A carboxyl esterase-responsive fluorescent dye, and preparation method and application thereof
By designing hydrogen bonds and hydrophobic interactions on fluorescent dyes, the problem of external interference with the detection of carboxylesterases by fluorescent dyes was solved, and high sensitivity and high accuracy of carboxylesterase detection were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHANGSHA UNIVERSITY OF SCIENCE AND TECHNOLOGY
- Filing Date
- 2023-12-15
- Publication Date
- 2026-07-28
AI Technical Summary
Existing fluorescent dyes are easily affected by external factors when detecting carboxylesterases, resulting in low sensitivity and low signal-to-background ratio.
A fluorescent dye responsive to carboxylesterase was designed. By introducing an oxygen atom onto the fluorescent dye as a hydrogen bond donor and acceptor, it forms hydrogen bonds with Met145 and Asp90 in carboxylesterase and forms hydrophobic interactions with multiple residues, thereby enhancing the binding effect.
It improves the binding stability of fluorescent dyes to carboxylesterases, enhances the fluorescence signal, reduces interference from external factors, and improves the sensitivity and accuracy of detection, especially maintaining a high signal-to-background ratio under different temperature and pH conditions.
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Figure CN117700346B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fluorescent dye technology, specifically to a responsive carboxylesterase fluorescent dye, its preparation method, and its application. Background Technology
[0002] Carboxylesterase (CE) is an important serine hydrolase that hydrolyzes ester, thioester, amide, and carbamate bonds in various endogenous and exogenous compounds, thus playing a crucial role in the metabolism of endogenous substances and the activation or detoxification of exogenous compounds. Furthermore, carboxylesterase can also metabolize cholesterol esters, triglycerides, and other endogenous lipids, playing an important physiological role in maintaining lipid homeostasis.
[0003] Currently, carboxylesterase detection methods include immunological methods, chemiluminescence methods, real-time quantitative PCR, and Western blotting. Real-time quantitative PCR is a commonly used method, but current techniques often suffer from low sensitivity and low signal-to-backflow ratios due to poor binding of the fluorescent dyes to carboxylesterases. For example, a research paper (RSC Analyst, 2012, 137, 716-721) reported a fluorescent probe based on a halogen (9-hydroxy-3-isophenoxazinone) derivative. This probe exhibits almost no fluorescence at 585 nm. In the presence of carboxylesterase, it catalyzes the breakage of the carboxylic acid ester bond in the probe molecule, causing the benzyl group to break and releasing the halogen. Under 550 nm laser irradiation, the probe molecule emits orange-red fluorescence at 585 nm, achieving fluorescence-enhanced detection of carboxylesterases. However, this fluorescent probe is susceptible to interference from external factors, sometimes hindering accurate analysis of carboxylesterases. Therefore, it is necessary to design and synthesize a fluorescence-enhancing dye with low background interference that can bind to carboxylesterases and respond rapidly to carboxylesterases. This has important research significance and practical value. Summary of the Invention
[0004] To address the problem that existing fluorescent dyes are easily affected by external factors during detection, this invention provides a fluorescent dye that responds to carboxylesterase, its preparation method, and its application.
[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows.
[0006] A first aspect of the present invention provides a fluorescent dye responsive to carboxylesterase, the fluorescent dye having the structural formula shown in formula (I):
[0007]
[0008] A second aspect of the present invention provides a method for preparing a responsive carboxylesterase fluorescent dye, wherein the responsive carboxylesterase fluorescent dye is the responsive carboxylesterase fluorescent dye provided in the first aspect of the present invention, comprising the following steps:
[0009] Compound 1 was prepared by dissolving 2,3,3-trimethyl-3H-indole and 3-iodopropionic acid in toluene and then reacting the mixture at 110–130 °C under a protective atmosphere.
[0010] Compound 1 was mixed with 3,4-dihydroxy-3-cyclobutene-1,2-dione and dissolved in a mixed solution of n-butanol, toluene and pyridine. The mixture was then heated to 110–130 °C under a protective atmosphere to prepare compound 2.
