Novel squaraine dye-peptide complex and novel squaraine dye

Abstract

To provide a novel squaraine dye-peptide complex that enables selective detection of esterase, being one of tumor markers.SOLUTION: A squaraine dye-peptide complex is prepared by incorporating an oligopeptide between two squaraine dyes represented by formula (1), where R1B and R2B each independently represent a C1 to C30 monovalent aliphatic hydrocarbon group, with at least one having a carboxyl group at a terminal, and R3B and R4B each independently represent H or a carboxyl group, with at least one being a carboxyl group.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a novel squaraine dye peptide complex and a novel squaraine dye.

Background Art

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

[0003] Also, detection and sensing using near-infrared fluorescent dyes have technical simplicity, very high sensitivity, can immediately measure molecular interactions in situ, and can effectively detect the target substance. On the other hand, although there are reports of near-infrared fluorescent dyes that 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 particular need for new near-infrared fluorescent dyes that fluoresce in buffer solutions (body fluids), and this is expected to lead to applications in biosensing and in vivo bioimaging.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a novel squarylium dye peptide complex capable of selectively detecting esterase, which is one of the tumor markers. Another object is to provide a novel squarylium dye having near-infrared fluorescence characteristics. [Means for Solving the Problems]

[0006] The present inventors have found that by introducing a predetermined oligopeptide between predetermined squarylium dyes, it is possible to selectively detect esterase, which is one of the tumor markers, by utilizing near-infrared fluorescence characteristics, and have completed the present invention.

[0007] In addition, the present inventors have found that a squarylium dye having a specific structure has excellent near-infrared fluorescence characteristics, and have completed the present invention.

[0008] That is, the present invention is as follows. [1] A squarylium dye peptide complex represented by the following formula (I). [Chemical Formula] (In formula (I), R 1A and R 2A each independently represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R 3A and R 4A each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, R 5A represents an amino group or a carboxy group, X represents a divalent linking group derived from any of the following amino acid sequences (where 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 derived from glycine, β-alanine, 6-aminohexanoic acid, Mini-PEG, and PEG-DGA-OH.) [2] In formula (I), R 1A and R 2A each independently represents an alkylene group having 3 to 18 carbon atoms, and R 3A and R 4A each independently represents an alkyl group having 1 to 18 carbon atoms, characterized in that the squalaine dye peptide complex according to [1] above.) [3] In formula (I), R 1A and R 2A represent an alkylene group having 3 to 6 carbon atoms, and R 3A and R 4A represent an alkyl group having 1 to 4 carbon atoms, characterized in that the squalaine dye peptide complex according to [2] above.) [4] In formula (I), X represents a divalent linking group derived from APA, and Y represents a divalent linking group derived from β-alanine, characterized in that the squalaine dye peptide complex according to any one of [1] to [3] above.) [5] A reagent for detecting elastase, characterized by containing the squalaine dye peptide complex according to any one of [1] to [4] above.) [6] A step of contacting the squalaine dye peptide complex according to any one of [1] to [4] above with a sample, and a step of detecting elastase by measuring a fluorescence emission spectrum, characterized in that it has a method for detecting elastase.) [7] A method for providing information necessary for predicting or diagnosing a disease related to elastase, a step of contacting the squalaine dye peptide complex according to any one of [1] to [4] above with a sample, and a step of detecting elastase by measuring a fluorescence emission spectrum, characterized in that it has a method for providing information.)

[0009] [8] A squarylium dye characterized by being represented by the following formula (1). [Chemical formula] (In formula (1), R 1B and R 2B each independently represent a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, and at least one of them has a carboxy group at the terminal. R 3B and R 4B represent a hydrogen atom or a carboxy group, and at least one of them is a carboxy group.) [9] In formula (1), when R 1B and R 2B have a carboxy group at the terminal, they each independently represent an alkyl group having 2 to 18 carbon atoms. The squarylium dye according to the above [8], wherein when R 1B and R 2B do not have a carboxy group at the terminal, they represent an alkyl group having 1 to 18 carbon atoms.

[10] The squarylium dye according to the above [8] or [9], wherein in formula (1), R 1B and R 2B have a carboxy group at the terminal.

[11] The squarylium dye according to the above

[10] , wherein in formula (1), R 1B and R 2B represent an alkyl group having 2 to 6 carbon atoms.

