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CN115872950B - Fluorescent compound, application thereof in detection of urease and in-vitro diagnostic reagent - Google Patents

Fluorescent compound, application thereof in detection of urease and in-vitro diagnostic reagent Download PDF

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Publication number
CN115872950B
CN115872950B CN202111125919.XA CN202111125919A CN115872950B CN 115872950 B CN115872950 B CN 115872950B CN 202111125919 A CN202111125919 A CN 202111125919A CN 115872950 B CN115872950 B CN 115872950B
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urease
fluorescent compound
detection
fluorescent
reaction
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CN115872950A (en
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傅秀军
裴庆
姚敏
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

A fluorescent compound has a structure shown in the structural formula I, wherein R 1 And R is 2 Independently selected from a fluorescent group or a quenching group. When the substrate is contacted with the urease, the reaction liquid generates fluorescence characteristics under the hydrolysis of the urease, and the activity of the urease can be detected by measuring the change of the fluorescence intensity value. The urease detection efficiency and reliability are improved, and the detection time is shortened.

Description

Fluorescent compound, application thereof in detection of urease and in-vitro diagnostic reagent
Technical Field
The invention relates to a compound with fluorescence characteristics, in particular to a compound with a similar structure with urea, which is used as a substrate of urease, is used for detecting the content of the urease by utilizing the fluorescence characteristics of the compound, and is applied to medical equipment for detecting the urease.
Background
Urease is a nickel-containing metal oligoenzyme capable of hydrolyzing urea to produce carbon dioxide and ammonia, and widely exists in plant seeds, animal urine and blood, and many microorganisms can synthesize and secrete urease. Such as streptococcus salivarius, helicobacter pylori, actinomyces endometricus and other microorganisms parasitic in the upper digestive tract of human body can produce urease, and the urease produced by these microorganisms is related to pathogenicity, and common related diseases include dental caries, periodontitis, atrophic gastritis, peptic ulcer and the like. Compared with other urease-producing microorganisms, the helicobacter pylori has large urease content and high activity, the urease content of the helicobacter pylori accounts for 5% -10% of the total protein of cells, the urease activity of the helicobacter pylori is 2 times of that of the proteus mirabilis, and the intestinal bacteria is 7 times of that of the proteus mirabilis. Therefore, the urease activity determination can be used as a rapid diagnosis basis for helicobacter pylori infection in clinic.
The urease detection method mainly comprises a phenol red indicator method, a Nahner reagent chromogenic method, a latex agglutination test method, an immunoblotting and enzyme-linked immunosorbent assay. The principle of the above detection method can be divided into two types, one is that the color change of the indicator phenol red or Nashi reagent occurs based on ammonia generated by hydrolysis of substrate urea by urease; the other is an immune reaction that utilizes urease antibodies to specifically recognize urease antigens. These detection and analysis methods have some drawbacks such as: the method through antigen-antibody binding reaction has better specificity, but the detection time is longer, and the method using the pH value change of the reaction system has more severe requirements on the reaction conditions. Therefore, the development of a more efficient, simple, convenient, rapid and reliable urease detection method has important social significance.
Disclosure of Invention
The invention aims to provide a fluorescent compound which has a molecular structure similar to urea, takes the fluorescent compound as a substrate of urease, and utilizes the fluorescent characteristic of the compound under the hydrolysis of the urease to generate specific wavelength so as to realize qualitative or quantitative detection of the urease.
Another object of the present invention is to provide a method for preparing a fluorescent compound, which has a molecular structure similar to urea, and uses the molecular structure as a substrate for qualitative and quantitative detection of urease.
It is a further object of the present invention to provide a fluorescent compound for use in the detection of urease which results in a more efficient, simple and reliable urease detection.
It is a further object of the present invention to provide the use of a fluorescent compound in the manufacture of a medical device for detecting urease, such that the urease detection is more efficient, simpler and more reliable.
A fifth object of the present invention is to provide the use of a fluorescent compound for the manufacture of an in vitro diagnostic reagent for the detection of urease.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a fluorescent compound having a structure represented by the following structural formula I,
wherein R is 1 And R is 2 Independently selected from a fluorescent group or a quenching group. Such as: r is R 1 And R is 2 Selected from the same fluorescent groups; the other steps are as follows: r is R 1 Selected from fluorescent groups, R 2 Selected from quenching groups; another example is: r is R 1 Selected from the group consisting of quenching groups, R 2 Selected from fluorescent groups; another example is: r is R 1 And R is 2 Selected from the group of fluorophores, but the two groups have different excitation wavelengths, and the emission wavelength of one of the fluorophores is within the excitation wavelength range of the other fluorophore.
Fluorescent compounds of the invention, fluorescent groups such as: but are not limited to toluidine blue, coumarin, or fluorescein.
Fluorescent compounds of the invention, quenching groups such as: but are not limited to black hole quenchers (Black Hole Quencher, BHQ).
When the fluorescent compound is not contacted with urease, fluorescence is inhibited by a mechanism of fluorescence energy resonance transfer (fluorescence resonance energy transfer, FRET). When the reaction solution is a substrate and contacts with urease, under the hydrolysis action of the urease, the reaction solution generates fluorescence, generates fluorescence or enhances the fluorescence, and the activity of the urease can be detected by measuring the change of the fluorescence intensity value, such as: qualitative or quantitative detection.
Compared with the prior art, the detection principle for detecting urease is different. The invention directly calculates the urease by detecting the change of the fluorescence quantity of the urease hydrolysate, and the detection is quick and simple. In the prior art, urease activity is indirectly calculated by measuring the pH value change of a solution after urease reaction, or the detection is slow and complex by antigen-antibody combination detection analysis with harsh reaction conditions and slow time.