[0011] Compound 2 and m-phenylenediol were mixed, and a crosslinking agent was added to carry out a crosslinking reaction to prepare the fluorescent dye.
[0012] The specific reaction equation is as follows:
[0013]
[0014] In a preferred embodiment, the molar ratio of 2,3,3-trimethyl-3H-indole to 3-iodopropionic acid is 1 to 1.2:1.
[0015] In a preferred embodiment, the molar ratio of compound 1 to 3,4-dihydroxy-3-cyclobutene-1,2-dione is 2:1 to 1.2;
[0016] The volume ratio of n-butanol, toluene, and pyridine is 12–15:12–15:7–8;
[0017] The ratio of compound 1 to n-butanol is 2 mmol: 13-15 ml.
[0018] In a preferred embodiment, the crosslinking agent is EDC and DMAP.
[0019] In a preferred embodiment, the protective atmosphere in the preparation of compound 1 and the protective atmosphere in the preparation of compound 2 are both nitrogen.
[0020] In a preferred embodiment, the preparation of compound 1 includes washing after the heating reaction, wherein the washing is performed using toluene.
[0021] In a preferred embodiment, the preparation process of compound 2 further includes vacuum distillation and chromatography after the heating reaction;
[0022] The chromatography is silica gel column chromatography, and the eluent for the chromatography is a mixture of methanol and dichloromethane in a volume ratio of 1:10-12.
[0023] In a preferred embodiment, the specific preparation process of the fluorescent dye is as follows:
[0024] Compound 2, isophthalic acid, EDC and DMAP were dissolved in dichloromethane and reacted at room temperature. After the reaction was completed, the mixture was distilled under reduced pressure and chromatographically analyzed to obtain the fluorescent dye.
[0025] The molar ratio of compound 2, isophthalic acid, EDC, and DMAP is 1–1.2:1–1.2:1–1.2:1–1.2;
[0026] The reaction time is 4 to 4.5 hours; the chromatography is silica gel column chromatography, and the eluent for the chromatography is a mixture of methanol and dichloromethane in a volume ratio of 1:28 to 30.
[0027] A third aspect of the present invention provides the use of a fluorescent dye in the preparation of a fluorescent probe for the specific recognition of carboxylesterases, wherein the fluorescent dye is the fluorescent dye provided in the first aspect of the present invention.
[0028] The fluorescent dye, as a fluorescent group in real-time quantitative PCR, can bind to carboxylesterase. At the binding site, steric hindrance complementarity is formed, resulting in hydrogen bonds and hydrophobic interactions between the carboxylesterase and the fluorescent dye, thereby improving the binding effect between the fluorescent dye and the carboxylesterase.
[0029] An oxygen atom in the fluorescent dye acts as both a hydrogen bond donor and acceptor, forming hydrogen bonds with Met145 and Asp90 in the carboxylesterase. The fluorescent dye also forms hydrophobic interactions with numerous residues in the carboxylesterase (Thr298, Val146, Leu304, Ala93, Lys92, Met361, Trp357, Leu367, Met458, Ler364, Les302, Phe302, and Ser305). Through hydrogen bonding and hydrophobic interactions, the binding between the fluorescent dye and the carboxylesterase is enhanced, making the binding of the fluorescent dye to the carboxylesterase more stable, enhancing the fluorescence signal, and thus improving the signal-to-background ratio of the carboxylesterase in real-time quantitative PCR.
[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0031] (1) The fluorescent dye in this invention can bind to carboxylesterase more strongly under different temperatures and pH conditions. The fluorescence signal is not affected by ambient temperature and pH, and the fluorescence signal is enhanced, thereby improving the signal-to-background ratio of carboxylesterase in real-time fluorescence quantitative detection.
[0032] (2) In this invention, the fluorescent dye and carboxylesterase form a suitable steric hindrance complementarity at the binding site. One oxygen atom of the fluorescent dye acts as a hydrogen bond donor and hydrogen bond acceptor, forming hydrogen bonds with Met145 and Asp90 in the carboxylesterase. The fluorescent dye forms hydrophobic interactions with a large number of residues in the carboxylesterase. Through the hydrogen bonds and hydrophobic interactions formed between the fluorescent dye and the carboxylesterase, the binding effect between the fluorescent dye and the carboxylesterase is enhanced, so that the detection is not affected by external factors, and the sensitivity and accuracy of the detection are improved.