[12] The squarylium dye according to any one of the above [8] to

[11] , wherein in formula (1), R 3B and R 4B represent a carboxy group. [Advantages of the Invention]

[0010] The novel squarylium dye - peptide complex of the present invention enables the selective detection of esterase, which is one of the tumor markers. Further, the novel squarylium dye of the present invention exhibits excellent near - infrared fluorescence characteristics effect. [Brief Description of the Drawings]

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0012] <Squaline Dye Peptide Complex> The squaline dye peptide complex according to the first invention is a compound represented by the following formula (I).

[0013]

Chemical formula

[0014] R 1A and R 2A each independently represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms. R 1A and R 2A may be a linear hydrocarbon group or a branched hydrocarbon group, but a linear hydrocarbon group is preferred. Also, it 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, as described above, 3 to 30, but 3 to 18 is preferred, 3 to 12 is more preferred, and 3 to 6 is even more preferred. R 1A and R 2A may be the same or different, but it is preferred that they are the same.

[0015] R 3A and R 4A each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms. R 3A and R 4A may be a linear hydrocarbon group or a branched hydrocarbon group, but a linear hydrocarbon group is preferred. Also, it 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, as described above, 1 to 30, but 1 to 18 is preferred, 1 to 12 is more preferred, and 1 to 4 is even more preferred. R 3A and R 4AIt may be the same or different, but it is preferably the same.

[0016] The aromatic skeleton in the above formula (I) may have a substituent. As the substituent, an electron-donating group is preferred. Specifically, for example, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a hydroxyl group, etc. can be mentioned. These substituents are preferably substituted at positions where they function as electron-donating groups. Also, it 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, D represents aspartic acid, the left side is the N-terminus linked to the linking group Y, and the right side is the C-terminus linked to lysine. As X, a divalent linking group derived from (1) APA is particularly preferred. The squaraine dye peptide complex of the present invention is cleaved by hydrolysis with 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 is a spacer between the carbon atom of the carbonyl group of the squaraine dye in formula (I) and the oligopeptide (X), and represents a divalent linking group derived from glycine, β-alanine, 6-aminohexanoic acid, Mini-PEG, and PEG-DGA-OH. As Y, a divalent linking group derived from β-alanine is particularly preferred.

[0020] The squalaine dye peptide complex of the present invention may be a symmetric squalaine dye peptide complex in which an oligopeptide is bound between the same squalaine dyes, or an asymmetric squalaine dye peptide complex in which an oligopeptide is bound between different squalaine dyes. However, from the viewpoint of easy production, etc., a symmetric squalaine dye peptide complex in which an oligopeptide is bound between the same squalaine dyes is preferred.

[0021] As the squalaine dye peptide complex of the present invention, in formula (I), R 1A and R 2A represent a tetramethylene group, R 3A and R 4A represent an ethyl group, R 5A represents an amino group, X represents a divalent linking group derived from APA, and Y represents a divalent linking group derived from β-alanine. A squalaine dye peptide complex is particularly preferred. That is, a squalaine dye peptide complex represented by the following formula (IA) (SQ-215-PC of Example 1) is preferred.

[0022]

Chemical formula

[0023] The squalaine dye peptide complex of the present invention is obtained by introducing an oligopeptide between two squalaine dyes. The squalaine dye peptide complex of the present invention exhibits quenched fluorescence alone in a polar solvent such as water or a buffer solution, but exhibits fluorescence emission after the peptide bond is cleaved by the hydrolysis of elastase.

[0024] The inventors of the present invention consider the reason as follows. First, as described above, the squalaine dye peptide complex of the present invention alone shows no fluorescence or very weak fluorescence due to fluorescence resonance energy transfer (FRET) or quenching by aggregation. However, when the peptide moiety is cleaved by elastase, the squalaine dye molecules having peptide fractions are outside the range where fluorescence resonance energy transfer occurs (usually the Förster radius of 2 to 5 nanometers), and it is presumed that the appearance or enhancement of fluorescence occurs. Further, after cleavage of the peptide bond by hydrolysis with elastase, the presence of the squalaine dye having a peptide fraction suppresses the aggregation of the squalaine dye having a peptide fraction and the other squalaine dye, so that fluorescence quenching due to the aggregation of both is prevented, and it is presumed that the appearance or enhancement of fluorescence occurs.

