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Hemicyanine-based fluorescent probe for detecting carboxypeptidase Y, preparation method and applications thereof

A technology of fluorescent probes and carboxypeptidases, applied in biochemical equipment and methods, fluorescence/phosphorescence, chemical instruments and methods, etc., can solve the problems of harsh storage and permission conditions, difficult synthesis of polypeptide substrates, and difficulty in substrate synthesis Large and other problems, to achieve the effect of high sensitivity and selectivity, low price and high sensitivity

Active Publication Date: 2018-06-22
HUAZHONG NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The substrate required by ultraviolet absorption spectroscopy has a simple structure and is easy to operate, and has been widely used at present, but the ultraviolet method has the following two defects: first, the large background interference of ultraviolet absorption spectroscopy leads to low analysis sensitivity; secondly, Most of the substrates used for analysis are peptides, which are difficult to synthesize and costly
Fluorescence spectroscopy mainly uses polypeptide fluorescent probes as substrates, and its reaction sensitivity is higher than that of ultraviolet spectroscopy. However, the synthesis of polypeptide substrates used for analysis is difficult and costly, and most of them have poor stability under normal conditions. Allowing conditions are harsh and atom utilization is low
In addition, the substrate of p-nitrophenyl trimethylacetate can be used without polypeptide substrate, but the detection of p-nitrophenyl trimethylacetate substrate is a UV-visible method with very limited sensitivity
[0004] At present, there are no reports of non-peptide near-infrared fluorescent probes used to detect the activity of carboxypeptidase Y at home and abroad.

Method used

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  • Hemicyanine-based fluorescent probe for detecting carboxypeptidase Y, preparation method and applications thereof
  • Hemicyanine-based fluorescent probe for detecting carboxypeptidase Y, preparation method and applications thereof
  • Hemicyanine-based fluorescent probe for detecting carboxypeptidase Y, preparation method and applications thereof

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preparation example Construction

[0027] In a second aspect, the present invention also provides a method for preparing the above-mentioned semicyanine fluorescent probe, the method comprising: in an organic solvent under the conditions of a nucleophilic reaction and the presence of an acid-binding agent, the compound represented by the formula (2) The structure compound is contacted with pivaloyl chloride;

[0028]

[0029] In the preparation method of the semicyanine fluorescent probe of the present invention, the nucleophilic reaction is the reaction between the hydroxyl group in formula (2) and pivaloyl chloride, and the conditions of the nucleophilic reaction include a temperature of minus 5°C to 30°C, the time is 10-200min, preferably, the condition of the nucleophilic reaction is that the temperature is 0-25°C, and the time is 80-160min.

[0030] In the preparation method of the semicyanine fluorescent probe of the present invention, the contact needs to be carried out in the presence of an acid-bind...

Embodiment 1

[0075] This example is used to illustrate the preparation method of the semicyanine fluorescent probe described in the present invention.

[0076] In an organic solvent (dichloromethane, 20mL), in the presence of an acid-binding agent (triethylamine, 2mmol), the compound (1mmol) represented by formula (2) and pivaloyl chloride (2mmol) were mixed at 5°C, The contact was maintained for 120 minutes to obtain the contacted material. Then, the contacted material was washed with water (50 mL) and saturated saline solution (50 mL) successively to obtain the washed material. The organic phase in the washed material was separated and dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product, which was purified by a silica gel column to obtain 0.27 g of a white solid product.

[0077] 1 H NMR (400MHz, DMSO-d 6 ): \ δ9.14(s,1H),8.60(d,J=8.0Hz,1H),7.79(dd,J 1 =J 2 =4.0Hz,2H),7.60(d,J=8.0Hz,1H),7.50(t,J=6.0Hz,1H),7.44(s,1H),7.38...

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Abstract

The invention relates to the field of detection of carboxypeptidase Y, particular to a hemicyanine-based fluorescent probe for detecting carboxypeptidase Y, a preparation method and applications thereof, and discloses a hemicyanine-based fluorescent probe and a preparation method thereof, wherein the hemicyanine-based fluorescent probe has a structure represented by a formula (1), and tert amyl isused as the recognition group of carboxypeptidase Y. The synthesis method comprises that a compound represented by a formula (2) contacts pivaloyl chloride in an organic solvent under a nucleophilicreaction condition in the presence of an acid binding agent. The invention further discloses applications of the probe in detection of the activity of carboxypeptidase Y, and a method and a detectionkit for detecting the activity of carboxypeptidase Y. According to the present invention, the hemicyanine-based fluorescent probe has advantages of high sensitivity, good selectivity, high fluorescence intensity change, easy detection, high stability, convenient use and low cost in the application related to the detection of the activity of carboxypeptidase Y. The formulas (1) and (2) are definedin the specification.

Description

technical field [0001] The present invention relates to the detection field of carboxypeptidase Y, in particular, to a semicyanine fluorescent probe, a method for preparing the semicyanine fluorescent probe and its application in detecting the activity of carboxypeptidase Y, and a A detection method for carboxypeptidase Y activity and a detection kit for carboxypeptidase Y activity. Background technique [0002] Carboxypeptidase Y (EC number: 3.4.16.1), which can hydrolyze all amino acids with a carboxyl terminal, has become a common tool enzyme in the analysis of the C-terminus of protein polypeptide chains. In addition, carboxypeptidase Y can also make other amino acid derivatives or nucleophiles replace the amino acid residues at the end of the peptide chain through the transpeptide reaction, thereby forming new peptides. [0003] At present, the main methods for measuring the activity of carboxypeptidase Y are ultraviolet absorption spectrometry and fluorescence spectro...

Claims

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Application Information

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IPC IPC(8): C07D405/06C09K11/06C12Q1/37G01N21/64
CPCC07D405/06C09K11/06C09K2211/1029C09K2211/1088C12Q1/37G01N21/6486G01N2333/948
Inventor 杨光富杨文超杨庶侯
Owner HUAZHONG NORMAL UNIV
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