[0033] (3) The fluorescent dye in this invention has excellent performance and can achieve a high specificity and high sensitivity response to carboxylesterase, and has potential application prospects in bioimaging. Attached Figure Description
[0034] Figure 1 This is a two-dimensional structural diagram of the binding mode between the fluorescent dye and carboxylesterase in Example 1 of the present invention.
[0035] Figure 2 This is a schematic diagram of the binding pattern between the fluorescent dye and the surface of the carboxylesterase molecule in Example 1 of the present invention.
[0036] Figure 3 This is a schematic diagram of the binding pattern of fluorescent dye and carboxylesterase in Example 1 of the present invention.
[0037] Figure 4 This is a bar chart showing the fluorescence intensity of the fluorescent dye in Example 1 of this invention binding to carboxylesterase at different temperatures.
[0038] Figure 5 This is a bar chart showing the fluorescence intensity of the fluorescent dye in Example 1 of the present invention binding to carboxylesterase under different pH conditions.
[0039] Figure 6 This is a graph showing the selectivity of the fluorescent dye for carboxylesterase in Example 1 of the present invention.
[0040] Figure 7 The fluorescent dye in Example 1 of this invention binds to different concentrations of carboxylesterase with fluorescence spectra.
[0041] Figure 8 This is an NMR spectrum of the fluorescent dye described in an embodiment of the present invention. Detailed Implementation
[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0043] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0044] Carboxylesterase (CE) is an important serine hydrolase that hydrolyzes ester, thioester, amide, and carbamate bonds in various endogenous and exogenous compounds. Therefore, it plays a crucial role in the metabolism of endogenous substances and the activation or detoxification of exogenous compounds. Furthermore, carboxylesterase can also metabolize cholesterol esters, triglycerides, and other endogenous lipids, playing an important physiological role in maintaining lipid homeostasis.
[0045] Currently, carboxylesterase detection methods include immunological methods, chemiluminescence methods, real-time quantitative PCR, and Western blotting. Real-time quantitative PCR is a commonly used method, but it suffers from drawbacks such as insufficient binding of fluorescent dyes to carboxylesterases, susceptibility to external interference, and difficulty in accurately analyzing carboxylesterases, resulting in low sensitivity and a low signal-to-backflow ratio.
[0046] This invention provides a fluorescent dye responsive to carboxylesterase, its preparation method, and its application. When detecting carboxylesterase, the fluorescent dye uses an oxygen atom as both a hydrogen bond donor and acceptor to form hydrogen bonds with Met145 and Asp90 residues in the carboxylesterase. Furthermore, the fluorescent dye forms hydrophobic interactions with numerous residues in the carboxylesterase, such as Thr298, Val146, Leu304, Ala93, Lys92, Met361, Trp357, Leu367, Met458, Ler364, Les302, Phe302, and Ser305. Through these hydrogen bonds and hydrophobic interactions, the binding effect between the fluorescent dye and the carboxylesterase is effectively improved, avoiding inaccurate detection results due to poor binding caused by external factors.
[0047] The fluorescent dyes in the following examples have the structural formula shown in formula (I):
[0048]
[0049] The fluorescent dye was subjected to NMR scanning to obtain an NMR image, such as... Figure 8 As shown in the figure, there are 3 H atoms at the chemical shift position of the fluorescent dye at 0.9, a total of 10 H atoms at positions 1.44 to 1.54, 6 H atoms at 1.79, 2 H atoms at 4.13, 4 H atoms at 4.42 and 4.61, 4 H atoms at 5.09, 5.20, and 5.27, and 8 H atoms at chemical shift positions 7.06 to 7.59 on the benzene ring.
[0050] The following describes in detail a method for preparing a fluorescent dye that responds to carboxylesterase.