[0025] Since the squalaine dye peptide complex of the present invention has the above characteristics, elastase can be detected with high sensitivity and selectivity by monitoring the near-infrared fluorescence characteristics. Thereby, elastase in biological fluids such as blood, urine, and saliva can be sensitively detected and used for disease prediction (early detection) and diagnosis.

[0026] Specifically, the squalaine dye peptide complex of the present invention can be used as a reagent for detecting elastase. Further, the squalaine dye peptide complex of the present invention can be used in a method for detecting elastase. Specifically, the method for detecting elastase using the squalaine dye peptide complex of the present invention includes a step of contacting the squalaine dye peptide complex of the present invention with a sample (first step) and a step of detecting elastase by measuring a fluorescence emission spectrum (second step). In the second step, elastase can be detected by measuring the fluorescence emission spectrum and detecting a change in fluorescence (change in absorption wavelength).

[0027] In addition, information (measurement data) for predicting or diagnosing diseases related to elastase can also be provided by using the above-described method for detecting esterase. Elastase is one of the tumor markers and can be used, for example, for detecting pancreatic cancer and pancreatitis.

[0028] The squalaine dye peptide complex of the present invention can be produced by introducing an oligopeptide between two predetermined squalaine dyes that are the same or different. As a method for introducing an oligopeptide between two squalaine dyes, a known method can be used. Specifically, for example, a peptide solid-phase synthesis method (SPPS method) using the Fmoc strategy can be mentioned.

[0029] <Squalaine dye> The squalaine dye according to the second invention is a compound represented by the following formula (1).

[0030]

Chemical formula

[0031] R 1B and R 2B each independently represent a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, and one or both of R 1B and R 2B have a carboxy group at the terminal of the aliphatic hydrocarbon group. R 1B and R 2B may be a linear hydrocarbon group or a branched-chain hydrocarbon group, but a linear hydrocarbon group is preferred. Also, it may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, but a saturated hydrocarbon group (alkyl group) is preferred.

[0032] R 1B and R 2B When have a carboxy group at the terminal, R 1B and R 2B are preferably alkyl groups having 2 to 18 carbon atoms, and more preferably alkyl groups having 2 to 6 carbon atoms. R 1B and R 2Bmay be the same or different, but it is preferably the same.

[0033] R 1B and R 2B When they do not have a carboxy group at the terminal, an alkyl group having 1 to 18 carbon atoms is preferable, and an alkyl group having 1 to 6 carbon atoms is more preferable.

[0034] R 3B and R 4B represent a hydrogen atom or a carboxy group, at least one of which is a carboxy group, and R 3B and R 4B are preferably carboxy groups.

[0035] The aromatic skeleton in the above formula (1) may have a substituent. As the substituent, an electron-donating group is preferable. Specifically, for example, an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, a hydroxyl group, etc. can be mentioned. These substituents are preferably substituted at positions where they function as electron-donating groups. Also, a halogen group may be used.

[0036] As the squarylium dye of the present invention, in formula (1), R 1B and R 2B represent an ethyl group (an alkyl group having 3 carbon atoms) having a carboxyl group at the terminal, and R 3B and R 4B represent a carboxy group, and a squarylium dye is particularly preferable. That is, a squarylium dye represented by the following formula (1A) (SQ-58 in Example 2) is preferable.

[0037] [Chemical formula]

[0038] The squaraine dye of the present invention is characterized by strongly absorbing light in the near-infrared region in solution. Therefore, for example, it can be used in the fields of photoconduction, data storage, light-emitting field-effect transistors, solar cells, nonlinear optical materials, optical bioimaging, NIR-fluorescent probes, and optical detection of labels.

[0039] In phosphate buffer (PB), the squaraine dye of the present invention shows a decrease in absorbance and fluorescence intensity due to the formation of dye aggregates compared to in an organic solvent (nonpolar solvent). However, in the presence of protein, dye aggregation is suppressed by the interaction with the protein, and the absorbance and fluorescence intensity increase. Also, by increasing the concentration of the protein, the absorbance and fluorescence intensity can be increased.

[0040] The squaraine dye of the present invention can be produced using known methods.

Examples

[0041] Hereinafter, the first aspect of the present invention will be described in more detail with reference to examples.

[0042] [Example 1] <Production of the squaraine dye peptide complex SQ-215-PC of the present invention>

[0043]

Chemical formula

[0044] (Production of squaraine dye SQ-215) First, the squaraine dye SQ-215 was produced as a raw material. The outline of the production process is shown below.

