Fluorescent probe for detecting gamma-glutamyl transpeptidase as well as preparation method and application of fluorescent probe

A glutamyl transpeptidase and fluorescent probe technology, applied in the field of molecular detection and molecular imaging, to achieve the effects of good selectivity, good solubility and low cost

Inactive Publication Date: 2015-09-09
INST OF CHEM CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Cresyl violet has been widely used in biological analysis due to its high quantum yield and good stability, but a highly sensitive fluorescent probe based on cresyl violet for the detection of γ-glutamyl transpeptidase has not been reported yet.

Method used

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  • Fluorescent probe for detecting gamma-glutamyl transpeptidase as well as preparation method and application of fluorescent probe
  • Fluorescent probe for detecting gamma-glutamyl transpeptidase as well as preparation method and application of fluorescent probe
  • Fluorescent probe for detecting gamma-glutamyl transpeptidase as well as preparation method and application of fluorescent probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The preparation of the fluorescent probe (formula I) of embodiment 1, gamma-glutamyl transpeptidase detection

[0057] according to figure 1 The chemical reaction scheme shown in the preparation was carried out.

[0058] Step 1): Dissolve 151mg Boc-L-glutamate-1-tert-butyl ester (0.5mmol) in 5mL dichloromethane, add 190mg HATU (0.5mmol) and 200μL N,N-diisopropylethylamine After mixing and stirring for 30 minutes, 160 mg of cresyl violet acetate (0.5 mmol) was added, and the reaction was continued for 2.5 hours at 25° C., filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. The intermediate product was purified by column chromatography (silica gel G, 200-300 mesh) and the intermediate product was an orange solid.

[0059] The results of structural confirmation are as follows: 1 H NMR (400MHz, 298K, DMSO-d 6 ):δ10.45(s,1H),8.59(d,1H,J=7.2Hz),8.14(d,1H,J=6.8Hz),7.88(m,4H),7.48(m,1H),7.19 (d,1H,J=8.0Hz),6.40(s,1H),3.90(...

Embodiment 2

[0064] Embodiment 2, preparation of γ-glutamyl transpeptidase detection kit

[0065] Weigh 3.9mg of fluorescent probe (formula I), dissolve in 40mL of methanol, and prepare fluorescent probe stock solution (also known as mother solution, 250 μM) to obtain γ-glutamyl transpeptidase detection kit.

[0066] The kit of the present invention detects gamma-glutamyl transpeptidase specifically according to the following steps:

[0067] 1) Make a standard curve:

[0068] With 585nm as the excitation wavelength, measure the concentration of 1, 5, 10, 25, 40 and 50U / L of γ-glutamyl transpeptidase standard reaction solution respectively 2mL, and the fluorescence intensity at the emission wavelength of 615nm, recorded as F; and measure the fluorescence intensity of the reagent blank at an emission wavelength of 615nm, denoted as F 0 , with the concentration C of γ-glutamyl transpeptidase as the abscissa, and the fluorescence intensity change value ΔF as the ordinate, draw a standard cur...

Embodiment 3

[0079] Example 3, Application of γ-Glutamyl Transpeptidase Detection Kit

[0080] 1. Detection of the content of γ-glutamyl transpeptidase in human serum

[0081] First make a standard curve according to the detection of the spectral properties in Example 2, then dilute the independent undiluted human serum sample 10 times and perform the test according to the above method, and substitute the test results into the standard curve to calculate the 10 times diluted serum The amount of γ-glutamyl transpeptidase, and then converted to obtain the amount of γ-glutamyl transpeptidase in the original serum sample. At the same time, the γ-glutamyl transpeptidase fluorescence assay kit of Sigma Company was used to measure the content of the enzyme in the serum, and the results are shown in Table 1 below. Comparing the results obtained by the two methods, there is no significant difference in the 90% confidence interval between the two methods, indicating that the patented kit can accura...

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Abstract

The invention discloses a fluorescent probe for detecting gamma-glutamyl transpeptidase as well as a preparation method and application of the fluorescent probe. The structure formula of a compound is shown by formula I, and a preparation method of the compound comprises the following steps: (1) in an organic solvent I, adding a condensing agent into Boc-L-glutamic acid-1-tert-butyl ester for activation, and then adding cresyl violet acetate for reaction to obtain an intermediate product; and (2) in an organic solvent II, mixing the intermediate product with a de-protection reagent for reaction. The compound provided by the invention is applied to preparation of the fluorescent probe for detecting gamma-glutamyl transpeptidase, and/or detection of gamma-glutamyl transpeptidase and imaging of gamma-glutamyl transpeptidase. A kit for detecting gamma-glutamyl transpeptidase comprises the fluorescent probe for detecting gamma-glutamyl transpeptidase and a solvent; the fluorescent probe for detecting gamma-glutamyl transpeptidase is the compound; and the kit for detecting gamma-glutamyl transpeptidase is applied to the detection of gamma-glutamyl transpeptidase and analysis of biological samples. The fluorescent probe disclosed by the invention has the advantages of simple and convenient operation, low cost, fastness, sensitivity and the like, and is easy to popularize and apply.

Description

technical field [0001] The present invention relates to a fluorescent probe for the detection of γ-glutamyl transpeptidase and its preparation method and application, in particular to a fluorescent probe for the detection of γ-glutamyl transpeptidase and its preparation method The invention relates to a gamma-glutamyl transpeptidase detection kit, belonging to the fields of molecular detection and molecular imaging. Background technique [0002] γ-glutamyl transpeptidase is a key enzyme in the glutamyl cycle, which can specifically catalyze the transfer reaction of γ-glutamyl group, and plays a vital role in various physiological processes. Clinically, γ-glutamyl transpeptidase is an important indicator for the diagnosis of hepatobiliary diseases, and is widely used in the diagnosis and prognosis of liver diseases and other organ diseases. In addition, γ-glutamyl transpeptidase is highly expressed in cervical cancer, ovarian cancer, liver cancer and other cancers, and is an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D265/34C09K11/06G01N21/64
CPCC07D265/34C09K11/06C09K2211/1033G01N21/6428
Inventor 史文马会民李丽红
Owner INST OF CHEM CHINESE ACAD OF SCI
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