Novel compounds and structures for detecting hydrogen sulfide

Abstract

To provide a novel compound that can detect hydrogen sulfide in a biological sample, with high sensitivity.SOLUTION: A compound comprises a xanthene dye represented by the constitutional formula 1 combined with a light absorber (dye) that has light absorption in a wavelength range of 350 to 700 nm and a highest molecular absorption coefficient of 104M-1 cm-1 or more.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to novel compounds and structures for the detection of hydrogen sulfide. [Background technology]

[0002] Detecting hydrogen sulfide in aqueous samples requires not only the detection of hydrogen sulfide itself, but also the detection of hydrogen sulfide as a product of enzymatic reactions. Examples include soil and water quality testing in factories, industrial waste treatment plants, sewage treatment plants, and surrounding areas; dynamic analysis of biologically active substances in living organisms and living cells; halitosis testing; and the detection and quantification of cysteine, methionine, homocysteine, and other compounds in body fluids. Cysteine, methionine, homocysteine, and other compounds can be reacted with specific enzymes, such as lyases, to determine the amount of hydrogen sulfide produced. In these cases, detection and quantification of hydrogen sulfide are necessary.

[0003] To detect hydrogen sulfide, functional dyes that undergo a detectable color change (absorption or emission) in the presence of hydrogen sulfide may be used.

[0004] A functional dye is a compound in which o-methylfluorescein is bonded to a methyl 2-cyano-3-phenylacrylate derivative that reacts with hydrogen sulfide. This compound is known to react with hydrogen sulfide and emit fluorescence in the visible region around 520 nm (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Org Lett.2012, 14, 2184-2187. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors aimed to detect hydrogen sulfide and its metabolites in aqueous specimens such as biological samples, and confirmed whether it was possible to detect hydrogen sulfide in neutral phosphate-buffered saline using the above-mentioned compound disclosed in Non-Patent Document 1 as a detection material. As a result, it was found that the detection material precipitated or aggregated in the solution, reducing its reactivity with hydrogen sulfide and making it difficult to detect hydrogen sulfide with high sensitivity. In other words, the present inventors discovered the problem that a method for increasing the water solubility of the detection material was needed to improve the detection sensitivity of hydrogen sulfide.

[0007] Our research has shown that fluorescein, which has a hydrophilic hydroxyl group, is more water-soluble than o-methylfluorescein. However, a compound combining fluorescein with a methyl 2-cyano-3-phenylacrylate derivative constantly emits fluorescence regardless of the presence or absence of hydrogen sulfide, making it difficult to detect hydrogen sulfide.

[0008] Therefore, an object of the present invention is to provide a novel material for detecting hydrogen sulfide in aqueous specimens such as biological samples with high sensitivity. [Means for solving the problem]

[0009] The compound according to the present invention is represented by the following structural formula 1.

[0010] [ka]

[0011] In the above structural formula 1 The dye has light absorption in the wavelength range of 350 nm to 700 nm and has a maximum molecular extinction coefficient of 10 4 M -1 cm -1 represents a light absorber having the above structure, A1 represents a substituted or unsubstituted alkylene group having 3 to 12 carbon atoms or an alkoxy group; B1 represents any one of -C(=O)-, -S(=O)2-, and -CH2-; B2 is -H, -NHC(=S)NH-(CH2CH2O) n represents either -CH3 or -NHC(=S)-amino acid, and n is an integer of 1 to 25, B3 represents any one of -OH, -NH2, -NHCH3, -NHCH2CH3, -N(CH3)2, and -N(CH2CH3)2. [Effects of the Invention]

[0012] The novel compounds of the present invention enable highly sensitive detection of hydrogen sulfide in aqueous specimens such as biological samples. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 2 is a diagram showing an example of a structure for detecting hydrogen sulfide according to the present embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0014] An embodiment of the present invention will be described below, but the present invention is not limited to the following.

[0015] (First embodiment) (compound) The compound according to this embodiment is represented by the following structural formula 1.

[0016] [ka]

[0017] Structural Formula 1 will be described in detail below.

[0018] (Dye) The dye in the above structural formula 1 has light absorption in the wavelength region of 350 nm or more and 700 nm or less, and has a maximum molecular extinction coefficient of 10 4 M -1 cm -1 The dye according to this embodiment more preferably has light absorption in the wavelength region of 365 nm or more and 650 nm or less.

[0019] As the dye in this embodiment, any of dyes having an ionic or nonionic functional group, pigments, fluorescent substances, non-fluorescent substances, dyes, gold nanoparticles, gold colloids, and silver nanoparticles can be used.