[0051] Example 1
[0052] A method for preparing a fluorescent dye responsive to carboxylesterase includes the following steps:
[0053] S1. 12 mmol of 2,3,3-trimethyl-3H-indole and 12 mmol of 3-iodopropionic acid were dissolved in a round-bottom flask containing 5 mL of toluene at a molar ratio of 1:1. The mixture was reacted under a nitrogen atmosphere at 110 °C and refluxed for 14 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed several times with toluene, and the solid was collected and dried under vacuum to give 1.67 g of red solid compound 1.
[0054] The yield of compound 1 was 72%, with an ESI-MS calculated value of 232.13 and a mass spectrometry result of 232.0.
[0055] S2. Compound 1 from S1 (molar ratio 2:1, 4 mmol) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (volume ratio 15:15:8) were dissolved in a mixed solution of 15 mL n-butanol, 15 mL toluene, and 8 mL pyridine. The mixture was reacted at 110 °C under a nitrogen atmosphere and refluxed for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by vacuum distillation to obtain a blue-green crude product. The blue-green crude product was then subjected to silica gel column chromatography, eluted with methanol and dichloromethane (volume ratio 1:10) to obtain 0.14 g of blue-green compound 2.
[0056] The yield of compound 2 was 12%, with a calculated value of 597.72 by MALDI-TOF-MS and a mass spectrometry result of 597.0.
[0057] S3. Compound 2 from S2 (0.1 mmol), 0.1 mmol of isophthalic acid, 0.1 mmol of EDC, and 0.1 mmol of DMAP in a molar ratio of 1:1:1:1 were dissolved in a round-bottom flask containing 7 mL of dichloromethane and reacted at room temperature for 4 h. The extent of the reaction was detected by TLC. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a crude blue-green product. The crude blue-green product was subjected to silica gel column chromatography and eluted with methanol and dichloromethane in a volume ratio of 1:30 to obtain 38 mg of blue-green solid, i.e., fluorescent dye.
[0058] The yield of the fluorescent dye was determined to be 53%; the calculated ESI-MS value of the fluorescent dye was 718.36, and the mass spectrometry result was 728.0.
[0059] Example 2
[0060] A method for preparing a fluorescent dye responsive to carboxylesterase includes the following steps:
[0061] S1. 11 mmol of 2,3,3-trimethyl-3H-indole and 10 mmol of 3-iodopropionic acid in a molar ratio of 1.1:1 were dissolved in a round-bottom flask containing 5 mL of toluene. The mixture was reacted at 120 °C under a nitrogen atmosphere and refluxed for 15 h. After the reaction was completed, the mixture was cooled to room temperature, filtered, washed several times with toluene, and the solid was collected and dried under vacuum to give 1.67 g of red solid compound 1.
[0062] The yield of compound 1 was 72%, and the calculated ESI-MS value of compound 1 was 232.13, while the mass spectrometry result was 232.0.
[0063] S2. 2 mmol of compound 1 from S1 and 1.1 mmol of 3,4-dihydroxy-3-cyclobutene-1,2-dione in a molar ratio of 2:1.1 were dissolved in a mixed solution of 15 mL n-butanol, 15 mL toluene, and 7 mL pyridine in a volume ratio of 15:15:7. The mixture was reacted at 120 °C under a nitrogen atmosphere and refluxed for 11 h. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was removed by vacuum distillation to obtain a blue-green crude product. The blue-green crude product was then subjected to silica gel column chromatography, eluted with methanol and dichloromethane in a volume ratio of 1:12, to obtain 0.14 g of blue-green compound 2.
[0064] The yield of compound 2 was 12%; the calculated value of compound 2 by MALDI-TOF-MS was 597.72, and the mass spectrometry result was 597.0.
[0065] S3. Compound 2 from S2 (0.1 mmol), 0.1 mmol of isophthalic acid, 0.1 mmol of EDC, and 0.12 mmol of DMAP in a molar ratio of 1:1:1:1.2 were dissolved in a round-bottom flask containing 7 mL of dichloromethane and reacted at room temperature for 4.2 h. The extent of the reaction was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a crude blue-green product. The crude blue-green product was subjected to silica gel column chromatography and eluted with methanol and dichloromethane in a volume ratio of 1:35 to obtain 38 mg of blue-green solid, i.e., fluorescent dye.
