Novel squaraine dye-peptide complex

Abstract

A squaraine dye-peptide complex according to the present invention is represented by formula (I). (In formula (I), R1A represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R2A to R4A each independently represent a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, R5A represents an amino group or a carboxy group, and X represents any of 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).)

Description

Novel squaraine dye-peptide conjugates

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

[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 substances. 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, new near-infrared fluorescent dyes that fluoresce, especially in buffer solutions (body fluids), are in demand, and this is expected to lead to applications in biosensing and in vivo bioimaging.

[0004] Special Publication No. 2021-504538

[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.

[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 have 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 have 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 provides the following: [1] A squaraine dye peptide conjugate represented by the following formula (I): (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 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 [3] In the formula (I), R １Ａ represents an alkylene group having 3 to 6 carbon atoms, and R ２Ａ ~R ４Ａeach independently represent an alkyl group having 1 to 4 carbon atoms. [4] The squaraine dye peptide conjugate according to [1] above, wherein in formula (I), Y 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] A method for detecting elastase, comprising the steps of: contacting a sample with the squaraine dye peptide conjugate of any one of [1] to [6] above; and detecting elastase by measuring the fluorescence emission spectrum. [9] A method for providing information necessary for predicting or diagnosing elastase-related diseases, comprising the steps of: contacting a sample with the squaraine dye peptide conjugate of any one of [1] to [6] above; and detecting elastase by measuring the fluorescence emission spectrum.

[0009]

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

[11] The squaraine dye peptide conjugate according to

[10] above, characterized in that 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, characterized in that in formula (1), W represents a divalent linking group based on a small molecule or PEG.

[13] The squaraine dye peptide conjugate according to

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

[14] The squaraine dye peptide conjugate according to formula (1), １Ｂ represents an alkylene group having 3 to 18 carbon atoms, and R ２Ｂ

[15] The squaraine dye peptide conjugate according to any one of

[10] to

[13] above, wherein R represents an alkyl group having 1 to 18 carbon atoms. １Ｂ represents an alkylene group having 3 to 6 carbon atoms, and R ２Ｂ

[16] A 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 method for detecting elastase, comprising the steps of contacting a sample with the squaraine dye peptide conjugate according to any one of

[10] to

[15] above, and detecting elastase by measuring the fluorescence emission spectrum.

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

[10] to

[15] above, and detecting elastase by measuring the fluorescence emission spectrum.

[0010] The novel squaraine dye-peptide conjugate of the present invention is capable of selectively detecting elastase, which is one of the biomarkers.

[0011] 1 is a diagram showing the results of a HomoFRET imaging test of a squaraine dye peptide conjugate according to the first invention.

[0034] FIG. 1 is a diagram showing the results of measuring fluorescence kinetics after 1 minute and 60 minutes in a HomoFRET imaging test of a squaraine dye peptide conjugate according to the first invention.

[0035] FIG. 1 is a diagram showing an outline of an in vivo imaging test of a squaraine dye peptide conjugate according to the first invention using an air pouch model.

[0036] FIG. 1 is a diagram showing the results of an in vivo imaging test of a squaraine dye peptide conjugate according to the first invention using an air pouch model.

[0037] FIG. 1 is a diagram showing the results of measuring fluorescence kinetics in an in vivo test of a squaraine dye peptide conjugate according to the first invention using gout-induced mice.

[0038] FIG. 1 is a diagram showing the results of a HomoFRET imaging test of a squaraine dye peptide conjugate according to the second invention.

[0039] FIG. 1 is a diagram showing the results of measuring fluorescence kinetics after 1 minute and 60 minutes in a HomoFRET imaging test of a squaraine dye peptide conjugate according to the second invention.

[0039] FIG. 1 is a diagram showing the results of measuring fluorescence kinetics in an in vivo test of a squaraine dye peptide conjugate according to the second invention using gout-induced mice.

[0039] FIG. 2 is a fluorescence microscope photograph of NETs derived from human thrombus in the presence of a squaraine dye peptide conjugate according to the second invention. Figure 1 shows a fluorescence micrograph of NETs derived from a mucosal surface in the presence of the squaraine dye peptide conjugate of the second invention. Figure 2 shows a flow cytometry analysis of mouse bone marrow after mild induction of NETs using the squaraine dye peptide conjugate of the second invention. Figure 3 shows a flow cytometry analysis of mouse bone marrow after strong induction of NETs using the squaraine dye peptide conjugate of the second invention.

[0012] <Squaraine Dye Peptide Conjugate of the First Invention> The squaraine dye peptide conjugate of the first invention is a compound represented by the following formula (I).

[0013]

[0014] R １Ａrepresents 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. The number of carbon atoms is 3 to 30, as described above, but is preferably 3 to 18, more preferably 3 to 12, and even more preferably 3 to 6.