[0020] For example, when the dye according to the present embodiment is a dye, any one of an azo dye, a xanthene dye, a coumarin dye, a triarylmethane dye, and ethidium bromide can be used. Note that the dye according to the present embodiment is preferably at least one selected from the group consisting of an azo dye, a xanthene dye, and a coumarin dye, and is particularly preferably an azo dye that functions as a quencher.

[0021] Furthermore, the dye of the above structural formula 1 is preferably represented by the following structural formula 2.

[0022] [ka]

[0023] * in the above structural formula 2 represents the position of N in NH of the structural formula 1.

[0024] Specific examples of preferred structures of Dye in Structural Formula 1 that can be used in this embodiment are shown in the following formulae (d1) to (d18). The * below indicates the position of N in NH in Structural Formula 1. In other words, * can also be said to represent the bond bonding to N in -NH in Structural Formula 1. The same applies hereinafter to the meaning of *.

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[0043] (A1) In this embodiment, A1 represents a substituted or unsubstituted alkylene group or alkoxy group having 3 to 12 carbon atoms. This is because the shorter the distance, the higher the efficiency of energy transfer between the xanthene dye and the dye, allowing for greater quenching. In this embodiment, A1 is preferably an unsubstituted alkylene group having 3 carbon atoms.

[0044] (B1) In this embodiment, B1 represents any one of -C(=O)-, -S(=O)2-, and -CH2-. In an aqueous solution with a neutral pH, such as under physiological conditions, the ring structure opens to form -CO2 - , -SO3 - , or -CHOH. As a result, the water solubility of the compound represented by the structural formula 1 can be increased, and a decrease in detection sensitivity due to precipitation or aggregation can be suppressed. From the viewpoint of high water solubility, B1 in this embodiment is preferably represented by -C(=O)-.

[0045] (B2) In this embodiment, B2 is -H, -NHC(=S)NH-(CH2CH2O) n In this embodiment, B2 is represented by either -H, -NHC(=S)NH-(CH2CH2O). ... n It is preferably represented by -CH3 or -NHC(=S)NH-CHR-COOH, where R represents an amino acid side chain. It is more preferable that the above n is 4 or more and 22 or less. In this embodiment, when B2 is -NHC(=S)NH-CH2-COOH, the amino acid of -NHC(=S)-amino acid is glycine.

[0046] (B3) In this embodiment, B3 represents any one of -OH, -NH2, -NHCH3, -NHCH2CH3, -N(CH3)2, and -N(CH2CH3)2. In this embodiment, B3 is preferably -OH or -NH2, and more preferably -OH. This is because these are water-soluble functional groups and can increase the water solubility of the compound, thereby preventing a decrease in detection sensitivity due to precipitation or aggregation. From the perspective of high water solubility, B3 in this embodiment is preferably -OH or -NH2, and more preferably -OH.

[0047] (About the effects) As described above, the compound according to this embodiment has a high water solubility because it contains many water-soluble functional groups.

[0048] As shown in structural formula 1, the xanthene dye and dye are linked via a 2-cyano-3-phenylacrylate derivative as a linker. Therefore, fluorescence resonance energy transfer (FRET) occurs between the xanthene dye and dye. As a result, when irradiated with excitation light of a specific wavelength, quenching or the generation of fluorescence at two different wavelengths is observed. Upon reaction of the 2-cyano-3-phenylacrylate derivative with hydrogen sulfide, the xanthene dye and dye separate. This separation results in the FRET phenomenon disappearing, and the fluorescence intensity increases or the fluorescence intensity ratio of the two different wavelengths changes. The amount of hydrogen sulfide can be estimated by the increase in fluorescence intensity, the increase ratio, or the change in the intensity ratio before and after reaction with hydrogen sulfide. In the FRET phenomenon, the xanthene dye can act as the donor and the dye as the acceptor, or alternatively, the xanthene dye can act as the acceptor and the dye as the donor. The above-mentioned Non-Patent Document 1 discloses a method that utilizes the Michael addition reaction of hydrogen sulfide to the vinyl carbon moiety of a methyl 2-cyano-3-phenylacrylate derivative as an initiating reaction, followed by intramolecular cyclization that cleaves the ester bond, resulting in decomposition.

