Novel squaraine dye-peptide conjugates

Abstract

To provide a new squaraine pigment peptide complex capable of selectively detecting elastase which is one of biomarkers.SOLUTION: Novel squaraine dye-peptide conjugates are provided that are 4-membered ring systems with structural rigidity. By introducing a predetermined oligopeptide between predetermined squaraine dyes, elastase can be selectively detected using near-infrared fluorescence characteristics.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel squaraine dye-peptide conjugates. [Background technology]

[0002] Squaraine dyes, which are four-membered ring systems with structural rigidity, possess unique photoelectric properties, characterized by very sharp and strong absorption associated with intense fluorescence emission in solution. These favorable properties are expected to be utilized in a variety of applications, including photoconductivity, data storage, light-emitting field-effect transistors, solar cells, and fluorescent histological probes. Specifically, squaraine dyes have been proposed for use in filters for display devices (see, for example, Patent Document 1).

[0003] Furthermore, detection and sensing using near-infrared fluorescent dyes is technically simple and highly sensitive, allowing for the immediate measurement of molecular interactions and the effective detection of target molecules. While some near-infrared fluorescent dyes have been reported to exhibit fluorescence in nonpolar solvents, these conventional near-infrared fluorescent dyes suffer from fluorescence quenching due to aggregation in polar solvents, water, and buffer solutions. Therefore, there is a demand for new near-infrared fluorescent dyes that fluoresce, especially in buffer solutions (body fluids), which are expected to be useful for biosensing and in vivo bioimaging. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2021-504538 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel squaraine dye-peptide conjugate that enables selective detection of elastase, which is one of the biomarkers. [Means for solving the problem]

[0006] The present inventors have discovered that by incorporating a specific oligopeptide between a specific squaraine dye, it is possible to selectively detect elastase, a biomarker, by utilizing its near-infrared fluorescence properties. In particular, they have found that neutrophil elastase (NE) in vivo and neutrophil extracellular traps (NETs) that persist for a long period in inflamed tissues can be detected, leading to the completion of the present invention.

[0007] Furthermore, the present inventors discovered that by coupling an oligopeptide that specifically binds to elastase to a specific squaraine dye, it is possible to selectively detect elastase, a biomarker, by utilizing its near-infrared fluorescence properties. In particular, they found that neutrophil elastase (NE) in vivo and neutrophil extracellular traps (NETs) that persist for a long period in inflamed tissues can be continuously detected, leading to the completion of the present invention.

[0008] That is, the present invention is as follows. [1] A squaraine dye-peptide conjugate represented by the following formula (I): [ka] (In formula (I), R 1A represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R 2A ~R 4A each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, R 5A represents an amino group, a carboxy group, or an ester group, X represents a divalent linking group derived from any one of the following amino acid sequences (1) to (7) (A represents alanine, V represents valine, P represents proline, I represents isoleucine, S represents serine, L represents leucine, G represents glycine, and D represents aspartic acid), (1) APA (2) VPV (3) APV (4) AAPI (SEQ ID NO: 1) (5) AAPV (SEQ ID NO: 2) (6) AAPA (SEQ ID NO: 3) (7) AVSLGD (SEQ ID NO: 4) Y represents a divalent linking group as a spacer. [2] In formula (I), R 1A represents an alkylene group having 3 to 18 carbon atoms, and R 2A ~R 4A and each independently represent an alkyl group having 1 to 18 carbon atoms. [3] In formula (I), R 1A represents an alkylene group having 3 to 6 carbon atoms, and R 2A ~R 4A and each independently represent an alkyl group having 1 to 4 carbon atoms. [4] The squaraine dye peptide conjugate according to [1] above, wherein Y in formula (I) is a divalent linking group based on a small molecule or PEG. [5] The squaraine dye peptide conjugate according to any one of [1] to [4] above, wherein in formula (I), X represents a divalent linking group derived from APA, and Y represents a divalent linking group derived from β-alanine. [6] The squaraine dye peptide conjugate according to any one of [1] to [4] above, wherein in formula (I), X represents a divalent linking group derived from VPV, and Y represents a divalent linking group derived from β-alanine. [7] A reagent for detecting elastase, comprising the squaraine dye peptide conjugate according to any one of [1] to [6] above. [8] contacting the squaraine dye-peptide conjugate of any one of [1] to [6] above with a sample; detecting elastase by measuring the fluorescence emission spectrum; A method for detecting elastase, comprising: [9] A method for providing information necessary for predicting or diagnosing an elastase-related disease, comprising: contacting a sample with the squaraine dye-peptide conjugate according to any one of [1] to [6] above; detecting elastase by measuring the fluorescence emission spectrum; 1. A method for providing information, comprising:

[0009]

[10] A squaraine dye-peptide conjugate represented by the following formula (1): [ka] (In formula (1), R 1B represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R 2B represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, W represents a divalent linking group as a spacer; Z represents a monovalent group derived from an oligopeptide that specifically binds to elastase.

[11] The squaraine dye peptide conjugate according to

[10] above, wherein in formula (1), Z represents a monovalent group derived from the amino acid sequence of SEQ ID NO: 5, or a monovalent group derived from the amino acid sequence of SEQ ID NO: 5 in which one or several amino acids have been deleted, substituted and / or added while retaining methionine.

[12] The squaraine dye peptide conjugate according to

[10] or

[11] above, wherein in formula (1), W is a divalent linking group based on a small molecule or PEG.

[13] The squaraine dye-peptide conjugate according to

[12] above, wherein in formula (1), W represents a divalent spacer derived from PEG-DGA-OH.

[14] In equation (1), R 1B represents an alkylene group having 3 to 18 carbon atoms, and R 2BThe squaraine dye peptide conjugate according to any one of the above

[10] to

[13] , wherein represents an alkyl group having 1 to 18 carbon atoms.

[15] In equation (1), R 1B represents an alkylene group having 3 to 6 carbon atoms, and R 2B The squaraine dye peptide conjugate according to

[14] above, wherein represents an alkyl group having 1 to 4 carbon atoms.

[16] A reagent for detecting elastase, comprising the squaraine dye peptide conjugate according to any one of

[10] to

[15] above.

