Methoxyfluorescein derivative and its preparation method, carboxypeptidase y detection method and detection kit

A detection method, fluorescein technology, applied in the direction of fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of harsh storage and permission conditions, difficult peptide substrate synthesis, and difficult substrate synthesis, etc., to achieve High sensitivity and selectivity, low price, high sensitivity effect

Active Publication Date: 2019-10-08
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main method for measuring the activity of carboxypeptidase Y mainly contains ultraviolet absorption spectrometry. The substrate required by this method is simple in structure and easy to operate, and has been widely used at present. However, the ultraviolet method has the following two defects: first, ultraviolet absorption The large background interference of the spectroscopic method leads to low analysis sensitivity; secondly, most of the substrates used for analysis are peptides, which are difficult to synthesize and costly
The second commonly used method is fluorescence spectrometry, which mainly uses polypeptide fluorescent probes as substrates, and its reaction sensitivity is higher than that of ultraviolet spectrometry, but the synthesis of polypeptide substrates for analysis is difficult and costly Most of them have poor stability under normal conditions, harsh storage and permissive conditions, and low atomic utilization
[0003] At present, there are no reports of non-peptide fluorescent probes used to detect the activity of carboxypeptidase Y at home and abroad.

Method used

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  • Methoxyfluorescein derivative and its preparation method, carboxypeptidase y detection method and detection kit
  • Methoxyfluorescein derivative and its preparation method, carboxypeptidase y detection method and detection kit
  • Methoxyfluorescein derivative and its preparation method, carboxypeptidase y detection method and detection kit

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preparation example Construction

[0033] In a second aspect, the present invention also provides a method for preparing the above-mentioned methoxyfluorescein derivatives, the method comprising: in an organic solvent under the conditions of a nucleophilic reaction and the presence of an acid-binding agent, the The structure compound is contacted with one of acid chloride or acid anhydride;

[0034]

[0035] Wherein, the general formula of the acid chloride is R'-C(=O)Cl, R' has the same selection as R; that is, R' is a C1-C10 alkyl or cycloalkyl group; preferably, R' It is an alkyl group of C2-C6; more preferably, R' is selected from one of ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; more Preferably, R' is selected from one of ethyl, propyl, butyl and cyclohexyl, more preferably, R' is selected from ethyl, n-propyl, isopropyl, tert-butyl and cyclohexyl (that is, the acid chloride is selected from one of propionyl chloride, n-butyryl chloride, isobutyryl chlorid...

Embodiment 1

[0076] This example is used to illustrate the preparation method of the methoxyfluorescein derivatives of the present invention.

[0077] In an organic solvent (dichloromethane, 20mL), in the presence of an acid-binding agent (triethylamine, 2mmol), the compound represented by formula (2) (methoxyfluorescein, 1mmol) and acid chloride (trimethyl Acetyl chloride, 2 mmol) was maintained at 5°C for 120 min 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.

[0078] The white solid product was characterized by NMR and HRMS, and the data are as follows. 1 H NMR (600MHz, DMSO-d 6 ): δ8.02(d, J=11.4Hz, 1H)...

Embodiment 2

[0082] This example is used to illustrate the preparation method of the methoxyfluorescein derivatives of the present invention.

[0083] The preparation of methoxyfluorescein derivatives was carried out according to the method of Example 1, except that cyclohexylformyl chloride was used as the acid chloride, and finally 0.31 g of white solid was obtained.

[0084] Detect and characterize this white solid product with NMR and HRMS, data: 1H NMR (600MHz, DMSO-d 6 ): δ8.05(d, J=7.2Hz, 1H), 7.81(t, J=7.2Hz, 1H), 7.75(t, J=7.2Hz, 1H), 7.33(d, J=7.2Hz, 1H ), 7.23(s, 1H), 6.96(s, 1H), 6.92-6.86(m, 2H), 6.74(q, J=8.8Hz, 2H), 3.82(s, 3H), 2.61(quintuplet, J= 9.6Hz,1H),1.99-1.97(m,2H),1.73-1.71(m,2H),1.51-1.46(m,2H),1.36-1.30(m,2H),1.25-1.22(m,2H) .HRMS calcd for [M+H] + :457.1646. Found: 457.1648..

[0085] This data completely agrees with the theoretical value of the compound shown in formula (1), proves that this product is the compound shown in formula (4).

[0086]

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Abstract

The invention relates to the detection field of carboxypeptidase Y, in particular to a methoxy fluorescein derivative and a preparation method of the methoxy fluorescein derivative and a detection method and detection kit of carboxypeptidase Y. According to the methoxy fluorescein derivative and the preparation method of the methoxy fluorescein derivative disclosed by the invention, the methoxy fluorescein derivative has the structure of the formula (1), wherein R is alkyl of C1-C10 or cycloalkyl; the method comprises the step of contacting a compound of the formula (2) with one of acyl chloride and anhydride in an organic solvent in the presence of nucleophilic reaction conditions and an acid binding agent. The invention further discloses application of methoxy fluorescein derivative in detection of activity of carboxypeptidase Y and the method for detecting the activity of carboxypeptidase Y. The methoxy fluorescein derivative disclosed by the invention is high in sensitivity, good in selectivity, large in change of fluorescence intensity and easy in detecting fluorescence intensity, higher in stability, more convenient to use and lower in price while used in the carboxypeptidaseY activity related detection.

Description

technical field [0001] The invention relates to the detection field of carboxypeptidase Y, in particular to a methoxyfluorescein derivative, a preparation method thereof, a carboxypeptidase Y detection method and a detection kit. Background technique [0002] Carboxypeptidase Y (EC number: 3.4.16.1), which can hydrolyze all amino acids with a carboxyl terminal, has become a commonly used 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. At present, the main method for measuring the activity of carboxypeptidase Y mainly contains ultraviolet absorption spectrometry. The substrate required by this method is simple in structure and easy to operate, and has been widely used at present. However, the ultraviolet method has the following tw...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D493/10C09K11/06G01N21/64
Inventor 杨光富杨文超杨庶侯伍磊
Owner HUAZHONG NORMAL UNIV
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