[0049] When the amount of hydrogen sulfide is small, the amounts of xanthene dye and dye separated are small. On the other hand, when the amount of hydrogen sulfide is large, the amounts of xanthene dye and dye separated are large. In other words, the amount of hydrogen sulfide can be estimated by detecting the amount of liberated xanthene dye or dye. The amount of hydrogen sulfide can also be estimated by measuring the fluorescence and absorbance of the xanthene dye or dye, or both the xanthene dye and dye. Therefore, the mechanism for detecting hydrogen sulfide in the compounds according to the present embodiments is different from the conventional method using only the xanthene dye structure, such as that described in Non-Patent Document 1, and is less susceptible to precipitation and aggregation.

[0050] Therefore, the compound according to this embodiment can detect hydrogen sulfide in an aqueous specimen such as a biological sample with high sensitivity.

[0051] (biological samples) The biological sample in this embodiment corresponds to urine, blood, sweat, tears, saliva, mucus, and liquid samples based on these, as well as diluted or concentrated solutions containing increased or decreased amounts of water. By contacting the biological sample with an enzyme and a hydrogen sulfide detection device for a detection target believed to be contained in such a sample, the amount and concentration of the detection target metabolite in the biological sample can be measured.

[0052] (Detection target) In this embodiment, the detection target may be a biomarker related to a disease, physical condition, or degree of stress on a living body. Examples include hydrogen sulfide, methanethiol, cysteine, and homocysteine, but the detection target according to the embodiment of the present invention is not limited to these substances. The detection target is preferably one contained in urine, blood, sweat, tears, saliva, or the like.

[0053] (Application) The compound according to this embodiment may be used in the structure according to the second embodiment, or in other devices or measuring instruments. Examples of devices or measuring instruments that can be used include automated clinical blood analyzers, simple medical testing devices, rapid diagnostic testing devices, and biochemical testing devices. Other examples that can be used include, but are not limited to, lateral flow test chips, flow-through test chips, dipsticks, microfluidic chips, microchemical chips, and biochips.

[0054] (Second embodiment) (structure) The structure according to this embodiment has a substrate and the compound according to the first embodiment provided on the substrate.

[0055] (base material) In this embodiment, the substrate may be a solid material, preferably one having low reactivity with hydrogen sulfide. Desirable substrates in this embodiment include glass, ceramic, silicone resin, paper using cellulose or μfiber, felt, knitted fabric, nonwoven fabric, porous material, filter paper, etc., and paper materials are preferred in terms of availability.

[0056] The substrate in this embodiment may be glass with an etched channel or circle, or a paper material with a channel or circular frame printed with a hydrophobic material. The hydrophobic material in this embodiment may be at least one selected from the group consisting of wax, crayon, paraffin, SU-8, silicone, oil-based marker, polyacrylic acid, acrylic lacquer, alkyl ketone dimer, polystyrene, octadecyltrichlorosilane, polydimethylsiloxane, polyacrylate, and cyclic olefin copolymer, but is not limited to these. The substrate in this embodiment may be paper with a circular frame printed with a material containing a cyclic olefin copolymer and a plasticizer. By applying the compound according to the first embodiment to the channel or circular frame, the compound according to the first embodiment is retained within the frame, allowing for highly sensitive detection of hydrogen sulfide on the substrate. In the case of a circular structure, the area within the frame can serve as a hydrogen sulfide sensing region. In the structure according to this embodiment, the compound according to the first embodiment applied to the substrate is released into an aqueous biological sample, or the aqueous biological sample easily penetrates the substrate to which the compound is applied. Therefore, if the material for detecting hydrogen sulfide has low water solubility, the material will strongly adsorb to the substrate due to hydrophobic interaction, making it difficult for the material to come into contact with hydrogen sulfide and resulting in a decrease in detection sensitivity. On the other hand, the compound according to this embodiment has high water solubility, so that a large amount is released from the substrate or the compound is weakly adsorbed, making it easy for the material to come into contact with hydrogen sulfide, thereby reducing the decrease in detection sensitivity. In other words, the structure according to this embodiment can detect hydrogen sulfide on a substrate with high sensitivity due to the high water solubility of the compound.

[0057] (Example of a structure) An example of a structure according to this embodiment is shown in Fig. 1. In Fig. 1, a structure 101 for detecting hydrogen sulfide has a hydrogen sulfide detection material (the compound according to the first embodiment) 102 and a substrate 103. If necessary, the structure 101 may include a support 104.

[0058] Upon addition of a biological sample 105, the detection material 102 adsorbed to the substrate 103 is released from the substrate into the specimen, or penetrates and is mixed into the aqueous specimen (biological sample). In the presence of hydrogen sulfide, the detection material 102 mixed in the substrate or aqueous specimen reacts with the hydrogen sulfide and separates into xanthene dye 106 and dye 107. The presence of hydrogen sulfide can be confirmed by observing the fluorescence intensity or brightness in the aqueous specimen or on the substrate.