[17] A step of contacting the squaraine dye-peptide conjugate according to any one of

[10] to

[15] above with a sample; detecting elastase by measuring the fluorescence emission spectrum; A method for detecting elastase, comprising:

[18] A method for providing information necessary for predicting or diagnosing an elastase-related disease, comprising: contacting a sample with the squaraine dye-peptide conjugate according to any one of

[10] to

[15] above; detecting elastase by measuring the fluorescence emission spectrum; 1. A method for providing information, comprising: [Effects of the Invention]

[0010] The novel squaraine dye-peptide conjugate of the present invention enables the selective detection of elastase, which is one of the biomarkers. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of a HomoFRET imaging test of a squaraine dye-peptide conjugate according to the first invention. [Figure 2] FIG. 1 shows the fluorescence dynamics measurement results after 1 minute and 60 minutes of a HomoFRET imaging test of the squaraine dye-peptide conjugate according to the first invention. [Figure 3]FIG. 1 shows an outline of an in vivo imaging test using an air pouch model of a squaraine dye-peptide conjugate according to the first invention. [Figure 4] FIG. 1 shows the results of an in vivo imaging test using an air pouch model of the squaraine dye-peptide conjugate according to the first invention. [Figure 5] FIG. 1 shows the results of measuring the fluorescence kinetics of the squaraine dye-peptide conjugate according to the first invention in an in vivo test using gout-induced mice. [Figure 6] FIG. 10 shows the results of a HomoFRET imaging test of a squaraine dye-peptide conjugate according to the second invention. [Figure 7] FIG. 10 shows the fluorescence dynamics measurement results after 1 minute and 60 minutes of a HomoFRET imaging test of a squaraine dye-peptide conjugate according to the second invention. [Figure 8] FIG. 10 is a graph showing the results of measuring the fluorescence kinetics of the squaraine dye-peptide conjugate according to the second invention in an in vivo test using gout-induced mice. [Figure 9] FIG. 10 shows a fluorescence micrograph of NETs derived from human thrombus in the presence of a squaraine dye-peptide conjugate according to the second invention. [Figure 10] FIG. 10 shows a fluorescence micrograph of NETs derived from a mucosal surface in the presence of a squaraine dye-peptide conjugate according to the second invention. [Figure 11] FIG. 10 shows flow cytometry analysis of mouse bone marrow after mild induction of NETs using the squaraine dye-peptide conjugate of the second invention. [Figure 12] FIG. 10 shows flow cytometry analysis of mouse bone marrow after potent induction of NETs using the squaraine dye-peptide conjugate of the second invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Squaraine dye-peptide conjugate according to the first aspect of the invention> The squaraine dye peptide conjugate according to the first aspect of the present invention is a compound represented by the following formula (I):

[0013] [ka]

[0014] R 1A represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms. 1A may be a linear hydrocarbon group or a branched hydrocarbon group, but a linear hydrocarbon group is preferred. Also, may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, but a saturated hydrocarbon group (alkylene group) is preferred. As mentioned above, the number of carbon atoms is 3 to 30, but 3 to 18 is preferred, 3 to 12 is more preferred, and 3 to 6 is even more preferred.

[0015] R 2A ~R 4A R each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms. 2A ~R 4A R may be a linear hydrocarbon group or a branched hydrocarbon group, but is preferably a linear hydrocarbon group. It may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, but is preferably a saturated hydrocarbon group (alkyl group). The number of carbon atoms is 1 to 30, as described above, but is preferably 1 to 18, more preferably 1 to 12, and even more preferably 1 to 4. 5A represents an amino group, a carboxy group, or an ester group.

[0016] The aromatic skeleton in the above formula (I) may have a substituent. The substituent is preferably an electron-donating group. Specific examples include an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, and a hydroxyl group. These substituents are preferably substituted at a position that functions as an electron-donating group. Alternatively, the substituent may be a halogen group.

[0017] X is an oligopeptide introduced between two squaraine dyes and represents a divalent linking group derived from any of the following amino acid sequences (1) to (7). In the amino acid sequence, A represents alanine, V represents valine, P represents proline, I represents isoleucine, S represents serine, L represents leucine, G represents glycine, and D represents aspartic acid. The left side represents the N-terminus linked to the linking group Y, and the right side represents the C-terminus linked to lysine. The amino acids contained in the sequence may be either L- or D-configuration. X is particularly preferably (1) a divalent linking group derived from APA. In the squaraine dye peptide conjugate according to the first invention, X is cleaved by hydrolysis with an elastase enzyme.

[0018] (1) APA (2) VPV (3) APV (4) AAPI (SEQ ID NO: 1) (5) AAPV (SEQ ID NO: 2) (6) AAPA (SEQ ID NO: 3) (7) AVSLGD (SEQ ID NO: 4)

[0019] Y represents a divalent linking group serving as a spacer between the carbon atom of the carbonyl group of the squaraine dye in formula (I) and the oligopeptide (X). Y is preferably a low-molecular-weight or PEG-based divalent linking group.

[0020] Examples of low-molecular-weight linking groups include those with a molecular weight of approximately 75 to 600, such as D- or L-amino acids (AAs), 3-amino-3-(2-nitrophenyl)propionic acid (ANP), β-alanine, 4-aminobutyric acid (GABA), 5-aminovaleric acid (GAva), 6-aminohexanoic acid (ACA / AHA / Ahx), (2-aminomethoxy)acetic acid (AEA), trioxatridecane-succinic acid (Ttds), and 12-aminododecanoic acid.

[0021] The PEG-based linking group is not particularly limited as long as it is a divalent group derived from a PEG derivative capable of linking a squaraine dye to an oligopeptide, and may be linear or branched, although linear is preferred. Examples of PEG derivatives include Mini-PEG and PEG-DGA-OH.

[0022] Y is preferably a divalent linking group derived from glycine, β-alanine, 6-aminohexane, Mini-PEG, or PEG-DGA-OH, and particularly preferably a divalent linking group derived from β-alanine.

[0023] The squaraine dye peptide conjugate according to the first aspect of the present invention is a compound represented by the formula (I): 1A represents a tetramethylene group, and R 2A and R 4A represents an ethyl group, and R 3A represents a butyl group, and R 5A Particularly preferred is a squaraine dye peptide conjugate in which X represents an amino group, X represents a divalent linking group derived from APA, and Y represents a divalent linking group derived from β-alanine, i.e., a squaraine dye peptide conjugate represented by the following formula (IA) (Hetero-SQ215-APA-SQ46 in Example 1A):

[0024] [ka]

[0025] The squaraine dye peptide conjugate of the first invention comprises two squaraine dyes and an oligopeptide introduced between them. The squaraine dye peptide conjugate of the first invention exhibits quenched fluorescence when used alone in polar solvents such as water or buffer solutions, but exhibits fluorescence emission after the peptide bond is cleaved by hydrolysis with elastase.