The fluorescent compound of the invention is used as a substrate to prepare urease medical devices, such as: and in-vitro diagnostic reagent to realize rapid quantitative and qualitative detection of urease.
A method for preparing the fluorescent compound provided by the invention comprises the following steps:
mixing urea containing two reaction functional groups with fluorescent molecules (such as toluidine blue) serving as a reaction raw material in a molar ratio of 1:2, dissolving the mixture in an organic solvent (such as triethylamine and dimethylformamide), and reacting at room temperature under a dark condition; after the reaction, the solvent was removed to obtain a solid, which was redissolved in methanol. Purifying by chromatography with methanol and dichloromethane at a volume ratio of 20:1 to obtain fluorescent compound.
Another method for preparing the fluorescent compound provided by the invention comprises the following steps:
mixing urea containing two reaction functional groups with fluorescent molecules (such as toluidine blue) and quenching molecules (such as BHQ 3) serving as reaction raw materials in a molar ratio of 1:1:1, dissolving in an organic solvent, and reacting at room temperature under a dark condition; after the reaction, the solvent was removed to obtain a solid, which was redissolved in methanol. Purifying by chromatography with methanol and dichloromethane at a volume ratio of 20:1 to obtain fluorescent compound.
Another method for preparing the fluorescent compound provided by the invention comprises the following steps:
with urea containing two reactive functional groups, with two different fluorescent molecules, such as: coumarin and fluorescein are mixed according to the mol ratio of 1:1:1 as reaction raw materials, dissolved in an organic solvent and reacted at room temperature under the condition of avoiding light; after the reaction, the solvent was removed to obtain a solid, which was redissolved in methanol. Purifying by chromatography with methanol and dichloromethane at a volume ratio of 20:1 to obtain fluorescent compound.
Drawings
FIG. 1 is a scheme showing the synthesis of fluorescent compounds in example 1 of the present invention;
FIG. 2 shows the nuclear magnetic resonance hydrogen spectrum 1H NMR of a fluorescent compound of example 1 of the present invention;
FIG. 3 is a linear relationship between urease concentration and amount of fluorescence measured by the fluorescent compound of example 1 of the present invention.
Detailed Description
The technical scheme of the present invention is described in detail below. The embodiments of the present invention are only for illustrating the technical scheme of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical scheme of the present invention, which is intended to be covered by the scope of the claims of the present invention.
In the examples below, all temperatures are in degrees Celsius (C.) unless otherwise indicated. Unless otherwise indicated, various starting materials and reagents are commercially available, such as Sigma-Aldrich, sammer feier technology (ThermoFisher Scientific). The starting materials and reagents obtained were used without further purification.
The glassware is oven dried and/or heat dried. The reaction was followed on a glass silica gel-60F 254 plate (0.25 mm) (TLC). Analytical thin layer chromatography and development was performed in the appropriate solvent ratio (v/v).
1 The H NMR spectrum was determined with a Bruker instrument (400 MHz) and the chemical shifts were expressed in ppm. Tetramethylsilane internal standard (0.00 ppm) was used. 1 H NMR representation method: s=singlet, d=doublet, t=triplet, m=multiplet, br=broadened, dd=doublet of doublet. If coupling constants are provided, they are in Hz.
The molecular weight of the product is determined by an LC/MS mass spectrometer, and the ionization mode is ESI or APCI.
The preparation methods provided by the present invention should be understood as examples given for the necessity of full disclosure and not as limitations on the scope of protection as claimed. The person skilled in the art will be able to prepare the various compounds provided by the invention according to the textbook, the laboratory manual or the guidance of the examples listed in the present invention. The preparation of these compounds is also within the ordinary skill of one of ordinary skill in the art and can be accomplished under the guidance of the prior art. Specifically, the preparation method of part of the compounds provided by the invention is shown in the following figure 1.
4,4'- (carbonyldiimino) bis-, 1' -bis (2, 5-dioxo-1-pyrrolidinyl) ester and toluidine blue are taken as reaction raw materials, and are mixed and dissolved in N, N-Dimethylformamide (DMF) solvent according to the mol ratio of 1:2, and triethylamine (triethylenediamine) is added for reaction at room temperature under the condition of light shielding; after the reaction, removing the solvent to obtain a black oily solid, dissolving the black oily solid in methanol, and further carrying out chromatographic purification by using methanol to dichloromethane 20:1 to obtain the fluorescent compound.
1H NMR (400 MHz, DMSO). Delta.10.13 (s, 1H), 9.90 (s, 1H), 8.59 (s, 1H), 8.31 (s, 1H), 8.15 (s, 1H), 7.93 (s, 1H), 7.27 (s, 2H), 7.14 (s, 1H), 6.91 (s, 2H), 6.65 (s, 2H), 3.58 (s, 8H), 3.17 (s, 2H), 3.10 (s, 2H), 3.04 (s, 2H), 2.97 (s, 3H), 2.89 (m, 1H), 2.73 (s, 1H), 2.58 (s, 6H), 2.24 (s, 2H), 2.10 (s, 2H), 1.98 (s, 2H), 1.56 (s, 4H), 1.23 (s, 2H), 0.95 (s, 2H) (FIG. 2). ES-MS (m/z): 838.23 (MH + )
The urease detection step comprises the following steps:
(1) Urease solutions with different concentrations were prepared with phosphate buffer at pH 7.4 to final concentrations of 0, 1.25, 2.5, 5, 10 and 20U/ml in 100. Mu.l assay system; adding a fluorescent compound with a final concentration of 5mmol/L into the urease solution, reacting for 3-5 minutes at 37 , measuring the fluorescence amount in the solution, and preparing a standard curve according to the urease concentration and the corresponding fluorescence amount, referring to fig. 3, so as to obtain a linear fitting equation y=1.029+0.078x.
(2) Adding a sample to be detected (such as saliva, gastric juice, urine, soil, gastric mucosa biopsy tissue sample and the like) into 100 mu L of a detection system, adding a fluorescent compound with a final concentration of 5mmol/L, reacting for 3-5 minutes at 37 , measuring the fluorescence amount in the solution, and calculating the urease enzyme activity in the sample according to a standard curve. Using a saliva sample as an example, the relative fluorescence was measured to be 1.411, and the urease activity was measured to be 1.68U/ml.
In the whole process, the detection can be completed in about 10 minutes, and compared with the prior art, the detection time is obviously shortened.