[0059] It should be noted that the example of the structure shown in FIG. 1 is merely an example, and the present invention is not limited to this.

[0060] Alternatively, the structure 101 may be immersed in a solution containing hydrogen sulfide. The xanthene dye 106 or dye 107 separated by the hydrogen sulfide dissolves in the solution. The presence of hydrogen sulfide can also be confirmed by observing the amount of color development of the xanthene dye or dye dissolved in the solution. Here, the amount of color development refers to the signal intensity of color intensity or fluorescence intensity.

[0061] (Application) The structure according to this embodiment includes the compound according to the first embodiment, and therefore is capable of detecting hydrogen sulfide. Alternatively, an enzyme that generates hydrogen sulfide using a metabolite to be detected as a substrate may be provided on the substrate. For example, homocysteine α,γ-lyase can be used as the enzyme to detect homocysteine. The amount of the substance to be detected can be determined by detecting hydrogen sulfide, which is the product of the enzyme reaction. The substance to be detected can also be detected by using an aqueous sample to which an enzyme has been added in advance, in which hydrogen sulfide has been generated from the substance to be detected. The substance to be detected can also be an enzyme. That is, the enzyme can be detected by containing a substrate for the enzyme to be detected.

[0062] (enzyme) Other examples of the enzyme that can be used in this embodiment include, but are not limited to, at least one selected from the group consisting of cystathinine γ-lyase, 3-mercaptopyruvate sulfurtransferase, thiosulfate reductase, methanethiol oxidase, sulfhydrogenase, sulfur oxygenase / reductase, methylated-thiol-coenzyme M methyltransferase, O-phosphoserine sulfhydrylase, carbon disulfide hydrolase, carbonyl sulfide hydrolase, homocysteine desulfhydrase (homocysteine α,γ-lyase), L-3-cyanoalanine synthase, D-cysteine desulfhydrase, L-cysteine desulfhydrase, L-methionine γ-lyase, and cystathionine β-synthase.

[0063] (Third embodiment) (detection device) A detection device according to a third embodiment of the present invention has a blood cell separation membrane provided on the substrate of the structure according to the second embodiment. The provision of the blood cell separation membrane allows detection of hydrogen sulfide contained in blood. Furthermore, as in the second embodiment, homocysteine α,γ-lyase is provided on the substrate, allowing detection of homocysteine contained in blood.

[0064] (Fourth embodiment) A microchannel device according to a fourth embodiment of the present invention includes the substrate and the compound according to the first embodiment. The substrate has a sensing area coated with the compound according to the first embodiment, a blood drop area provided with a blood cell separation membrane, and a channel through which plasma or serum can move from the blood drop area to the sensing area.

[0065] (flow path) The flow channel in this embodiment is preferably a development flow channel through which plasma or serum can move by capillary action.

[0066] In this embodiment, the substrate may be a paper material containing a cyclic olefin copolymer and a plastic component, on which a dumbbell-shaped frame is printed, and the sensing area and blood drop area may be located at both ends of the dumbbell shape.

[0067] (Fifth embodiment) A detection method according to a fifth embodiment of the present invention includes a contacting step of contacting a biological sample with a dye for detecting hydrogen sulfide, and a measuring step of measuring the amount of color development or fluorescence generated by the contacting step. The dye for detecting hydrogen sulfide can be the compound according to the first embodiment.

[0068] In this embodiment, a solid containing a dye for detecting hydrogen sulfide, or a solution in which the dye is dispersed, may be brought into contact with an aqueous specimen such as a biological sample suspected of containing hydrogen sulfide.

[0069] Alternatively, the sensing area of the device may be immersed in a container containing a biological sample to which an enzyme that generates hydrogen sulfide has been added, or the biological sample may be spread on a substrate using capillary action, as in paper chromatography, and brought into contact with the sensing area. The enzyme may also be applied to the substrate in advance.

[0070] By using the above method, a biological sample is brought into contact with the sensing area on the substrate, and after a certain period of time has passed, the amount of change in the color intensity or fluorescence signal intensity of the sensing area is measured by visual observation or with a reflection densitometer or a fluorescence spectrodensitometer, thereby allowing the amount of the substance to be detected to be calculated.

[0071] When the amount of the target substance to be detected is small, the amount of change in the sensing area is small. When the amount of the target substance to be detected is large, the amount of change in the sensing area is large. The amount of change may be evaluated by the color intensity of the sensing area or the fluorescence intensity of the sensing area emitted by irradiation with ultraviolet or visible light using an ultraviolet lamp or the like.