[0026] The present inventors speculate that the reason for this is as follows. First, as described above, the squaraine dye-peptide conjugate of the first invention alone exhibits very weak or no fluorescence due to quenching caused by fluorescence resonance energy transfer (FRET) and aggregation. However, when the peptide portion is cleaved by elastase, the squaraine dye molecules bearing the peptide fraction are outside the range where fluorescence resonance energy transfer occurs (usually a Förster radius of 2 to 5 nanometers), resulting in the appearance or enhancement of fluorescence. Furthermore, after the peptide bond is cleaved by hydrolysis with elastase, the presence of the squaraine dye bearing the peptide fraction inhibits aggregation of the squaraine dye bearing the peptide fraction and the other squaraine dye, thereby preventing fluorescence quenching caused by aggregation of both squaraine dyes, resulting in the appearance or enhancement of fluorescence.

[0027] For example, we speculate that in tissues where inflammation is occurring, NE in NETs undergoes hydrolysis, and interaction with the tissue results in the appearance or enhancement of fluorescence.

[0028] The squaraine dye-peptide conjugate of the first invention, having the above-mentioned characteristics, can detect elastase with high sensitivity and selectivity by monitoring its near-infrared fluorescence characteristics, which allows for the sensitive detection of elastase in biological fluids such as blood, urine, and saliva, as well as in pathological tissues, and can be useful for the prediction (early detection) and diagnosis of diseases.

[0029] For example, the squaraine dye-peptide conjugate of the first invention can be used as a probe for neutrophil elastase (NE), which causes lung injury in COVID-19 patients and plays an important role in chronic inflammation and acute responses to infection and injury, and for neutrophil extracellular traps (NETs), which contribute to tissue damage in inflammatory diseases and some autoimmune diseases.

[0030] Specifically, the squaraine dye peptide conjugate of the first invention can be used as a reagent for detecting elastase. Furthermore, the squaraine dye peptide conjugate of the first invention can be used in a method for detecting elastase. Specifically, a method for detecting elastase using the squaraine dye peptide conjugate of the first invention comprises the steps of contacting the squaraine dye peptide conjugate of the first invention with a sample (step 1) and detecting elastase by measuring the fluorescence emission spectrum (step 2). In step 2, the fluorescence emission spectrum is measured to detect a change in fluorescence (change in absorption wavelength), thereby detecting elastase. The elastase to be detected is preferably NE contained in NETs.

[0031] Furthermore, the above-mentioned method for detecting elastase can also be used to provide information (measurement data) for predicting or diagnosing elastase-related diseases. Elastase is a prognostic biomarker and can be used to detect, for example, inflammatory bowel disease, chronic pancreatitis, colon cancer, lung cancer, chronic obstructive pulmonary disease, pancreatic cancer, etc.

[0032] The squaraine dye peptide conjugate according to the first aspect of the present invention can be produced by introducing an oligopeptide between two identical or different squaraine dyes. The introduction of an oligopeptide between the two squaraine dyes can be achieved by a known method. A specific example is solid-phase peptide synthesis (SPPS) using the Fmoc strategy.

[0033] <Squaraine dye peptide conjugate according to the second invention> The squaraine dye peptide conjugate according to the second invention is a compound represented by the following formula (1):

[0034] [ka]

[0035] R 1Brepresents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms. 1B may be a linear hydrocarbon group or a branched hydrocarbon group, but a linear hydrocarbon group is preferred. Also, may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, but a saturated hydrocarbon group (alkylene group) is preferred. As mentioned above, the number of carbon atoms is 3 to 30, but 3 to 18 is preferred, 3 to 12 is more preferred, and 3 to 6 is even more preferred.

[0036] R 2B represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms. 2B may be a linear hydrocarbon group or a branched hydrocarbon group, but a linear hydrocarbon group is preferred. Also, may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, but a saturated hydrocarbon group (alkyl group) is preferred. As mentioned above, the number of carbon atoms is 1 to 30, but 1 to 18 is preferred, 1 to 12 is more preferred, and 1 to 4 is even more preferred.

[0037] The aromatic skeleton in the above formula (1) may have a substituent. The substituent is preferably an electron-donating group. Specific examples include an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, and a hydroxyl group. These substituents are preferably substituted at a position that functions as an electron-donating group. Alternatively, the substituent may be a halogen group.

[0038] W represents a divalent linking group serving as a spacer between the carbon atom of the carbonyl group of the squaraine dye in formula (1) and the oligopeptide (Z). W is preferably a low-molecular-weight or PEG-based divalent linking group.

[0039] Examples of low-molecular-weight linking groups include those with a molecular weight of approximately 75 to 600, such as D- or L-amino acids (AAs), 3-amino-3-(2-nitrophenyl)propionic acid (ANP), β-alanine, 4-aminobutyric acid (GABA), 5-aminovaleric acid (GAva), 6-aminohexanoic acid (ACA / AHA / Ahx), (2-aminomethoxy)acetic acid (AEA), trioxatridecane-succinic acid (Ttds), and 12-aminododecanoic acid.

[0040] The PEG-based linking group is not particularly limited as long as it is a divalent group derived from a PEG derivative capable of linking a squaraine dye to an oligopeptide, and may be linear or branched, although linear is preferred. Examples of PEG derivatives include Mini-PEG and PEG-DGA-OH.

[0041] W is preferably a divalent linking group derived from glycine, β-alanine, 6-aminohexane, Mini-PEG, or PEG-DGA-OH, and particularly preferably a divalent linking group derived from PEG-DGA-OH.

[0042] Z represents a monovalent group derived from an oligopeptide that specifically binds to elastase (preferably neutrophil elastase). Z functions as an NE-recognition sequence having a sequence that recognizes NE, and contains a methionine (Met / M) in the sequence that forms a covalent bond with NE. Specifically, Z is preferably an oligopeptide consisting of 5 to 20 amino acids and containing methionine in the sequence, more preferably an oligopeptide consisting of 9 to 17 amino acids, and even more preferably an oligopeptide consisting of 11 to 15 amino acids. More specifically, Z is preferably a monovalent group derived from the amino acid sequence (GEAIPMSIPPEVK) set forth in SEQ ID NO: 5, or a monovalent group derived from an amino acid sequence obtained by deleting, substituting, and / or adding one or several (preferably one or two) amino acids in the amino acid sequence set forth in SEQ ID NO: 5 while retaining the methionine. G (glycine) is linked to a spacer (W).