[0066] The yield of the fluorescent dye was found to be 53%; the calculated ESI-MS value of the fluorescent dye was 718.36, and the mass spectrometry result was 728.
[0067] Example 3
[0068] A method for preparing a fluorescent dye responsive to carboxylesterase includes the following steps:
[0069] S1. 12 mmol of 2,3,3-trimethyl-3H-indole and 10 mmol of 3-iodopropionic acid in a molar ratio of 1.2:1 were dissolved in a round-bottom flask containing 5 mL of toluene and reacted under a nitrogen atmosphere at 130 °C. The mixture was refluxed for 16 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The solid was collected after washing several times with toluene and dried under vacuum to give 1.67 g of red solid compound 1.
[0070] The yield of compound 1 was 72%, with an ESI-MS calculated value of 232.13 and a mass spectrometry result of 232.0.
[0071] S2. Compound 1 from S1 (2 mmol, molar ratio 2:1.2) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (1.2 mmol, volume ratio 13:13:8) were dissolved in a mixed solution of 13 mL n-butanol, 13 mL toluene, and 8 mL pyridine. The mixture was reacted at 130 °C under a nitrogen atmosphere and refluxed for 10 h. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by vacuum distillation to obtain a blue-green crude product. The blue-green crude product was then subjected to silica gel column chromatography and eluted with methanol and dichloromethane (volume ratio 1:11) to obtain 0.14 g of blue-green compound 2.
[0072] The yield of compound 2 was 12%; the calculated value by MALDI-TOF-MS was 597.72, and the mass spectrometry result was 597.0.
[0073] S3. Compound 2 from S2 (0.1 mmol), 0.12 mmol of isophthalic acid, 0.12 mmol of EDC, and 0.12 mmol of DMAP in a molar ratio of 1:1.2:1.2:1.2 were dissolved in a round-bottom flask containing 7 mL of dichloromethane and reacted at room temperature for 3.5 h. The extent of the reaction was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a blue-green crude product. The blue-green crude product was subjected to silica gel column chromatography and eluted with methanol and dichloromethane in a volume ratio of 1:32 to obtain 38 mg of blue-green solid, i.e., fluorescent dye.
[0074] The yield of the fluorescent dye was determined to be 53%; the calculated value by ESI-MS was 718.36, and the mass spectrometry result was 728.
[0075] Example 4
[0076] A method for preparing a fluorescent dye responsive to carboxylesterase includes the following steps:
[0077] S1. 11 mmol of 2,3,3-trimethyl-3H-indole and 10 mmol of 3-iodopropionic acid in a molar ratio of 1.1:1 were dissolved in a round-bottom flask containing 5 mL of toluene. The mixture was reacted at 120 °C under a nitrogen atmosphere and refluxed for 16 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The solid was collected after washing several times with toluene and dried under vacuum to give 1.67 g of red solid compound 1.
[0078] The yield of compound 1 was 72%, with an ESI-MS calculated value of 232.13 and a mass spectrometry result of 232.0.
[0079] S2. Compound 1 from S1 (2 mmol in a molar ratio of 2:1) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (1 mmol in a volume ratio of 14:14:8) were dissolved in a mixed solution of 14 mL n-butanol, 14 mL toluene, and 8 mL pyridine. The mixture was reacted at 130 °C under a nitrogen atmosphere and refluxed for 11 h. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by vacuum distillation to obtain a blue-green crude product. The blue-green crude product was then subjected to silica gel column chromatography and eluted with methanol and dichloromethane in a volume ratio of 1:11 to obtain 0.14 g of blue-green compound 2.
[0080] The yield of compound 2 was 12%; the calculated value by MALDI-TOF-MS was 597.72, and the mass spectrometry result was 597.0.
[0081] S3. Compound 2 from S2 (0.12 mmol), 0.12 mmol of isophthalic acid, 0.12 mmol of EDC, and 0.12 mmol of DMAP (molar ratio 1.2:1.2:1.2:1.2) were dissolved in a round-bottom flask containing 7 mL of dichloromethane and reacted at room temperature for 3.5 h. The extent of the reaction was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a crude blue-green product. The crude blue-green product was then subjected to silica gel column chromatography and eluted with methanol and dichloromethane in a volume ratio of 1:32 to obtain 38 mg of blue-green solid, which is the fluorescent dye.