[0072] Furthermore, as a fourth embodiment of the present invention, there is provided a hydrogen sulfide detection structure containing the compound of the first embodiment, a substrate, and an enzyme that generates hydrogen sulfide using a metabolite to be detected as a substrate.

[0073] (Sixth embodiment) (detection kit) A detection kit according to a sixth embodiment of the present invention includes the compound according to the first embodiment or a solution containing the compound, and the substrate according to the second embodiment. The detection kit according to this embodiment may include a substrate and a separate container containing the compound or a solution containing the compound. When detecting hydrogen sulfide, the compound is applied to the substrate. A biological sample suspected of containing hydrogen sulfide can be directly dropped onto the coated substrate (which can also be referred to as a structure) to measure the amount and concentration of hydrogen sulfide. A separate container may also be provided for allowing the biological sample to permeate the structure. By providing a separate container, the biological sample can be uniformly permeated into the structure.

[0074] Alternatively, the solid enzyme or an aqueous solution containing the enzyme may be contained in a separate container in advance. The biological sample may be added to the container and the structure may be permeated at the same time. Alternatively, the biological sample may be added to the container, and after a certain period of time has elapsed to generate hydrogen sulfide, the structure may be permeated.

[0075] The detection kit according to this embodiment may include a color sample showing the hue, brightness, and saturation of the structure to observe the color (fluorescence) change according to the amount or concentration of hydrogen sulfide or its metabolites. This may be printed on a separate surface such as paper or a plastic plate. By visually comparing the color of the structure after the change with the color sample, the amount or concentration of hydrogen sulfide or its metabolites in the biological sample can be determined semi-quantitatively.

[0076] The detection kit according to this embodiment may be equipped with a separate LED light, preferably an LED lamp emitting light in the wavelength range of 365 nm to 500 nm. By illuminating the sensing area with the LED light as excitation light, changes in fluorescence in the sensing area can be visually observed. Alternatively, the sensing area can be captured as image data using a compact digital camera or smartphone camera, and the color and fluorescence changes in the sensing area can be measured as intensity values using an application. This allows for quantitative determination of the amount or concentration of hydrogen sulfide and metabolites in a biological sample.

[0077] The test kit according to this embodiment may include a long-pass filter, preferably with a cut-on wavelength in the wavelength range of 400 nm to 650 nm. By observing or detecting changes in fluorescence in the sensing region through the long-pass filter, scattering and reflection of the excitation light can be cut off, allowing for selective detection of only the fluorescence. Alternatively, the material may be colored cellophane, which has low transmittance for excitation light and high transmittance for fluorescence.

[0078] The test kit according to this embodiment may include a small chamber into which a biological sample, the compound according to the first embodiment, or the structure according to the second embodiment can be added or inserted. The test kit according to this embodiment preferably includes a small chamber with an internal structure capable of blocking external light such as sunlight or fluorescent light. This is because fluorescence can be selectively detected without being affected by external light. The small chamber may also be integrated with an LED light or a long-pass filter, and preferably has a window that allows the interior of the small chamber to be viewed from the outside using the naked eye or the camera function of a compact digital camera or smartphone. It is also preferable that the test kit have a holder function that allows a compact digital camera or smartphone to be attached. [Example]

[0079] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples as long as it does not depart from the gist of the invention.

[0080] <Identification of compounds> The compound (dye) synthesized below is: 1 Identification was carried out using a 1 H-NMR (Bruker Avance500, resonance frequency: 500 MHz) measuring device.

[0081] (Production Example 1) Synthesis of Compound 1-1 2.15 g of tert-butyl 2-formylbenzoate (synthesized with reference to Chem. Commun. 2019, 55, pp. 13610-13613 (Non-Patent Document 2)) was dissolved in 30 mL of tetrahydrofuran, and 3.11 g of 3-[[(tert-butoxy)carbonyl]amino]propyl 2-cyanoacetate (synthesized with reference to Org Biomol Chem. 2011, 9, pp. 3530-3540 (Non-Patent Document 3)) was added dropwise. 2 mL of diazabicycloundecene was added dropwise, and the mixture was stirred overnight at room temperature. After distilling off the solvent, the mixture was purified by silica gel column chromatography to obtain 1.60 g of a colorless oil.