[0043] Z may be cyclized. Examples of cyclized forms include those cyclized by introducing cystine (Cys / C) to both ends of the amino acid sequence set forth in SEQ ID NO: 5, and those cyclized by bonding the NH2 and OH of an amino acid. The amino acids contained in the sequence may be either L- or D-form.

[0044] The squaraine dye peptide conjugate according to the second aspect of the present invention is a compound represented by the formula (1), 1B represents a tetramethylene group, and R 2B Particularly preferred is a squaraine dye peptide conjugate in which represents an ethyl group, Y represents a divalent linking group derived from PEG-DGA-OH, and Z represents a monovalent group derived from the amino acid sequence set forth in SEQ ID NO: 5 (GEAIPMSIPPEVK). That is, a squaraine dye peptide conjugate represented by the following formula (1A) (SQ-215-NETP of Example 3) is preferred.

[0045] [ka]

[0046] The squaraine dye-peptide conjugate of the second invention is characterized by a specific squaraine dye bound to an oligopeptide specifically recognized by elastase. In particular, the squaraine dye-peptide conjugate of the second invention targets neutrophil elastase (NE), enabling it to distinguish between activated and resting neutrophils. The oligopeptide in this squaraine dye-peptide conjugate can be designed by utilizing the property of NE to bind to a 20-amino acid sequence within the reactive center loop (RCL) of α1-antitrypsin (AAT).

[0047] The inventors believe the reason for this is as follows: The squaraine dye-peptide conjugate of the second invention contains hydrophilic amino acids, which improves the solubility of the probe. In the hydrophilic tissue environment, molecular aggregation occurs, resulting in fluorescence quenching and very weak fluorescence. However, we speculate that upon reaching and accumulating in inflamed tissue, the conjugate covalently binds to NE in NETs, ​​allowing proteins and other large molecules in the tissue to enter the dye molecules, disrupting the dye aggregation and resulting in the appearance or enhancement of fluorescence.

[0048] The squaraine dye-peptide conjugate of the second invention, having the above-mentioned characteristics, can detect elastase with high sensitivity and selectivity by monitoring its near-infrared fluorescence characteristics, which allows for the sensitive detection of elastase in biological fluids such as blood, urine, and saliva, as well as in pathological tissues, and can be useful for the prediction (early detection) and diagnosis of diseases.

[0049] For example, the squaraine dye-peptide conjugate of the second invention can be used as a probe for neutrophil elastase (NE), which causes lung injury in COVID-19 patients and plays an important role in chronic inflammation and acute responses to infection and injury, and for neutrophil extracellular traps (NETs), which contribute to tissue damage in inflammatory diseases and some autoimmune diseases.

[0050] Specifically, the squaraine dye peptide conjugate of the second invention can be used as a reagent for detecting elastase. Furthermore, the squaraine dye peptide conjugate of the second invention can be used in a method for detecting elastase. Specifically, a method for detecting elastase using the squaraine dye peptide conjugate of the second invention includes a step (first step) of contacting the squaraine dye peptide conjugate of the second invention with a sample, and a step (second step) of detecting elastase by measuring the fluorescence emission spectrum. In the second step, elastase can be detected by measuring the fluorescence emission spectrum and detecting a change in fluorescence (change in absorption wavelength). The elastase to be detected is preferably NE contained in NETs.

[0051] Furthermore, the above-mentioned method for detecting elastase can also be used to provide information (measurement data) for predicting or diagnosing elastase-related diseases. Elastase is a prognostic biomarker and can be used to detect, for example, inflammatory bowel disease, chronic pancreatitis, colon cancer, lung cancer, chronic obstructive pulmonary disease, pancreatic cancer, etc.

[0052] The squaraine dye peptide conjugate according to the second aspect of the present invention can be produced by binding an oligopeptide specifically recognized by elastase to a specific squaraine dye. A known method can be used to bind an oligopeptide to the specific squaraine dye. A specific example is solid-phase peptide synthesis (SPPS) using the Fmoc strategy. [Example]

[0053] The first invention will be described in more detail below with reference to examples.

[0054] Example 1A <Preparation of the squaraine dye peptide conjugate Hetero-SQ215-APA-SQ46 of the present invention>

[0055] [ka]

[0056] (Production of squaraine dye SQ-215) The squaraine dye SQ-215 was manufactured as a raw material. The manufacturing process is outlined below.

[0057] [ka]

[0058] Each step will be specifically described below. [Process 1-1] (Production of Compound 2)

[0059] [ka]

[0060] To a solution of compound 1 (1,1,2-trimethyl-1H-benzo[e]indole) (50 mmol) in 150 mL of acetonitrile, 125 mmol of 1-iodoethane was added and refluxed for approximately 18 hours. The reaction was monitored by TLC, and after completion of the reaction, most of the solvent was evaporated. Ethyl acetate was added to the remaining solvent to reprecipitate the desired product. The solid was filtered, washed with sufficient ethyl acetate, and dried under vacuum to obtain compound 2 (90% yield).

[0061] Mass spectrometry (HRMS) of compound 2 was performed. FAB-Mass (m / z: calculated: 238.1590 for C 17 H 20 N + , observed: 238.1596).

[0062] [Step 1-2] (Production of Compound i)

[0063] [ka]

[0064] In a two-necked round-bottom flask placed in an ice bath, equimolar amounts of compound 2 obtained from the above reaction and dibutyl squarate (10 mmol) were dissolved in 15 mL of ethanol. Triethylamine (40 mmol) was added dropwise with stirring. The reaction mixture was stirred at room temperature under an inert atmosphere for 24 hours. The solid was filtered, washed with a minimum amount of ethanol, and dried under vacuum to give the half-dye butyl ester (60% yield) as a bright yellow solid.

[0065] Mass spectrometry (HRMS) of the half dye butyl ester was performed. FAB-Mass(m / z: Calculated: 389.1991 for C 25 H 27 NO3, Observed: 389.1996).

[0066] Next, because the hydrolyzed half-dye butyl ester is unstable, the ester was hydrolyzed immediately before proceeding to the final dye synthesis step. 2 mmol of half-dye butyl ester was dissolved in 20 mL of ethanol. 2 mL of 10% NaOH was added, refluxed for 30 minutes, and then cooled to room temperature. The reaction mixture was neutralized with 1.5 mL of 20% HCl, and water was added to precipitate the product. The solid was filtered, washed with water, and dried under vacuum to obtain compound i.