[0045]

Chemical formula

[0046] Hereinafter, each step will be specifically described. [Step 1-1] (Production of Compound 2)

[0047]

Chem.

[0048] To a solution of Compound 1 (1,1,2-trimethyl-1H-benzo[e]indole) (50 mmol) in 150 mL of acetonitrile, 125 mmol of iodoethane was added and the mixture was refluxed for about 18 hours. The reaction was monitored by TLC. 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 thoroughly with ethyl acetate, and dried under vacuum to obtain Compound 2 (yield 90%).

[0049] High-resolution mass spectrometry (HRMS) of Compound 2 was performed. FAB-Mass (m / z: calculated: 238.1590 for C 17 H 20 N + , observed: 238.1596).

[0050] [Step 1-2] (Production of Compound i)

[0051]

Chem.

[0052] To a two-necked round-bottom flask placed in an ice bath, Compound 2 obtained from the above reaction and dibutyl squarate (10 mmol) in equimolar amounts were dissolved in 15 mL of ethanol. Triethylamine (40 mmol) was added dropwise with stirring. The reaction mixture was continuously stirred at room temperature for 24 hours under an inert atmosphere. The solid was filtered, washed with a minimal amount of ethanol, and dried under vacuum to obtain the half-dye butyl ester as a bright yellow solid (yield 60%).

[0053] High-resolution mass spectrometry (HRMS) of the half-dye butyl ester was performed. FAB-Mass (m / z: Calculated: 389.1991 for C 25 H 27 NO 3 , Observed: 389.1996).

[0054] Subsequently, since the hydrolyzed half-dye butyl ester becomes unstable, hydrolysis of the ester form was carried out immediately before proceeding to the final dye synthesis step. 2 mmol of the half-dye butyl ester was dissolved in 20 mL of ethanol. 2 mL of 10% NaOH was added thereto, and after refluxing for 30 minutes, it was 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.

[0055] [Step 1-3] (Production of Compound 3)

[0056] [Chemical Structure]

[0057] To a solution of Compound 1 (15 mmol) dissolved in 50 mL of propionitrile, 37 mmol of ethyl-6-iodohexanoate was added, and it was refluxed for about 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. Subsequently, the solid was filtered, washed thoroughly with ethyl acetate, and dried under vacuum to obtain Compound 3 (3-(6-ethoxy-6-oxohexyl)-1,1,2-trimethyl-1H-benzo[e]indolium iodide) (yield 80%) as a pale green hygroscopic solid.

[0058] Mass spectrometry (HRMS) of Compound 3 was performed. FAB-Mass (m / z: Calculated: 352.2271 for C 23 H 30 NO 2 + , Observed: 352.2276).

[0059] [Project 1 - 4] (Production of Compound SQ - 215)

[0060]

Chem.

[0061] An equimolar amount (~2 mmol) of hydrolyzed Compound i and Compound 3 (3-(6 - ethoxy - 6 - oxohexyl)-1,1,2 - trimethyl - 1H - benzo[e]indolium iodide) was dissolved in 1:1 butanol:benzene (40 mL) and refluxed for 18 - 24 hours under an inert atmosphere. The reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated, and the crude dye was purified by silica gel (C - 200 mesh) column chromatography using chloroform and methanol as the elution system to obtain Compound SQ - 215 (yield 80%) as a dark blue solid. Note that before proceeding to column purification, the crude ester form of the dye was hydrolyzed under the same conditions as the hydrolysis of Compound i.

[0062] For Compound SQ - 215 1 1H - NMR analysis and mass spectrometry (HRMS) were performed. 1H NMR: 1δH (CDCl3, 500 MHz), 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 N 2 O 4 .

[0063] Using the above compound SQ-215, SQ-215-PC of the present invention was produced. The outline of the production process is shown below.