[0082] (Production Example 2) Synthesis of Compound 1-2 0.89 g of compound 1-1 was added to 12 mL of a mixture of trifluoroacetic acid and dichloromethane (v / v = 1 / 1) and stirred at room temperature for 3 hours. After distilling off the solvent, 25 mL of N,N-dimethylformamide and 2 mL of triethylamine were added. 0.07 g of dimethylaminopyridine and 0.76 g of N-succinimidyl 4-[4-(Dimethylamino)phenylazo]benzoate (Tokyo Chemical Industry Co., Ltd.) were added, and the mixture was stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 0.60 g of a red solid.

[0083] (Production Example 3) Synthesis of Compound 1 To 20 mL of N,N-dimethylformamide, 280 mg of compound 1-2 and 336 mg of water-soluble carbodiimide were added and stirred at room temperature for 1 hour. To the solution, 73 mg of dimethylaminopyridine (Tokyo Chemical Industry Co., Ltd.) and 392 mg of fluorescein (Tokyo Chemical Industry Co., Ltd.) were added and stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 19 mg of a red solid. 1H-NMR(CD3OD)(ppm):9.02(s, 1H), 8.32(m, 1H), 8.02-7.75(m, 9H), 7.68-7.60(m, 2H), 7.20-7.11 (m, 2H), 6.87-6.55(m, 8H), 4.48-4.42(m, 2H), 3.66-3.63(m, 2H), 3.10(s, 6H), 2.12-2.10(m, 2H).

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[0085] (Production Example 4) Synthesis of Compound 2 With reference to the above-mentioned Non-Patent Document 1, 64 mg of a white solid was obtained.

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[0087] (Production Example 5) Synthesis of Compound 3-1 To 5 mL of N,N-dimethylformamide, 79 mg of fluorescein isothiocyanate isomer I (Sigma-Aldrich), 39 mg of glycine tert-butyl ester hydrochloride (Tokyo Chemical Industry Co., Ltd.), and 0.1 mL of triethylamine were added and stirred at room temperature for 3 hours. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 80 mg of an orange solid.

[0088] (Production Example 6) Synthesis of Compound 3-2 To 5 mL of N,N-dimethylformamide, 20 mg of compound 1-2, 25 mg of water-soluble carbodiimide, and 5 mg of dimethylaminopyridine were added and stirred at room temperature for 1 hour. 46 mg of compound 3-1 was added to the solution and stirred overnight at room temperature. After distilling off the solvent, ethyl acetate was added and the mixture was washed with water. The ethyl acetate layer was recovered, dehydrated by adding sodium sulfate, and then filtered. After distilling off the solvent from the filtrate, the residue was purified by silica gel column chromatography to obtain 10 mg of a red solid.

[0089] (Production Example 7) Synthesis of Compound 3 Trifluoroacetic acid (2 mL) was added to 10 mg of compound 3-2 and stirred at room temperature for 3 hours. The solvent was evaporated to give 10 mg of a red solid. 1 H-NMR(CD3OD / CDCl3=1 / 1)(ppm):9.02(s, 1H), 7.96-7.84(m, 11H), 7.35-7.32(m, 1H), 7.20-7.15 (m, 1H), 6.93-6.73(m, 8H), 4.43-4.41(m, 4H), 3.73-3.69(m, 2H), 3.16(s, 6H), 2.14-2.11(m, 2H).

[0090] [ka]

[0091] (Production Example 8) Synthesis of Compound 4 To 5 mL of N,N-dimethylformamide, 19 mg of compound 1-2, 23 mg of water-soluble carbodiimide, and 5 mg of dimethylaminopyridine were added and stirred at room temperature for 30 minutes. 32 mg of 2',7'-dichlorofluorescein (Tokyo Chemical Industry Co., Ltd.) was added to the solution and stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 20 mg of a red solid. 1 H-NMR(CDCl3)(ppm):9.04(s, 1H), 8.08-7.82(m, 12H), 7.75-7.66(m, 2H), 7.20-7.11(m, 2H) ), 6.77-6.67(m, 6H), 4.49-4.42(m, 2H), 3.66-3.62(m, 2H), 3.11(s, 6H), 2.14-2.11(m, 2H).

[0092] [ka]

[0093] (Production Example 9) Synthesis of Compound 5-1 To 1 mL of N,N-dimethylformamide, 70 mg of fluorescein isothiocyanate isomer I (Sigma-Aldrich), 38 mg of 3,6,9,12-tetraoxadecanamine (Tokyo Chemical Industry Co., Ltd.), and 0.1 mL of triethylamine were added and stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 95 mg of an orange solid.