[0082] The yield of the fluorescent dye was determined to be 53%; the calculated value by ESI-MS was 718.36, and the mass spectrometry result was 728.0.
[0083] Example 5
[0084] A method for preparing a fluorescent dye responsive to carboxylesterase includes the following steps:
[0085] S1. 12 mmol of 2,3,3-trimethyl-3H-indole and 10 mmol of 3-iodopropionic acid in a molar ratio of 1.1:1 were dissolved in a round-bottom flask containing 5 mL of toluene. The mixture was reacted at 110 °C under a nitrogen atmosphere and refluxed for 14 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The solid was collected after washing several times with toluene and dried under vacuum to give 1.67 g of red solid compound 1.
[0086] The yield of compound 1 was 72%, with an ESI-MS calculated value of 232.12 and a mass spectrometry result of 232.0.
[0087] S2. Compound 1 from S1 (2 mmol) and 3,4-dihydroxy-3-cyclobutene-1,2-dione (1.1 mmol) were dissolved in a mixed solution of 14 mL n-butanol, 13 mL toluene, and 8 mL pyridine (14:13:8, v / v). The mixture was reacted at 125 °C under a nitrogen atmosphere and refluxed for 11 h. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by vacuum distillation to obtain a blue-green crude product. The blue-green crude product was then subjected to silica gel column chromatography and eluted with methanol and dichloromethane (v / v) at a ratio of 1:11 to obtain 0.14 g of blue-green compound 2.
[0088] The yield of compound 2 was 12%; the calculated value by MALDI-TOF-MS was 597.72, and the mass spectrometry result was 597.0.
[0089] S3. Compound 2 from S2 (0.11 mmol), 0.11 mmol of isophthalic acid, 0.11 mmol of EDC, and 0.11 mmol of DMAP (molar ratio 1.1:1.1:1.1:1.1) were dissolved in a round-bottom flask containing 7 mL of dichloromethane and reacted at room temperature for 3.5 h. The extent of the reaction was monitored by TLC. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a crude blue-green product. The crude blue-green product was subjected to silica gel column chromatography and eluted with methanol and dichloromethane in a volume ratio of 1:32 to obtain 38 mg of blue-green solid, which is the fluorescent dye.
[0090] The yield of the fluorescent dye was determined to be 53%; the calculated value by ESI-MS was 718.36, and the mass spectrometry result was 728.0.
[0091] A fluorescent dye was prepared in all of the above Examples 1 to 5. The following experiment was conducted using the fluorescent dye prepared in Example 1 as an example.
[0092] 1. Principle analysis of fluorescent dye response to CE enzyme
[0093] like Figures 1-3 As shown, Figure 1This is a two-dimensional structural diagram of the binding mode between the fluorescent dye and carboxylesterase in Example 1. Figure 2 This illustrates the binding mode of the fluorescent dye to the surface of the carboxylesterase molecule in Example 1. Figure 3 This represents the binding mode of the fluorescent dye and carboxylesterase in Example 1.
[0094] from Figures 1-3 As can be seen, in Example 1, the fluorescent dye and carboxylesterase form suitable steric hindrance complementarity at the binding site. One oxygen atom of the fluorescent dye acts as both a hydrogen bond donor and acceptor, forming hydrogen bonds with Met145 and Asp90 of the carboxylesterase. The fluorescent dye and carboxylesterase form hydrophobic interactions with numerous residues (Thr298, Val146, Leu304, Ala93, Lys92, Met361, Trp357, Leu367, Met458, Ler364, Les302, Phe302, and Ser305) in the carboxylesterase. Through hydrogen bonds and hydrophobic interactions between the carboxylesterase and the fluorescent dye, the binding of the fluorescent dye and carboxylesterase is promoted, improving the binding efficiency and effectively reducing interference from external factors, thus improving detection accuracy.