[0015] R ２Ａ ~R ４Ａ R each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms. ２Ａ ~R ４Ａ 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. R 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 the lysine. The amino acids contained in the sequence may be either L- or D-configuration. Particularly preferred as X is (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 molecules for the low-molecule-based linking group include those with a molecular weight of about 75 to 600, and specific examples include 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, with linear being 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. That is, a squaraine dye peptide conjugate represented by the following formula (IA) (Hetero-SQ215-APA-SQ46 in Example 1A) is preferred.

[0024]

[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 a polar solvent such as water or a buffer solution, 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 according to 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, it is speculated that in tissues where inflammation has occurred, the NE in NETs undergoes hydrolysis, and interaction with the tissue causes the appearance or enhancement of fluorescence.

[0028] The squaraine dye-peptide conjugate of the first invention, having the above-mentioned characteristics, enables highly sensitive and selective detection of elastase by monitoring its near-infrared fluorescence characteristics, thereby enabling sensitive detection of elastase in biological fluids such as blood, urine, and saliva, as well as in pathological tissues, and thus useful for disease prediction (early detection) and diagnosis.

[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 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 includes a step (step 1) of contacting the squaraine dye peptide conjugate of the first invention with a sample, and a step (step 2) of detecting elastase by measuring the fluorescence emission spectrum. In step 2, the fluorescence emission spectrum is measured to detect a change in fluorescence (change in absorption wavelength), thereby enabling detection of elastase. The elastase to be detected is preferably NE contained in NETs.

[0031] Furthermore, the above-described 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, colorectal 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 of such a method is solid-phase peptide synthesis (SPPS) using the Fmoc strategy.

[0033] <Squaraine Dye Peptide Conjugate of the Second Invention> The squaraine dye peptide conjugate of the second invention is a compound represented by the following formula (1).

[0034]

[0035] R １Ｂ represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms. １Ｂmay 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. The number of carbon atoms is 3 to 30, as described above, but is preferably 3 to 18, more preferably 3 to 12, and even more preferably 3 to 6.

[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. 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.

[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 molecules for the low-molecule-based linking group include those with a molecular weight of about 75 to 600, and specific examples include 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, with linear being 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 acts as an NE recognition sequence having a sequence that recognizes NE and has 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 having a 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 in which cystine (Cys / C) is introduced at both ends of the amino acid sequence shown in SEQ ID NO: 5, or those in which NH of an amino acid is introduced. ２ and OH bonded to form a cyclized amino acid. The amino acids contained in the sequence may be either L- or D-amino acids.

[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 ２ＢParticularly preferred is a squaraine dye peptide conjugate in which Y 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 (GEAIPMSIPPEVK) set forth in SEQ ID NO: 5. That is, a squaraine dye peptide conjugate represented by the following formula (1A) (SQ-215-NETP in Example 3) is preferred.

[0045]

[0046] The squaraine dye-peptide conjugate of the second invention is characterized by conjugating a specific squaraine dye 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 of 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 according to the second invention also 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 the NEs in the 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 according to the second aspect of the present invention, having the above-mentioned characteristics, allows for highly sensitive and selective detection of elastase 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 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 (step 1) of contacting the squaraine dye peptide conjugate of the second invention with a sample, and a step (step 2) of detecting elastase by measuring the fluorescence emission spectrum. In step 2, the fluorescence emission spectrum is measured to detect a change in fluorescence (change in absorption wavelength), thereby enabling detection of elastase. The elastase to be detected is preferably NE contained in NETs.

[0051] Furthermore, the above-described 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, colorectal 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 conjugating a specific squaraine dye with an oligopeptide that is specifically recognized by elastase. A known method can be used to conjugate an oligopeptide to the specific squaraine dye. A specific example of such a method is solid-phase peptide synthesis (SPPS) using the Fmoc strategy.

[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]

[0056] (Production of squaraine dye SQ-215) The squaraine dye SQ-215 was produced as a raw material. The outline of the production process is shown below.

[0057]

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

[0059]

[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]

[0064] In a two-necked round-bottom flask placed in an ice bath, equimolar amounts of compound 2 obtained in 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] Subsequently, 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, and the mixture was 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]

[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 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 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]

[0073] Equimolar amounts (approximately 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 to 24 hours. 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 (yield 80%) as a dark blue solid. Prior to column purification, the crude ester of the dye was hydrolyzed under the same conditions as for the hydrolysis of Compound i.

[0074] Compound SQ-215 １H-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) Squaraine dye SQ-46 was produced. The outline of the production process is shown below.

[0076]

[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]

[0079] 2,3,3-Trimethyl-benzoindole-6-carboxylic acid methyl ester (420 mg, 1.5 mmol) and iodobutane (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 performed. FAB-Mass m / z: Observed: 338.2126. Calculated: 338.2115 for C 22 H 28 NO2 +

[0081] [Step 2-2] (Preparation 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 to 48 hours. After completion of the reaction (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 minutes, 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 an 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 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.H were used as coupling agents to couple the squaraine dyes to the amino acids (AA). ２ O was used, 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] The above compounds SQ-215 and SQ-46 were used to produce Hetero-SQ215-APA-SQ46 of Example 1A of the present invention. The outline of the production process is shown below. The reaction conditions are shown in Table 1.