[0094] (Production Example 10) Synthesis of Compound 5 To 3 mL of N,N-dimethylformamide, 23 mg of compound 1-2, 25 mg of water-soluble carbodiimide, and 6 mg of dimethylaminopyridine were added and stirred at room temperature for 1 hour. To the solution, 47 mg of compound 5-1 was added and stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 11 mg of a red solid. 1 H-NMR(CDCl3)(ppm):9.01(s, 1H), 8.78(br, 1H), 8.32-8.31(m, 1H), 8.07(b r, 1H), 7.99-7.97(m, 1H), 7.90-7.77(m, 9H), 7.10-7.05(m, 3H), 6.87-6.85( m, 1H), 6.77-6.66(m, 6H), 6.57-6.55(m, 2H), 4.42(t, 2H, J=5.5Hz), 3.88-3. 83(m, 2H), 3.72-3.58(m, 16H), 3.32(s, 3H), 3.10(s, 6H), 2.12-2.08(m, 2H).

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[0096] (Production Example 11) Synthesis of Compound 6 To 1 mL of N,N-dimethylformamide, 12 mg of compound 1-2, 16 mg of water-soluble carbodiimide, and 3 mg of dimethylaminopyridine were added and stirred at room temperature for 30 minutes. 10 mg of 2',7'-difluorofluorescein (Thermo Fisher Scientific) was added to the solution and stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 2 mg of a red solid.

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[0098] (Production Example 12) Synthesis of Compound 7 To 2 mL of N,N-dimethylformamide, 27 mg of compound 1-2, 30 mg of water-soluble carbodiimide, and 7 mg of dimethylaminopyridine were added and stirred at room temperature for 2 hours. 48 mg of 3,4,5,6-tetrachlorofluorescein (Tokyo Chemical Industry Co., Ltd.) was added to the solution and stirred overnight at room temperature. After distilling off the solvent, the residue was purified by silica gel column chromatography to obtain 9 mg of a red solid. 1 H-NMR(CDCl3)(ppm):9.04(s, 1H), 8.58(br, 1H), 7.90-7.85(m, 7H), 7.83( m, 1H), 7.78(m, 1H), 7.13-7.12(m, 1H), 6.98-6.94(m, 1H), 6.90-6.88(m, 1 H), 6.77-6.71(m, 4H), 6.62-6.60(m, 1H), 6.57-6.54(m, 1H), 5.76(br, 1H) , 4.45(t, 2H, J=6.0Hz), 3.68-3.64(m, 2H), 3.11(s, 6H), 2.15-2.10(m, 2H).

[0099] [ka]

[0100] [Example 1] Compound 1 was added to 1x phosphate-buffered saline (3 mL, pH 7.4) to a concentration of 5 μM, and the fluorescence intensity of the solution was immediately measured using a spectrofluorometer F-4500 (Hitachi High-Tech Technologies). Sodium sulfide, a hydrogen sulfide donor, was then added to the solution to a concentration of 0.2 mM, and the solution was allowed to stand for 10 minutes before measurement. The fluorescence intensity before the addition of sodium sulfide was designated F1, and the fluorescence intensity 10 minutes after the addition of sodium sulfide was designated F2. The change in fluorescence intensity (ΔF) before and after the addition of sodium sulfide was calculated using the formula F2 - F1. The change in fluorescence intensity (ΔF0) of the solution was calculated in the same manner, except that the sodium sulfide concentration was changed to 0 mM. The difference in fluorescence intensity between the presence and absence of sodium sulfide (ΔF - ΔF0) was calculated. The calculated values are listed in Table 1.

[0101] [Example 2] The same procedure as in Example 1 was carried out, except that Compound 1 was replaced with Compound 3.

[0102] [Example 3] The same procedure as in Example 1 was carried out, except that Compound 1 was replaced with Compound 5.

[0103] [Comparative Example 1] The same procedure as in Example 1 was carried out, except that Compound 1 was replaced with Compound 2.

[0104] Comparative Example 2 The same procedure as in Example 1 was carried out, except that Compound 1 was replaced with Compound 4.

[0105] Comparative Example 3 The same procedure as in Example 1 was carried out, except that Compound 1 was replaced with Compound 6.

[0106] Comparative Example 4 The same procedure as in Example 1 was carried out, except that Compound 1 was replaced with Compound 7.

[0107] [sensitivity] In the examples of the present invention, the evaluation was based on the following criteria, with A and B being acceptable levels and C and D being unacceptable levels. The larger ΔF-ΔF0, the higher the sensitivity of hydrogen sulfide detection. A: ΔF-ΔF0 is 40 or more B: ΔF-ΔF0 is 25 or more and less than 40 C: ΔF-ΔF0 is 15 or more and less than 25 D: ΔF-ΔF0 is less than 15

[0108] Table 1 shows the changes in fluorescence intensity ΔF and ΔF0 of the solutions in each example and comparative example, the difference in the changes in fluorescence intensity ΔF-ΔF0 in the presence or absence of sodium sulfide, and the evaluation results. As described above, it was found that when the compounds according to the examples of the present invention were used, hydrogen sulfide could be detected with high sensitivity.