[0067] [Step 1-3] (Production of Compound 3)

[0068] [ka]

[0069] To a solution of compound 1 (15 mmol) in 50 mL of propionitrile, 37 mmol of ethyl 6-iodohexanoate was added and refluxed for approximately 18 hours. The reaction was monitored by TLC, and upon completion, most of the solvent was evaporated. Ethyl acetate was added to the remaining solvent to reprecipitate the desired product. The solid was then filtered, washed with sufficient ethyl acetate, and dried under vacuum to obtain compound 3 (3-(6-ethoxy-6-oxohexyl)-1,1,2-trimethyl-1H-benzo[e]indolium iodide) (80% yield) as a pale green, hygroscopic solid.

[0070] Mass spectrometry (HRMS) of compound 3 was performed. FAB-Mass(m / z: Calculated: 352.2271 for C 23 H 30 NO2 + , Observed: 352.2276).

[0071] [Step 1-4] (Production of Compound SQ-215)

[0072] [ka]

[0073] Equimolar amounts (~2 mmol) of the hydrolyzed compound i and compound 3 (3-(6-ethoxy-6-oxohexyl)-1,1,2-trimethyl-1H-benzo[e]indolium iodide) were dissolved in 1:1 butanol:benzene (40 mL) and refluxed under an inert atmosphere for 18-24 h. The reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated and the crude dye was purified by silica gel (C-200 mesh) column chromatography using chloroform and methanol as an eluent to obtain compound SQ-215 (80% yield) as a dark blue solid. Prior to column purification, the crude ester of the dye was hydrolyzed under the same conditions as for compound i.

[0074] Compound SQ-215 1 H-NMR analysis and mass spectrometry (HRMS) were performed. 1H NMR: 1δH (CDCl3, 500MHz), 8.2 (2H, dd, Ar-H), 7.88 (4H, m, Ar-H), 7.58 (2H, m, Ar-H), 7.43 (2H, t, Ar-H), 7.29 (2H, dd, Ar-H), 6.1 (1H, s, methine proton), 5.9 (1H, s, methine proton), 4.17 (4H, m, N-methylene protons), 2.48 (2H, t, carboxy methylene protons), 2.07 (12H, s, methyl), 1.99-1.89 (4H, m, methylene), 1.75 (2H, p, methylene), 1.45 (3H, t, methyl) HRMS: Found m / z: 638.3148 - 100%, Calculated: 638.3145 for C 42 H 42 N2O4.

[0075] (Production of squaraine dye SQ-46) The squaraine dye SQ-46 was produced. The manufacturing process is outlined below.

[0076] [ka]

[0077] Each step will be specifically described below. [Step 2-1] (Synthesis of 3-butyl-1,1,2-trimethyl-1H-benzo[e]indole-6-carboxylic acid ethyl ester iodide (1b))

[0078] [ka]

[0079] 2,3,3-Trimethyl-benzoindole-6-carboxylic acid methyl ester (420 mg, 1.5 mmol) and iodine butane (0.70 mL, 6 mmol) were dissolved in acetonitrile (40 mL). The reaction mixture was refluxed until the reaction was complete. After completion of the reaction (as confirmed by TLC), most of the solvent was evaporated. Ethyl acetate was added to the remaining solvent to reprecipitate the desired product. The solid was filtered, washed with plenty of ethyl acetate, and dried under vacuum to give a light brown solid (65% yield).

[0080] Mass spectrometry (HRMS FAB) of compound 1b was carried out. FAB-Mass m / z: Observed: 338.2126. Calculated: 338.2115 for C 22 H 28 NO2 +

[0081] [Step 2-2] (Production of Compound SQ-46) Equimolar amounts (approximately 2 mmol) of compound 1b and compound 2 were dissolved in 1:1 butanol:benzene (40 mL) and refluxed under an inert atmosphere for 24–48 h. After completion of the reaction (as confirmed by TLC), the solvent was evaporated, and the crude ester form of the dye was dissolved in 20 mL of ethanol for hydrolysis. 2 mL of 10% NaOH was added, refluxed for 30 min, and cooled to room temperature. The reaction mixture was neutralized with 1.5 mL of 20% HCl, and the product was precipitated by adding water. The solid was filtered, washed with water, and dried under vacuum to obtain crude dye SQ-46. This was purified by silica gel (C-300 mesh) column chromatography using chloroform and methanol as the eluent to obtain pure SQ-46 as a light brown solid (65% yield), the identity of which was confirmed by NMR and HRMS.

[0082] Compound SQ-46 1 H-NMR analysis and mass spectrometry (HRMS) were performed. 1 H NMR: 1 δ H8.28 (1H, t, Ar-H); 8.2 (1H, d, Ar-H); 8.04 (1H, dd, Ar-H); 7.74 (H, d, Ar-H); 7.54 (1H, d, Ar-H); 7.35 7.20 (1H, m, Ar-H); 5.86 (2H, d, methine protons); 4.19 - 4.14 (4H, m, N-methylene protons); 1.89 (6H, s, methyl); 1.74-1.68 (8H, m, methylene & methyl); 1.43 - 1.39 (2H, m, methylene); 1.30 (3H, 0.93 (3H, t, methyl). HRMS: Found m / z: 574.2840 - 100%, Calculated: 574.2832 for C 37 H 38 N2O4.

[0083] (Preparation of squaraine dye-peptide conjugates) All squaraine dye-peptide conjugates described in the examples were synthesized by manual solid-phase peptide synthesis (SPPS) using Rink Amide MBHA resin (LR = 0.69 mmol / g) as the solid support in an SPPS reaction vessel rotated on an electric rotator. HBTU and HOBt.HO were used as coupling agents to couple the squaraine dye to the amino acid (AA), and DMF was selected as the solvent for all reactions. SPPS was performed according to established procedures. Fmoc-Lys(Boc)-COOH was loaded onto the resin as the first AA, followed by checking the loading ratio (LR). Free sites on the resin were capped using acetic anhydride (10 eq) in DCM with DIPEA as a catalyst. Peptide chain elongation was performed by repeated cycles of Fmoc deprotection followed by coupling of each AA.

[0084] Using the above compounds SQ-215 and SQ-46, Hetero-SQ215-APA-SQ46 of Example 1A of the present invention was produced. The outline of the production process is shown below. The reaction conditions are shown in Table 1.

[0085] [ka]

[0086] [Table 1]

[0087] After treatment with the cleavage cocktail, the cocktail mixture was filtered from the SPPS reaction vessel, evaporated under reduced pressure, and reprecipitated from diethyl ether to give crude Hetero-SQ215-APA-SQ46, which was purified by silica gel (C-300 mesh) column chromatography using chloroform and methanol as the eluent to give the pure squaraine dye-peptide conjugate Hetero-SQ215-APA-SQ46 of the first invention as a dark blue solid (31% yield).