[0064]

Chemical Structure

[0065] Homo-SQ-215-PC was synthesized using the solid-phase peptide synthesis method (SPPS method) with the Fmoc strategy. In this procedure, after coupling the following Fmoc-protected amino acids at room temperature using HBTU and HOBt, a cycle of Fmoc deprotection was performed. The first step was the Fmoc deprotection of Fmoc-Rink-Amide resin. Subsequently, Fmoc-Lys(Boc)-OH was coupled as the first amino acid. The cycle of deprotection and coupling was continued until the sequence of Fmoc-βAla-Ala-Pro-Ala-Lys(Boc)-Resin was reached. First, Fmoc was deprotected, and subsequently, Boc was deprotected with 2N hydrochloric acid in dioxane for 10 minutes. The next step was the coupling of SQ-215 with the peptide sequence. The unprotected sequence was coupled with 2.5-fold SQ-215 using the same coupling reagent. The squalaine dye peptide complex was cleaved from the resin using standard TFA cleavage conditions. The crude product was reprecipitated using cold diethyl ether and purified by silica gel chromatography with chloroform and methanol as the elution system to obtain the squalaine dye peptide complex SQ-215-PC of the present invention.

[0066] Mass spectrometry (TOF-Mass) of the squalaine dye peptide complex SQ-215-PC was performed. Found M / Z: [M+Na] +: 1718.88, calculated: 1695.89 for C 104 H 117 N 11 O 11

[0067] [Evaluation of Probe Specificity for Elastase] The effect of hydrolysis of elastase enzyme in phosphate buffer of the squalene dye peptide complex SQ-215-PC obtained in Example 1 was examined. Specifically, the squalene dye peptide complex SQ-215-PC (10 μM) was allowed to act on different concentrations of porcine pancreatic elastase in phosphate buffer (pH = 7.4), and the fluorescence emission spectrum was measured at time intervals.

[0068] The results are shown in FIGS. 1 to 3. As shown in FIG. 1, the addition of 1 unit / 210 pmole showed a two-fold fluorescence intensity. As shown in FIG. 2, the addition of 5 units / 1.05 nmole showed a three-fold fluorescence intensity. As shown in FIG. 3, the addition of 10 units / 2.1 nmole of elastase resulted in a more than ten-fold increase in fluorescence intensity. Also, the detection limit was found to be 136.1 picomoles.

[0069] [Evaluation of Probe Specificity for Other Serine Proteases] The squalene dye peptide complex SQ-215-PC (10 μM) obtained in Example 1 was treated with different serine proteases at 210 nM in phosphate buffer, incubated for 20 minutes, and evaluated.

[0070] The results are shown in FIG. 4. As shown in FIG. 4, trypsin, cathepsin B, and thrombin did not affect Homo-SQ-215-PC, and the protease had only a minimal effect. Therefore, it was found that the squalene dye peptide complex SQ-215-PC of the present invention is very specific for elastase.

[0071] [Evaluation of Optical Properties in Nonpolar Solvents] The optical properties of the squaraine dye peptide complex SQ-215-PC obtained in Example 1 above were evaluated in a nonpolar solvent. Specifically, using the squaraine dye peptide complex SQ-215-PC (10 μM), fluorescence emission spectra in chloroform and DMSO solutions were measured. For comparison, SQ-215 (10 μM) was used.

[0072] The results are shown in Fig. 5. As shown in Fig. 5, in chloroform and DMSO solutions, the squaraine dye peptide complex SQ-215-PC showed a decrease in fluorescence intensity compared to SQ-215. This indicates fluorescence quenching due to fluorescence resonance energy transfer (FRET).

[0073] Hereinafter, the second invention of the present invention will be described in more detail by way of examples. [Example 2] [Production of squaraine dye SQ-58 of the present invention] The squaraine dye SQ-58 of the present invention was produced. The outline of the production process is shown below.

[0074] [Chemical formula]

[0075] Hereinafter, each step will be specifically described. [Step 2-1] [Production of Compound 1]

[0076] [Chemical formula]

[0077] 2,3,3-Trimethyl-3H-indole-5-carboxylic acid (1) was synthesized according to the procedure reported in Saikiran M, Sato D, Pandey SS, Ohta T, Hayase S, Kato T. Photophysical characterization and BSA interaction of the direct ring carboxy functionalized unsymmetrical NIR cyanine dyes. Dye Pigment 2017; 140: 6 - 13. https: / / doi.org / 10.1016 / j.dyepig.2017.01.015.

[0078] The mass spectrometry (HRMS) of Compound 1 was performed. ESI-Mass (m / z: calculated 203.09463 for C 12 H 13 NO 2 , observed: [M+1] 204.10245).