[0095] 2. The binding of fluorescent dyes to carboxylesterases under different temperature and pH conditions
[0096] The 5 μM fluorescent dye from Example 1 and the 30 U / mL carboxylesterase test solution were bound at 25 °C, 30 °C, 35 °C, and 40 °C, respectively, and then fluorescence spectra were measured. The results are as follows: Figure 4 As shown. From Figure 4 The binding of fluorescent dye and carboxylesterase can be observed within the temperature range of 25–40℃. The signal-to-background ratio of fluorescence intensity at 650 nm was measured, showing that within the normal physiological range, the fluorescence signal is enhanced by about 200 times after the fluorescent dye binds to carboxylesterase.
[0097] The 5 μM fluorescent dye from Example 1 and the 30 U / mL carboxylesterase test solution were combined under conditions of pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, and pH 9.0, respectively, and then fluorescence spectroscopy was performed. The results are shown below. Figure 5 As shown, Figure 5 The results show the binding of fluorescent dyes to carboxylesterases in the pH range of 5.0 to 9.0. The signal-to-background ratio of fluorescence intensity at 650 nm was measured, and it can be seen that within the normal physiological range, the fluorescence signal is enhanced by about 200 times after the fluorescent dye binds to CE.
[0098] The binding effects of the fluorescent dye and carboxylesterase at different temperatures and pH conditions demonstrate that the fluorescent dye in Example 1 of this invention exhibits stable fluorescence signal within the normal physiological range, unaffected by ambient temperature and pH, when bound to carboxylesterase. This is primarily because the oxygen atoms in the fluorescent dye in Example 1 can form hydrogen bonds with carboxylesterase, and the hydrophobic interactions between the fluorescent dye and numerous residues in the carboxylesterase effectively enhance the binding effect between the fluorescent dye and carboxylesterase, ensuring stability during detection and avoiding the impact of external factors on detection accuracy.
[0099] 3. Selective response of fluorescent dyes to carboxylesterases
[0100] The fluorescent dye from Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM mixed solution of fluorescent dye and DMSO, which was stored in a cool pharmaceutical cabinet at 4°C. For testing, the 1 mM mixed solution of fluorescent dye and DMSO was diluted with DMSO to prepare a 5 μM mixed solution of fluorescent dye and DMSO. To the 5 μM mixed solution of fluorescent dye and DMSO, 30 U / mL of peroxidase (CAT), 30 U / mL of tyrosinase (TYR), 30 U / mL of invertase (Int), 30 U / mL of acetaldehyde dehydrogenase (ALDH), 30 U / mL of glucose oxidase (GOD), 30 U / mL of acetylcholine, 30 U / mL of esterase (AChE), 30 U / mL of leucine peptidase (LAP), 30 U / mL of acylase (Acyl), and 30 U / mL of carboxylesterase (CE) were added, respectively. The selectivity of the fluorescent dye for carboxylesterases was investigated, and the results are as follows: Figure 6 As shown.
[0101] from Figure 6 The results show that the fluorescence intensity of the fluorescent dye bound to carboxylesterase is much greater than that of the fluorescent dye bound to other enzymes, indicating that the fluorescent dye has high selectivity for carboxylesterase and can effectively improve the detection sensitivity without interference from other enzymes. This is mainly because the binding site of the fluorescent dye and carboxylesterase forms a suitable steric hindrance complementarity. One oxygen atom on the fluorescent dye acts as a hydrogen bond donor and acceptor, forming hydrogen bonds with Met145 and Asp90 in the carboxylesterase. Furthermore, the fluorescent dye forms hydrophobic interactions with a large number of residues in the carboxylesterase, such as Thr298, Val146, Leu304, Ala93, Lys92, Met361, Trp357, Leu367, Met458, Ler364, Les302, Phe302, and Ser305. Through the hydrogen bonds and hydrophobic interactions formed between the fluorescent dye and the carboxylesterase, the binding effect of the fluorescent dye and carboxylesterase is effectively improved.
[0102] 4. Binding of fluorescent dyes with different concentrations of carboxylesterases
[0103] Carboxylesterase at concentrations ranging from 0 to 40 U / mL was added to 5 μM of the fluorescent dye from Example 1, and fluorescent titration experiments were performed. The results are as follows: Figure 7 As shown. From Figure 7 The results show that the fluorescence intensity continuously increases with the increase of carboxylesterase concentration.