[0088] Mass spectrometry (HRMS) of the squaraine dye-peptide conjugate Hetero-SQ215-APA-SQ46 was performed. HRMS: Found m / z: [M+Na]+: 1654.8503(C 99 H 113 N 11 NaO 11 - 100%), Calculated: 1631.8621(C 99 H 113 N 11 O 11 ).

[0089] Example 2A <Preparation of the squaraine dye peptide conjugate Hetero-SQ-215-VPV-SQ-46 of the present invention>

[0090] The above-mentioned compound SQ-215 and compound SQ-46 were used to produce Hetero-SQ-215-VPV-SQ-46 of Example 2A of the present invention. The outline of the production process is shown below.

[0091] [ka]

[0092] Hetero-SQ-215-VPV-SQ-46 was synthesized and purified by the same procedure described for Hetero-SQ-215-APA-SQ-46, except that Ala was replaced with Val, to give the first invention squaraine dye-peptide conjugate Hetero-SQ-215-VPV-SQ-46 as a pure dark blue solid (yield: 25%).

[0093] Mass spectrometry (HRMS) of the squaraine dye-peptide conjugate Hetero-SQ-215-VPV-SQ-46 was performed. HRMS: Found m / z: [M+Na] + : 1710.9144 for C 103 H 121 N 11 NaO 11 - 100%, Calculated: 1687.9247 for C 103 H 121 N 11 O 11 .

[0094] [Reference Example 1A] <Preparation of squaraine dye-peptide conjugate Homo-SQ-215-APA>

[0095] Using the above compound SQ-215, Homo-SQ-215-APA of Reference Example 1A was produced. An outline of the production process is shown below. The reaction conditions are shown in Table 2. Note that Homo-SQ-215 of Reference Example 1A is a compound proposed by the present inventors in Japanese Patent Application No. 2023-189420.

[0096] [ka]

[0097] [Table 2]

[0098] After treatment with the cleavage cocktail, the cocktail mixture was filtered from the SPPS reaction vessel, evaporated under reduced pressure, and reprecipitated from diethyl ether to give crude Homo-SQ-215-APA, which was purified by silica gel (C-300 mesh) column chromatography using chloroform and methanol as the eluent to give the pure squaraine dye-peptide conjugate Homo-SQ-215-APA (Reference Example 1) as a blue solid (20% yield).

[0099] Mass spectrometry (TOF-Mass) of the squaraine dye-peptide conjugate Homo-SQ-215-APA was performed. Found m / z: [M+Na] + : 1718.88 - 100% for C 104 H 117 N 11 NaO 11 , Calculated: 1695.8968 for C 104 H 117 N 11 O 11 .

[0100] [HomoFRET imaging test] HomoFRET imaging was carried out using the squaraine dye-peptide conjugates of Examples 1A, 2A, and Reference Example 1A. The fluorescent dye was diluted to a final concentration of 10 μM in water or phosphate-buffered saline (PBS) supplemented with 1% DMSO. 200 μL of the solution containing the biologically active substance was prepared in a 96-well black plate. Either 1 μg of purified mouse neutrophil elastase (Lectinotest R&D) or 10 μg of fresh lysate of subcloned Nemeth-Kellner myeloblastoma cells overexpressing neutrophil elastase (NK / Ly-RB) was added to some wells, while other wells were imaged without NE to assess the photostability of the probe due to photodegradation or autodegradation. Fluorescence was analyzed using a PerkinElmer BioAssayreader HST7000 at 37°C, with excitation wavelengths of 680 / 10 nm and emission wavelengths of 720 / 20 nm, or a Li-CORPearl Trilogy In-Vivo imager (LI-CORBiosciences GmbH, Germany), with excitation wavelengths of 685 nm and emission wavelengths of 700 nm. The fluorescence kinetics was monitored over a 1 hour period and the results are shown in Figures 1 and 2.

[0101] As shown in Figures 1 and 2, all squaraine dye-peptide conjugates exhibited excellent activity against purified mouse neutrophil elastase (A) and intracellular elastase (B). In particular, Hetero-SQ215-APA-SQ46 (Example 1A) exhibited excellent activity. That is, it exhibited superior activity with alanine compared to valine.

[0102] [Cell permeability test] The squaraine dye-peptide conjugates of Examples 1A, 2A, and Reference Example 1A were subjected to a cell permeability test using NK / Ly-RB cells overexpressing NE. The results confirmed that the squaraine dye-peptide conjugates were cell impermeable.

[0103] [In vivo imaging study using an air pouch model] (Animal care and breeding conditions) Animal experiments were conducted in accordance with the 3R (Replacement, Reduction, Refinement) principle, based on protocols approved by the Ethics Committee of Danylo Halytsky Lviv National Medical University (20191216 / 10, 20210622 / 6). Mice were housed in a temperature-, humidity-, and light-controlled environment with free access to food and water.

[0104] (Test Procedure) Air pouch lavage fluid was isolated by injecting 5 ml of sterile PBS into the air pouch on days 1 and 3. Neutrophil infiltration was induced by injecting 1 mg of sterile MSU crystals on day 5, and imaging was performed on day 6 by injecting 10 μM of NE probe (squaraine dye-peptide conjugate) in 1% DMSO aqueous solution. A Li-CORPearl Trilogy In-vivo imager was used for excitation with 685 nm and 785 nm lasers, and emission was analyzed in the 700 nm and 800 nm channels at a resolution of 170 μm. Images were normalized using ImageStudio software. The outline of the test is shown in Figure 3, and the results are shown in Figure 4.

[0105] As shown in FIG. 4, Hetero-SQ-215-APA-SQ-46 of Example 1 showed the highest activity.

[0106] [In vivo test using gout-induced mice] One mg of needle-shaped MSU crystals was injected into the left paw, and saline was injected into the right paw, designated as the control. Another mouse also received an IP injection of 20 mg / kg of proprietary Neutrocure Compound 80 (NCure80) into the left paw. Both mice received an IP injection of 1 μM of the squaraine dye-peptide conjugate (Hetero-SQ215-APA-SQ46) according to Example 1A, and the fluorescence kinetics were measured 4 hours after administration. The fluorescence kinetics measurement results are shown in Figure 5. The measurements were performed using the same method as in the in vivo imaging study using the air pouch model.

[0107] As shown in Figure 5, a stable and strong signal was observed only in the gouty paw, demonstrating the probe's ability to selectively detect NETs and their accumulation in the inflamed area. No toxicity, behavioral changes, or weight changes were observed during the study.