[0079] [Step 2-2] (Production of Compound 2)

[0080]

Chemical Structure

[0081] In a round-bottom flask (RBF) equipped with a condenser, 1 (2 g, 10 mmol) and 1-iodopropionic acid (3 g, 15 mmol) were dissolved in 1,2-dichlorobenzene (20 ml), and the reaction mixture was heated at 140 °C for 18 hours. The reaction was monitored by HPLC. After the reaction was completed, the crude product was reprecipitated and washed with a sufficient amount of ethyl acetate to obtain Compound 2 (5-carboxy-1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indol-1-ium).

[0082] The mass spectrometry (HRMS) of Compound 2 was performed. ESI-Mass (m / z: calculated 277.13086 for C 15 H 19 NO 4 + , observed: [M-1] 276.12344 for C 15 H 18 NO 4 + ).

[0083] [Step 2-3] (Production of Compound 3)

[0084]

Chemical Structure

[0085] Compound 2 (2.5 mmol, 1 g) and 1-iodobutane (7.5 mmol, 0.85 mL) were dissolved in DMF, and cesium carbonate (7.5 mmol, 2.44 g) was added. The reaction mixture was stirred at 40 °C, and the reaction was monitored by TLC every 30 minutes. After completion of the reaction, DMF was evaporated, and the resulting solid was extracted with chloroform and washed twice with water and brine. The chloroform layer was dried over sodium sulfate, evaporated, and dried under vacuum to obtain Compound 3 (1-(3-butoxy-3-oxopropyl)-5-(butoxycarbonyl)-2,3,3-trimethyl-3H-indol-1-ium).

[0086] Mass spectrometry (HRMS) of Compound 3 was performed. ESI-Mass (m / z: calculated 388.2482 for C23H34NO4+, observed: 388.2497.)

[0087] [Step 2-4] (Production of Compound 4)

[0088]

Chemical Structure

[0089] Compound 3 (2 equivalents) and squaric acid (1 equivalent) were dissolved in 1-butanol:toluene (1:1, v / v, 30 mL) and refluxed for about 12 hours under an inert atmosphere. The reaction was monitored by TLC. After completion of the reaction, the solvent was evaporated and compound 4 (SQ-58-butyl ester) was purified by silica gel column chromatography using chloroform and methanol as eluting solvents.

[0090] For compound 4 1 1H-NMR analysis and mass spectrometry (HRMS) were performed. 1H NMR: 1δH (CDCl3, 500 MHz, TMS): 7.98 - 7.88 (4H, m, Ar-H), 7.17 (1H, d, Ar-H), 7.05 (1H, d, Ar-H), 5.9 (1H, s, methine proton), 5.6 (1H, s, methine proton), 4.2 - 3.9 (10H, m, methylene & N+-methylene protons), 2.76 (2H, t, N-methylene protons), 2.0 (4H, m, methylene protons), 1.70 (10H, m, methyl & methylene protons), 1.43 - 1.18 (18H, m, methyl & methylene protons), 0.94 - 0.78 (12H, m, methyl protons) HRMS ESI-Mass (m / z: calculated 852.4561 for C 50 H 64 N 2 O 10 , Observed: 852.4668).

[0091] [Step 2 - 5] (Production of compound SQ-58)

[0092]

Chemical Structure

[0093] Compound 4 was dissolved in 15 mL of ethanol, followed by the addition of 2 mL of 10% NaOH. The reaction mixture was refluxed until the reaction was monitored by TLC and completed. The reaction mixture was cooled and neutralized with 1.5 mL of 20% HCl. The following precipitate was filtered, washed thoroughly with water, and dried under vacuum to obtain the second squarylium dye SQ-58 of the present invention as a blue solid (yield 95%).

[0094] Mass spectrometry (HRMS) of squarylium dye SQ-58 was performed. HRMS: Found m / z: [M+1] + 629.21445, [M+Na] + : 651.19363, Calculated: 628.20570 for C 34 H 32 N 2 O 10 .

[0095] [Optical property evaluation] The optical properties of squarylium dye SQ-58 obtained in Example 2 were evaluated. Specifically, using squarylium dye SQ-58 (5 μM), UV-vis absorption spectra and fluorescence emission spectra in chloroform and phosphate buffer (PB) (pH 7.4) were measured.

[0096] The results are shown in Figures 6 and 7. As shown in Figures 6 and 7, SQ-58 showed fluorescence in the near-infrared region in chloroform. On the other hand, in phosphate buffer, the formation of dye aggregates was promoted, resulting in a blue shift and a decrease in absorption and fluorescence intensity.

