of NAC or NAL alone with sensitizers but are completely unreactive with non-sensitizers and can thus be satisfactorily used as detection reagents for sensitizers.

Two nucleophilic reagents (Compounds <NUM> and <NUM>) with a naphthylmethyl backbone (i.e., a structure in which naphthalene is disubstituted with amino acids) were evaluated. Compounds <NUM> and <NUM> are substances in which cysteine and lysine are attached at symmetrical substitution positions of a naphthalene ring with CH<NUM>-CO therebetween.

The residual fractions of the two nucleophilic reagents as prepared (<NUM> hours) and after <NUM> hours were calculated. The results are shown in <FIG>. The results show that the residual fractions of all nucleophilic reagents were about <NUM>% after <NUM> hours and that there was no nucleophilic reagent whose residual fraction decreased considerably after <NUM> hours.

The reactivity of the two nucleophilic reagents with three evaluation substances was calculated. The results are shown in <FIG>. Trimellitic anhydride, which is known to mainly react with lysine, exhibited a reactivity of almost <NUM>% for all nucleophilic reagents. This is comparable to the reactivity of NAL alone. In addition, <NUM>-methyl-<NUM>-isothiazol-<NUM>-one, which is known to mainly react with cysteine, exhibited a reactivity of about <NUM>%. The results show that the nucleophilic reagents were completely unreactive with salicylic acid, which is a non-sensitizer, and can thus be satisfactorily used as detection reagents for sensitizers.

Two reagents (NAC and NAL) are originally separately reacted with a test substance; it was investigated whether the two reagents can be simultaneously used as one reaction solution for testing.

Using the three substances shown in the following table, <NUM> mmol/L solutions were prepared and used for testing.

A solution prepared by dissolving NAC and NAL in a phosphate buffer solution (pH <NUM>) prepared in accordance with "(<NUM>-<NUM>) <NUM> mmol/L Phosphate Buffer Solution (pH <NUM>)" of "(<NUM>) Preparation of Various Solutions" above such that the concentration of each nucleophilic reagent was <NUM>µmol/L was used as a nucleophilic reagent stock solution.

Test substance solutions were prepared on a <NUM>-well plate (U96 PP-<NUM> NATURAL, Thermo (NUNC)), mainly using a <NUM>-channel pipette, and the reagent was added in the following amount:.

The procedures described in "(<NUM>-<NUM>) Reaction" and "(<NUM>-<NUM>) Reaction Stop" of "(<NUM>) Reaction" above were performed.

To simultaneously detect NAC and NAL, a measurement was performed under the HPLC conditions shown in the following table.

In addition, the nucleophilic reagent depletion (%) was determined by the method described in "(<NUM>) Data Analysis" above.

Because NAC and NAL were soluble in the same phosphate buffer solution, this solution was used for measurement for the three evaluation substances. The results are shown in <FIG>.

The depletions of the nucleophilic reagent prepared by mixing NAC and NAL together ("NAC/NAL mixture" in the figure) for the three evaluation substances were <NUM>%, <NUM>%, and <NUM>%. In contrast, the depletions of a conventional method in which NAC and NAL were separately prepared and used for measurement ("NAC/NAL separate" in the figure) were <NUM>%, <NUM>%, and <NUM>%, indicating that the depletion decreased when the nucleophilic reagents were mixed together. On the other hand, when measurements were performed with Compounds <NUM>, <NUM>, and <NUM> according to the present invention, their respective depletions were <NUM>%, <NUM>%, and <NUM>% for diphenylcyclopropenone, <NUM>%, <NUM>%, and <NUM>% for nonanoyl chloride, and <NUM>%, <NUM>%, and <NUM>% for <NUM>-methyl-<NUM>,<NUM>-hexanedione. In particular, the depletions of Compounds <NUM>, <NUM>, and <NUM> increased considerably for diphenylcyclopropenone and nonanoyl chloride as compared to the NAC/NAL mixture system and the NAC/NAL separate system. For <NUM>-methyl-<NUM>,<NUM>-hexanedione, the depletions of Compounds <NUM>, <NUM>, and <NUM> were similar to that of NAC/NAL separate system but increased considerably as compared to the NAC/NAL mixture system.

Claim 1:
A skin sensitization measuring reagent comprising, as a main measuring agent, an organic compound having at least one thiol group derived from an amino acid and at least one amino group derived from a side chain of an amino acid, the organic compound having an absorption spectrum in an ultraviolet, visible, or near-infrared region and having a molar absorption coefficient of <NUM>/mol·cm or more and <NUM>,<NUM>/mol·cm or less at a maximal absorption wavelength;
wherein the organic compound is represented by the following formula (<NUM>):

        W-[(L<NUM>-Xaa-L<NUM>)p-Z]q     (<NUM>)

wherein
W represents a q-valent group having an absorption spectrum in the ultraviolet, visible, or near-infrared region,
L<NUM> and L<NUM> each independently represent a single bond or a linking group,
Xaa represents an amino acid residue, provided that p × q Xaa moieties include at least one lysine residue and at least one cysteine residue,
Z represents a hydrogen atom or an alkyl group having <NUM> to <NUM> carbon atoms, wherein the alkyl group having <NUM> to <NUM> carbon atoms is optionally substituted with a substituent selected from the group consisting of a carboxyl group, an amino group, a hydroxy group, a guanidyl group, a pyridine ring, and an imidazole ring,
p represents an integer of <NUM> to <NUM>,
q represents an integer of <NUM> to <NUM>,
provided that p and q are not simultaneously <NUM>,
if p is <NUM> or more, L<NUM>-Xaa-L<NUM> moieties may be the same or different, and
if q is <NUM> or more, [(L<NUM>-Xaa-L<NUM>)p-Z] moieties may be the same or different.