[0108] The second invention will be described in more detail below with reference to examples.

[0109] Example 1B <Preparation of the squaraine dye peptide conjugate SQ-215-NETP of the present invention>

[0110] The compound SQ-215 was used to produce SQ-215-NETP of the second invention. The outline of the production process is shown below. The reaction conditions are shown in Table 3.

[0111] [ka]

[0112] [Table 3]

[0113] The synthesis was stopped at the Fmoc-GEAIPMSIPPEVK-Resin (side chain protection) step to confirm sequence identity, and the resin was treated with a cleavage cocktail, followed by filtration, evaporation under reduced pressure, and reprecipitation from diethyl ether to give crude Fmoc-GEAIPMSIPPEVK-NH2.

[0114] Crude Fmoc-GEAIPMSIPPEVK-NH2 1 H-NMR analysis and mass spectrometry (HRMS) were performed. Found m / z: [M+H] + : 1588.8057 for C 76 H 114 N 15 O 20 S1, [M+Na] + : 1610.7879 for C 76 H 113 N15 NaO 20 S1- 100%, Calculated: 1587.8007 for C 76 H 113 N 15 O 20 S1.

[0115] After confirming the desired peptide sequence, the next step was performed. After complete cleavage, the reaction mixture was filtered, evaporated under reduced pressure, and reprecipitated from diethyl ether to obtain crude SQ-215-NETP. Crude SQ-215-NETP was first semi-purified by size-exclusion chromatography on Sephadex LH-20 gel using methanol as the solvent. It was then further purified by preparative HPLC using an Xterra Prep MS C18 OBD 10 μm column. The mobile phase consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). A linear gradient of solvent B against solvent A (from 0% to 45% over 35 min) was used at a flow rate of 5.0 mL / min to obtain highly pure SQ-215-NETP.

[0116] High purity SQ-215-NETP of 1 H-NMR analysis and mass spectrometry (HRMS) were performed. Found m / z: [M+H] + : 1588.8057 for C 76 H 114 N 15 O 20 S1, [M+Na] + : 1610.7879 for C 76 H 113 N 15 NaO 20 S1- 100%, Calculated: 1587.8007 for C 76 H 113 N 15 O 20 S1.

[0117] [HomoFRET imaging test] HomoFRET imaging was performed using the squaraine dye-peptide conjugate of Example 1B in the same manner as in Example 1A, etc. The results are shown in Figures 6 and 7.

[0118] As shown in Figures 6 and 7, SQ-215-NETP of Example 1B exhibited superior activity to the squaraine dye-peptide conjugates of Examples 1A, 2A, and Reference Example 1A.

[0119] [Cell permeability test] The squaraine dye-peptide conjugate of Example 1B was subjected to a cell permeability test using NK / Ly-RB cells overexpressing NE, and the results confirmed that the squaraine dye-peptide conjugate was cell impermeable.

[0120] [In vivo study of hetero-APA probes in gout-induced mice] One mg of needle-shaped MSU crystals was injected into the left paw, and saline was injected into the right paw, designated as the control. Another mouse also received an IP injection of 20 mg / kg of proprietary Neutrocure Compound 80 (NCure80) into the left paw. 1 μM of the squaraine dye-peptide conjugate (SQ-215-NETP) of Example 1B was injected IP into both mice, and fluorescence kinetics were measured 4 hours after administration. The results are shown in Figure 8.

[0121] As shown in Figure 8, a stable and strong signal was observed only from the gouty paw, demonstrating the ability of the squaraine dye-peptide conjugate of Example 1B (SQ-215-NETP) to selectively detect NETs and accumulate in the inflamed area.

[0122] [Histopathological examination] The squaraine dye-peptide conjugate SQ-215-NETP of Example 1B was tested on histopathological samples of NETs derived from human aortic thrombi and human mucosal surfaces (human eye).

[0123] Histological evaluation of SQ-215-NETP was performed using human ocular NETs and human aortic thrombi. The former were smeared onto slides and fixed in methanol, while the latter were frozen into 7-μm-thick sections, fixed with PFA, and stained. An 800 nM aqueous solution of NETP-SQ215 was added to the tissue samples for 30 minutes, washed with water, and then 1 μg / ml PI was added for an additional 10 minutes. Slides were either wet-mounted in a fluorescent aqueous mounting medium or washed with water, air-stained, cleared with RotiClear (Carl Roth, DE) organic solution or butanol, and mounted with an organic-based, xylene-free RotiMount (Carl Roth, DE) polymeric mounting medium. Fluorescence microscopy was performed using a routine fluorescent microscope.

[0124] (Human thrombus-derived NETs) Figure 9 shows a fluorescence micrograph of NETs derived from human thrombi in the presence of 800 nM SQ-215-NETP. Figure 9B shows a fluorescence micrograph of the framed area in A with increased depth of field, and Figure 9C shows a confocal scan of the corresponding area. In Figure 9C, the faint signal is due to NE-positive granules specific to DNA fibers. Note that blue indicates 700 nM SQ-215-NETP, and red indicates PI staining, which indicates DNA.

[0125] As shown in Figure 9, NETosis observed around DNA released from neutrophil cells (as judged by nuclear morphology) was accompanied by a "cloud" of NE-positive signal derived from SQ-215-NETP. Several large NE-containing granules were observed inside the neutrophil or near the site of NETosis. Further analysis of the "NE-positive cloud" using confocal scanning microscopy revealed a collection of small NE granules adorning the externalized DNA, a feature generally considered a typical feature of NETosis. The resolution limit achieved was approximately 100 nm / pixel.

[0126] The squaraine dye-peptide conjugate SQ-215-NETP of Example 1B was found to be effective in staining NETs derived from human thrombi.

[0127] (NETs derived from mucosal surfaces) Figure 10 shows fluorescence micrographs of NETs derived from mucosal surfaces (human ocular NETs) in the presence of 800 nM SQ-215-NETP. (A) shows evaluation by fluorescence microscopy alone, and (B) shows evaluation in combination with optical sectioning. Green indicates SQ-215-NETP, and red indicates DNA. A structured illumination optical sectioning fluorescence microscope with a resolution of approximately 90 nm / pixel was used.

[0128] The squaraine dye-peptide conjugate SQ-215-NETP of Example 1B was found to be effective in staining NETs derived from mucosal surfaces.