[0104] This invention synthesizes a fluorescent dye through molecular structure design that enhances binding with carboxylesterase. This fluorescent dye exhibits high selectivity for carboxylesterase, and its fluorescence signal is unaffected by ambient temperature and pH, demonstrating stable function. One oxygen atom on the fluorescent dye acts as both a hydrogen bond donor and acceptor, forming hydrogen bonds with Met145 and Asp90 residues in the carboxylesterase. Furthermore, the fluorescent dye forms hydrophobic interactions with numerous residues in the carboxylesterase, such as Thr298, Val146, Leu304, Ala93, Lys92, Met361, Trp357, Leu367, Met458, Ler364, Les302, Phe302, and Ser305. Through these hydrogen bonds and hydrophobic interactions, the binding effect between the fluorescent dye and the carboxylesterase is effectively improved, avoiding inaccurate detection results caused by poor binding due to external factors.
[0105] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A fluorescent dye responsive to carboxylesterase, characterized in that, The structural formula of the fluorescent dye is shown in formula (I):
2. A method for preparing a fluorescent dye responsive to carboxylesterase as described in claim 1, characterized in that, Includes the following steps: Compound 1 was prepared by dissolving 2,3,3-trimethyl-3H-indole and 3-iodopropionic acid in toluene and then reacting the mixture at 110–130 °C under a protective atmosphere. Compound 1 was mixed with 3,4-dihydroxy-3-cyclobutene-1,2-dione and dissolved in a mixed solution of n-butanol, toluene and pyridine. The mixture was then heated to 110–130 °C under a protective atmosphere to prepare compound 2. Compound 2 was mixed with m-phenylenediol, and a crosslinking agent was added to carry out a crosslinking reaction to prepare the fluorescent dye. The specific reaction equation is as follows:
3. The method for preparing the fluorescent dye according to claim 2, characterized in that, The molar ratio of 2,3,3-trimethyl-3H-indole to 3-iodopropionic acid is 1 to 1.2:
1.
4. The method for preparing the fluorescent dye according to claim 2, characterized in that, The molar ratio of compound 1 to 3,4-dihydroxy-3-cyclobutene-1,2-dione is 2:1 to 1.2; The volume ratio of n-butanol, toluene, and pyridine is 12-15:12-15:7-8; The ratio of compound 1 to n-butanol is 2 mmol: 13-15 ml.
5. The method for preparing the fluorescent dye according to claim 2, characterized in that, The crosslinking agents are EDC and DMAP.
6. The method for preparing the fluorescent dye according to claim 2, characterized in that, The protective atmosphere used in the preparation of compound 1 and the preparation of compound 2 is nitrogen.
7. The method for preparing the fluorescent dye according to claim 2, characterized in that, The preparation of compound 1 includes washing after the heating reaction, and the washing is performed using toluene.
8. The method for preparing the fluorescent dye according to claim 2, characterized in that, The preparation process of compound 2 also includes vacuum distillation and chromatography after the heating reaction; The chromatography is silica gel column chromatography, and the eluent for the chromatography is a mixture of methanol and dichloromethane in a volume ratio of 1:10-12.
9. The method for preparing the fluorescent dye according to claim 5, characterized in that, The specific preparation process of the fluorescent dye is as follows: Compound 2, isophthalic acid, EDC and DMAP were dissolved in dichloromethane and reacted at room temperature. After the reaction was completed, the mixture was distilled under reduced pressure and chromatographically analyzed to obtain the fluorescent dye. The molar ratio of compound 2, isophthalic acid, EDC, and DMAP is 1–1.2:1–1.2:1–1.2:1–1.2; The reaction time is 4 to 4.5 hours; the chromatography is silica gel column chromatography, and the eluent for the chromatography is a mixture of methanol and dichloromethane in a volume ratio of 1:28 to 30.
10. The use of the fluorescent dye of claim 1 in the preparation of a fluorescent probe for the specific recognition of carboxylesterases.