A fluorescent probe for detecting glucuronyltransferase 1a1 and its application

A technology for glucuronic acid and fluorescent probes is applied in the field of detecting glucuronyltransferase 1A1 fluorescent probes, which can solve the problems of low efficiency, poor stability, and complicated detection, and achieve the effects of high efficiency, high speed and low noise.

Active Publication Date: 2022-07-19
DALIAN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the detection methods used in clinical practice have problems such as poor stability, cumbersome detection, and low efficiency.

Method used

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  • A fluorescent probe for detecting glucuronyltransferase 1a1 and its application
  • A fluorescent probe for detecting glucuronyltransferase 1a1 and its application
  • A fluorescent probe for detecting glucuronyltransferase 1a1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Confirmation of the structure of the dihydroxy probe

[0070] (1) Determination of the optimal fluorescent site in the probe

[0071]

[0072] Hydroxyl-substituted compounds 1-4, wherein the hydroxy group in compound 1 is substituted at the 6-position, the hydroxyl group in compound 2 is substituted at the 5-position, the hydroxyl group in compound 3 is substituted at the 7-position, and the hydroxyl group in compound 4 is substituted at the 8-position.

[0073] The absorption spectrum and fluorescence spectrum of compound 1-4 were measured. The absorption spectrum of compound 1-4 in Tris-HCl system was collected at 400-850 nm; the fluorescence emission spectrum of compound 1-4 in Tris-HCl system, excitation wavelength 670nm, acquisition band 696-850nm. The result is as figure 1 As shown, the maximum absorption of compound 1 is at 690 nm, while the maximum absorption of compounds 2-4 is around 580 nm; meanwhile, the fluorescence spectrum shows that only ...

Embodiment 2

[0077] The synthesis of embodiment 2 compound HHC

[0078] The synthetic route of HHC is as follows:

[0079]

[0080] 3,4-Dimethoxy-2-hydroxybenzaldehyde (1 mmol) and 2-bromo-1-cyclohexene-1-carbaldehyde (284 mg, 1.5 mmol) were dissolved in 10 mL of N,N-dimethyl In the formamide, cesium carbonate (815 mg, 2.5 mmol) was added and stirred at room temperature for 2 h. Then, the reaction solution was poured into deionized water, extracted with ethyl acetate, the organic phase was concentrated by distillation under reduced pressure, and the residue was separated by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain a yellow solid. 139mg, yield 51%. 1 H NMR (400MHz, CDCl 3 )δ10.44(s,1H),6.88(d,J=8.5Hz,1H),6.67(d,J=8.5Hz,1H),6.64(s,1H),3.92(s,3H),3.90( s, 3H), 2.58(dd, J=8.8, 3.6Hz, 2H), 2.46(t, J=6.1Hz, 2H), 1.76–1.70(m, 2H).

[0081] Compound 3 (0.1 mole) and compound 4 (32 mg, 0.1 mmol) were dissolved in 2 mL of acetic anhydride, potassium carbonate (21 m...

Embodiment 3

[0083] Example 3 In vitro determination of the selectivity of HHC to different UGTs single enzymes

[0084] (1) Prepare 190 μL of in vitro metabolic reaction system in advance, including Tris-HCl buffer (50 mM) at pH 7.4, different types of UGT single enzymes (0.1 mg / mL), HHC (final concentration 10 μM), and pre-incubate with shaking at 37 °C 3 minutes;

[0085] (2) Add 10 μL of UDPGA at a concentration of 40 mM (final concentration of 2 mM) to the reaction system to initiate the reaction;

[0086] (3) After 30 minutes, 100 μL of ice acetonitrile was added, and the reaction was terminated after vigorous shaking;

[0087] (4) Using a high-speed refrigerated centrifuge at 4°C and 20,000×g, after high-speed centrifugation for 20 minutes, take the supernatant for fluorescence detection (HHC-G: Ex=670nm, Em=720nm, the results are as follows: image 3 shown). Figure 4 It is shown that only UGT1A1 can catalyze the reaction, and the reaction rate is much higher than that of other ...

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Abstract

A fluorescent probe for detecting glucuronyl transferase 1A1 and its application belong to the technical field of biomedicine. It can be used to measure the activity of UGT1A1 in biological systems of different origins. Using HHC as the specific probe reaction substrate, with the help of the glucuronyl transferase in vitro reaction system, the glucuronidation binding reaction of the 5-hydroxyl group of HHC is used as the probe reaction, and the metabolite HHC-5- in unit time is quantitatively detected. β‑ The production of D-Glucuronoside (HHC-G) was used to measure the activity of UGT1A1 in each biological sample. The invention can be used for qualitative and quantitative determination of UGT1A1 activity in human and animal tissue samples from different individual sources, cells of different species and cell preparations, as well as various plants and microorganisms, and can also realize in vivo imaging research on UGT1A1. A rapid assessment of hepatic bile excretion function. By evaluating the activity of UGT1A1 in different individuals, it can be used to guide the rational use of clinical drugs, such as irinotecan, and high-throughput screening of UGT1A1 inhibitors.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a fluorescent probe for detecting glucuronyl transferase 1A1 (UGT1A1) and an application thereof. Background technique [0002] Glucuronosyltransferase 1A1 (UGT1A1) is a two-phase detoxification enzyme widely expressed in the human liver, small intestine, kidney and other organs, mainly distributed in the endoplasmic reticulum, especially in the liver. UGT1A1 can mediate the process of metabolism and detoxification of a variety of endogenous and endogenous substances. Most importantly, UGT1A1 is the most important enzyme in the metabolism and clearance of bilirubin in humans, and its activity changes are closely related to neonatal jaundice and clinical drug-induced jaundice, Hyperbilirubinemia is inextricably linked. [0003] The function and expression of UGT1A1 are also easily affected by a variety of environmental factors, and changes in its activity can dire...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D405/06C09K11/06G01N21/64A61K31/4745A61K31/366A61P35/00
CPCC07D405/06C09K11/06G01N21/6428A61K31/4745A61K31/366A61P35/00C09K2211/1029C09K2211/1088G01N2021/6439A61K2300/00
Inventor 马骁驰冯磊田象阁崔京南刘涛
Owner DALIAN MEDICAL UNIVERSITY
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