[0129] As described above, the squaraine dye-peptide conjugate SQ-215-NETP of Example 1B of the second invention has a low molecular weight, allowing it to provide high-resolution imaging. Furthermore, because the squaraine dye-peptide conjugate SQ-215-NETP of Example 1B has a molecular weight below the 5 kDa filtration limit of the kidney, it specifically binds to elastase after systemic injection, while the unbound squaraine dye-peptide conjugate is filtered out by the kidney.

[0130] [Flow cytometry analysis] Flow cytometry analysis of mouse bone marrow after NETs induction was performed using the squaraine dye-peptide conjugate according to the second invention.

[0131] The experiment was carried out under the following conditions. Mouse bone marrow was stained with SQ-215-NETP before and after induction of mild NETosis using 48 mM bicarbonate for 30 minutes. SQ-215-NETP-positive particles correspond to PMN cells (high SSC) or NETs (low FSC). A 632 nm laser was used for excitation, and detection was performed using the standard APC channel (650 / 20 nm). The results are shown in Figure 11.

[0132] Next, we performed similar analysis on mouse bone marrow stained with SQ-215-NETP after potently inducing NETosis using PMA for 3 hours. Excitation was performed using a 632 nm laser and detection was performed using the standard APC channel (650 / 20 nm). The results are shown in Figure 12.

[0133] As shown in Figures 11 and 12, SQ-215-NETP was confirmed to be capable of detecting NETs-positive particles in flow cytometry analysis. SQ-215-NETP can be used to detect NETs in clinical blood samples, which makes it possible to design an NIR detection kit for NETs. [Industrial Applicability]

[0134] The novel squaraine dye-peptide conjugate of the present invention is industrially useful since it can be used for detecting elastase, etc. [Sequence List Free Text]

[0135] [SEQ ID NO: 1] 1 shows the amino acid sequence (AAPI) of the squaraine dye peptide conjugate of the present invention. [SEQ ID NO: 2] 1 shows the amino acid sequence (AAPV) of the squaraine dye peptide conjugate of the present invention. [SEQ ID NO: 3] 1 shows the amino acid sequence (AAPA) of the squaraine dye peptide conjugate of the present invention. [SEQ ID NO: 4] 1 shows the amino acid sequence (AVSLGD) of the squaraine dye peptide conjugate of the present invention. [SEQ ID NO: 5] 1 shows the amino acid sequence (GEAIPMSIPPEVK) of the squaraine dye peptide conjugate of the present invention.

Claims

1. A squaraine dye peptide conjugate represented by formula (I): 【Chemistry 1】 (In formula (I), R １Ａ represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R ２Ａ ~R ４Ａ each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, R ５Ａ represents an amino group, a carboxy group, or an ester group, X represents a divalent linking group derived from any one of the following amino acid sequences (1) to (7) (A represents alanine, V represents valine, P represents proline, I represents isoleucine, S represents serine, L represents leucine, G represents glycine, and D represents aspartic acid), (1) APA (2) VPV (3) APV (4) AAPI (SEQ ID NO: 1) (5) AAPV (SEQ ID NO: 2) (6) AAPA (SEQ ID NO: 3) (7) AVSLGD (SEQ ID NO: 4) Y represents a divalent linking group as a spacer.

2. In formula (I), R １Ａ represents an alkylene group having 3 to 18 carbon atoms, and R ２Ａ ~R ４Ａ 2. The squaraine dye peptide conjugate according to claim 1, wherein each independently represents an alkyl group having 1 to 18 carbon atoms.

3. In formula (I), R １Ａ represents an alkylene group having 3 to 6 carbon atoms, and R ２Ａ ~R ４Ａ 3. The squaraine dye peptide conjugate according to claim 2, wherein each independently represents an alkyl group having 1 to 4 carbon atoms.

4. 2. The squaraine dye peptide conjugate of claim 1, wherein in formula (I), Y is a small molecule-based or PEG-based divalent linking group.

5. 5. The squaraine dye peptide conjugate according to claim 1, wherein in formula (I), X represents a divalent linking group derived from APA, and Y represents a divalent linking group derived from β-alanine.

6. 5. The squaraine dye peptide conjugate according to claim 1, wherein in formula (I), X represents a divalent linking group derived from VPV, and Y represents a divalent linking group derived from β-alanine.

7. A reagent for detecting elastase, comprising the squaraine dye-peptide complex of claim 1.

8. contacting a sample with the squaraine dye-peptide conjugate of claim 1; detecting elastase by measuring the fluorescence emission spectrum; A method for detecting elastase, comprising:

9. A method for providing information necessary for predicting or diagnosing an elastase-related disease, comprising: contacting a sample with the squaraine dye-peptide conjugate of claim 1; detecting elastase by measuring the fluorescence emission spectrum; 1. A method for providing information, comprising:

10. A squaraine dye peptide conjugate represented by the following formula (1): 【Chemistry 2】 (In formula (1), R １Ｂ represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R ２Ｂ represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, W represents a divalent linking group as a spacer; Z represents a monovalent group derived from an oligopeptide that specifically binds to elastase.

11. 11. The squaraine dye peptide conjugate according to claim 10, wherein Z in formula (1) represents a monovalent group derived from the amino acid sequence set forth in SEQ ID NO: 5, or a monovalent group derived from the amino acid sequence set forth in SEQ ID NO: 5 in which one or several amino acids have been deleted, substituted, and / or added while retaining methionine.

12. 12. The squaraine dye peptide conjugate of claim 10, wherein in formula (1), W is a divalent linking group based on a small molecule or PEG.

13. 13. The squaraine dye peptide conjugate according to claim 12, wherein in formula (1), W represents a divalent linking group derived from PEG-DGA-OH.

14. In formula (1), R １Ｂ represents an alkylene group having 3 to 18 carbon atoms, and R ２Ｂ 11. The squaraine dye peptide conjugate according to claim 10, wherein represents an alkyl group having 1 to 18 carbon atoms.

15. In formula (1), R １Ｂ represents an alkylene group having 3 to 6 carbon atoms, and R ２Ｂ 15. The squaraine dye peptide conjugate according to claim 14, wherein represents an alkyl group having 1 to 4 carbon atoms.

16. A reagent for detecting elastase, comprising the squaraine dye-peptide complex according to claim 10.

17. contacting a sample with the squaraine dye-peptide conjugate of claim 10; detecting elastase by measuring the fluorescence emission spectrum; A method for detecting elastase, comprising:

18. A method for providing information necessary for predicting or diagnosing an elastase-related disease, comprising: contacting a sample with the squaraine dye-peptide conjugate of claim 10; detecting elastase by measuring the fluorescence emission spectrum; 1. A method for providing information, comprising:

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