[0085]

[0086]

[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 compounds SQ-215 and 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]

[0092] Hetero-SQ-215-VPV-SQ-46 was synthesized and purified by the same procedure as described for Hetero-SQ-215-APA-SQ-46, except that Ala was replaced with Val, to give the squaraine dye-peptide conjugate of the first invention, 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]

[0097]

[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 (20% yield) of Reference Example 1 as a blue solid.

[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 Experiments: HomoFRET imaging was performed using the squaraine dye-peptide conjugates of Examples 1A, 2A, and Reference Example 1A. The fluorescent dyes were diluted to a final concentration of 10 μM in water or phosphate-buffered saline (PBS) containing 1% DMSO. 200 μL of a 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 a 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 probes due to photo / 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 with a Li-CORPearl Trilogy In-Vivo Imager (LI-CORBiosciences GmbH, Germany) with excitation wavelengths of 685 nm and emission wavelengths of 700 nm. Fluorescence kinetics was monitored over a 1-hour period. 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] A cell permeability test was performed on NK / Ly-RB cells overexpressing NE for the squaraine dye-peptide conjugates of Examples 1A, 2A, and Reference Example 1A. The results confirmed that the squaraine dye-peptide conjugates were cell impermeable.

[0103] [In vivo imaging test using the air pouch model] (Animal care and breeding conditions) Animal experiments were conducted in compliance with the 3R (replacement, reduction, refinement) principles based on protocols (20191216 / 10, 20210622 / 6) approved by the Ethics Committee of Danylo Halytsky Lviv National Medical University. Mice were kept in a temperature-, humidity-, and light-controlled environment with free access to food and water.

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

[0107] As shown in Figure 5, a stable and strong signal was observed only in the gouty paw, indicating the probe's ability to selectively detect NETs and accumulate 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]

[0112]

[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 N15 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.

[0115] After confirming the desired peptide sequence, the next step was carried out. 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 relative to solvent A (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 carried out 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] A cell permeability test was performed on the squaraine dye-peptide conjugate of Example 1B using NK / Ly-RB cells overexpressing NE. The results confirmed that the squaraine dye-peptide conjugate was cell impermeable.

[0120] In vivo testing of the hetero-APA probe 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 the 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, indicating that the squaraine dye-peptide conjugate of Example 1B (SQ-215-NETP) has the ability to selectively detect NETs and accumulate in the inflamed area.

[0122] Histopathological Testing The squaraine dye peptide conjugate SQ-215-NETP of Example 1B was used to test histopathological samples for NETs derived from human aortic thrombi and human mucosal surfaces (human eyeballs).

[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 in 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] (NETs derived from human thrombi) 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 an increased depth of field, and Figure 9C shows a confocal scan of the corresponding area. In Figure 9C, the blurred signal is due to specific NE-positive granules of 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 (as judged by nuclear morphology) observed around DNA released from neutrophil cells was accompanied by a "cloud" of NE-positive signal derived from SQ-215-NETP, and several large NE-containing granules were observed inside the neutrophil or near the NETosis site. Further analysis of the "NE-positive cloud" using confocal scanning microscopy revealed a collection of small NE granules decorating the externalized DNA, which is generally considered a typical feature of NETosis. The achieved resolution limit 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 (NETs derived from human ocular balls) 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 was carried out on mouse bone marrow after inducing NETs using the squaraine dye-peptide conjugate according to the second invention.

[0131] The experiment was performed 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] Subsequently, mouse bone marrow stained with SQ-215-NETP was analyzed in the same manner 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, making it possible to design an NIR detection kit for NETs.

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

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

Claims

1. A squaraine dye peptide conjugate represented by formula (I): (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 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. 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. The squaraine dye peptide conjugate according to any one of claims 1 to 4, 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 claims 1 to 4, 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. A method for detecting elastase, comprising the steps of: bringing the squaraine dye-peptide complex of claim 1 into contact with a sample; and detecting elastase by measuring the fluorescence emission spectrum.

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

10. A squaraine dye-peptide conjugate represented by the following formula (1): (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, and Z represents a monovalent group derived from an oligopeptide that specifically binds to elastase.

11. The squaraine dye peptide conjugate according to claim 10, characterized in that in formula (1), Z 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 more amino acids have been deleted, substituted and / or added while retaining methionine.

12. The squaraine dye peptide conjugate of claim 10 or 11, wherein in formula (1), W is a small molecule-based or PEG-based divalent linking group.

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 of claim 10.

17. A method for detecting elastase, comprising the steps of: bringing the squaraine dye-peptide complex of claim 10 into contact with a sample; and detecting elastase by measuring the fluorescence emission spectrum.

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

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