[0097] [Biocompatibility evaluation] The biocompatibility of squarylium dye SQ-58 obtained in Example 2 was evaluated. Specifically, at a fixed dye concentration of 2 μM, electronic absorption spectra and fluorescence emission spectra of SQ-58 at different concentrations of bovine serum albumin (BSA) in 0.1 M phosphate buffer were measured.

[0098] The results are shown in FIGS. 8 and 9. As shown in FIG. 8, two different strong electron absorption bands corresponding to the absorption of BSA and the dye SQ-58 were observed at 280 nm and 650 nm - 700 nm, respectively. As the concentration of BSA increased, the intensity of the 280 nm band gradually increased, indicating that there was no denaturation of BSA associated with the interaction with the dye. As shown in FIG. 9, as the concentration of BSA increased, the fluorescence intensity increased with a red shift. This is considered to be due to the destruction of the dye aggregates by the dye-BSA interaction.

[0099] Subsequently, as shown in FIG. 10, at a fixed dye concentration of 2 μM, by plotting (F ∞ -F 0 ) / (F X -F 0 ) as a function of the reciprocal of the BSA concentration, the apparent binding constant (K a ) was calculated using Equation 1 below. The value of K a was 6.74×10 7 M from the slope of FIG. 10, indicating that SQ-58 showed a very high binding affinity for BSA. This is considered to be due to the hydrophilic carboxylic acid (COOH) functional group with a direct ring substitution that promotes hydrogen bonding with the binding site of BSA.

[0100] F ∞ -F 0 / F x -F 0 =1+(1 / Ka[BSA])···(Equation 1)

[0101] In Equation 1, F 0 is the fluorescence intensity of the dye at each concentration in the absence of BSA, F x is the fluorescence intensity of the dye at each concentration in the presence of BSA, and F ∞ is the fluorescence intensity of the dye at the concentration when complete interaction occurs.

Industrial Applicability

[0102] The novel squarylium dye peptide complex of the present invention is industrially useful because it can be used for the detection of elastase and the like.

Sequence Listing Free-Text

[0103] [SEQ ID NO: 1] It is the amino acid sequence (AAPI) possessed by the squaraine dye peptide complex of the present invention. [SEQ ID NO: 2] It is the amino acid sequence (AAPV) possessed by the squaraine dye peptide complex of the present invention. [SEQ ID NO: 3] It is the amino acid sequence (AAPA) possessed by the squaraine dye peptide complex of the present invention. [SEQ ID NO: 4] It is the amino acid sequence (AVSLGD) possessed by the squaraine dye peptide complex of the present invention.

Claims

1. A squaraine dye-peptide conjugate represented by formula (I): 【Chemistry 1】 (In formula (I), R １Ａ and R ２Ａ each independently represents a divalent aliphatic hydrocarbon group having 3 to 30 carbon atoms, R ３Ａ and R ４Ａ each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, R ５Ａ represents an amino group or a carboxy 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 derived from glycine, β-alanine, 6-aminohexanoic acid, Mini-PEG, or PEG-DGA-OH.

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

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

4. 4. 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.

5. A reagent for detecting elastase, comprising the squaraine dye-peptide complex according to claim 1.

6. 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 the steps of:

7. 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:

8. A squaraine dye represented by the following formula (1): 【Chemistry 2】 (In formula (1), R １Ｂ and R ２Ｂ each independently represents a monovalent aliphatic hydrocarbon group having 1 to 30 carbon atoms, at least one of which has a carboxy group at its terminal; R ３Ｂ and R ４Ｂ represents a hydrogen atom or a carboxy group, and at least one of them is a carboxy group.

9. In formula (1), R １Ｂ and R ２Ｂ each independently represents an alkyl group having 2 to 18 carbon atoms when the group has a carboxy group at its terminal; R １Ｂ and R ２Ｂ The squaraine dye according to claim 8, wherein when the group does not have a terminal carboxyl group, the group represents an alkyl group having 1 to 18 carbon atoms.

10. In formula (1), R １Ｂ and R ２Ｂ The squaraine dye according to claim 8, characterized in that it has a carboxy group at its terminal.

11. In formula (1), R １Ｂ and R ２Ｂ 11. The squaraine dye according to claim 10, wherein represents an alkyl group having 2 to 6 carbon atoms.

12. In formula (1), R ３Ｂ and R ４Ｂ 9. The squaraine dye according to claim 8, characterized in that: represents a carboxy group.

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