[0109] [Table 1] [Explanation of symbols]

[0110] 101 Structure 102 Detection Materials 103 Base material 104 Support 105 Biological Samples 106 Xanthene dyes 107 Dye

Claims

1. A compound represented by the following structural formula 1. 【Chemical 1】 In the above structural formula 1 The dye has light absorption in the wavelength region of 350 nm or more and 700 nm or less, and the maximum value of the molecular extinction coefficient is 10 ４ M －１ cm －１ represents a light absorber having the above structure, A １ represents a substituted or unsubstituted alkylene group or alkoxy group having from 3 to 12 carbon atoms, B １ -C(=O)-, -S(=O) ２ -, -CH ２ - represents either B ２ is -H, -NHC(=S)NH-(CH ２ CH ２ O) ｎ -CH ３ , -NHC(=S)-amino acid, and n is an integer of 1 or more and 25 or less; B ３ -OH, -NH ２ , -NHCH ３ , -NHCH ２ CH ３ , -N(CH ３ ) ２ , -N(CH ２ CH ３ ) ２ It represents one of the following.

2. B in the structural formula 1 ３ The compound according to claim 1, wherein:

3. B in the structural formula 1 １ The compound according to claim 1 or 2, wherein is represented by -C(=O)-.

4. B in the structural formula 1 ２ is -H, -NHC(=S)NH-(CH ２ CH ２ O) ４ -CH ３、 -NHC(=S)NH-CH ２ The compound according to any one of claims 1 to 3, represented by -COOH.

5. A in the structural formula 1 １ The compound according to any one of claims 1 to 4, wherein is an alkylene group having 3 carbon atoms.

6. The compound according to any one of claims 1 to 5, wherein Dye in the structural formula 1 is represented by the following structural formula 2: 【Chemistry 2】 * in the above structural formula 2 represents the position of N in NH of the above structural formula 1.

7. A structure comprising a substrate and the compound according to claim 1 provided on the substrate.

8. 8. The structure according to claim 7, wherein an enzyme that generates hydrogen sulfide using a metabolite to be detected as a substrate is provided on the substrate.

9. 9. The structure of claim 8, wherein the enzyme is homocysteine α,γ-lyase or L-methionine γ-lyase.

10. A device for detecting hydrogen sulfide contained in blood, comprising the structure according to claim 7 or 8, on which a blood cell separation membrane is provided.

11. A device for detecting homocysteine contained in blood, comprising: A detection device in which a blood cell separation membrane is provided on the structure according to claim 9.

12. 12. The detection device of claim 10 or 11, wherein the substrate comprises a paper material having a circular frame printed thereon with a material comprising a cyclic olefin copolymer and a plastic component.

13. 7. A microfluidic device comprising: a substrate; and the compound according to claim 1 provided on the substrate, wherein the substrate has a sensing region where the compound is applied to the substrate; a blood drop region where a blood cell separation membrane is provided on the substrate; and a flow channel configured to allow plasma or serum to move from the blood drop region to the sensing region.

14. 14. The microfluidic device according to claim 13, wherein the sensing region is provided on a substrate coated with either homocysteine α,γ-lyase or L-methionine γ-lyase.

15. 14. The microfluidic device according to claim 13, wherein the blood drop area is provided on a substrate coated with either homocysteine α,γ-lyase or L-methionine γ-lyase.

16. 16. The microfluidic device according to claim 13, wherein the substrate comprises a paper material on which a dumbbell-shaped frame is printed using a material containing a cyclic olefin copolymer and a plastic component, and the sensing area and the blood drop area are located at both ends of the dumbbell shape.

17. A detection method comprising: a contacting step of contacting a biological sample with the compound according to claim 1; and a measuring step of measuring the amount of color development or fluorescence produced by the contacting step.

18. 18. The detection method according to claim 17, wherein the contacting step comprises a step of mixing the biological sample with either homocysteine α,γ-lyase or L-methionine γ-lyase to generate hydrogen sulfide.

19. The contacting step comprises:

19. The detection method according to claim 18, comprising the step of contacting a biological sample with a substrate to which the compound and either homocysteine α,γ-lyase or L-methionine γ-lyase have been applied.

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