Claims (4)

1. A fluorescent compound characterized by having the structure shown below.
2. A method for detecting urease using the fluorescent compound of claim 1 as a substrate, wherein the reaction solution generates fluorescent characteristics under the hydrolysis of urease after the substrate is contacted with urease, and the activity of urease can be detected by measuring the change of the fluorescent intensity value.
3. The use of a fluorescent compound of claim 1 for the preparation of a medical device for detecting urease.
4. An in vitro diagnostic reagent for detecting the enzymatic activity of urease, characterized in that it comprises the fluorescent compound according to claim 1.
CN202111125919.XA 2021-09-25 2021-09-25 Fluorescent compound, application thereof in detection of urease and in-vitro diagnostic reagent Active CN115872950B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070072929A1 (en) * 2003-10-28 2007-03-29 Silva Antonio Antibacterial and/or antiprotozoal nitromidazole derivative compounds with urease inhibitor activity, process for preparing these compounds and use in pharmaceutical compositions and medicines
CN108135903A (en) * 2015-09-09 2018-06-08 目标实验室有限责任公司 NIR dyes targeting PSMA and their applications

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070072929A1 (en) * 2003-10-28 2007-03-29 Silva Antonio Antibacterial and/or antiprotozoal nitromidazole derivative compounds with urease inhibitor activity, process for preparing these compounds and use in pharmaceutical compositions and medicines
CN108135903A (en) * 2015-09-09 2018-06-08 目标实验室有限责任公司 NIR dyes targeting PSMA and their applications

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
A Chemically Induced Vaccine Strategy for Prostate Cancer;Anna Dubrovska等;ACS Chem. Biol.(第6期);第1223–1231页 *
Development of Tumor-Targeted Near Infrared Probes for Fluorescence Guided Surgery;Lindsay E. Kelderhouse等;Bioconjugate Chem.;第24卷;第1075−1080页 *
Novel solid-phase strategy for the synthesis of ligand-targeted fluorescent-labelled chelating peptide conjugates as a theranostic tool for cancer;Sagnik Sengupta等;Beilstein J. Org. Chem.;第14卷;第2665–2679页 *
Prostate-Specific Membrane Antigen Targeted Imaging and Therapy of Prostate Cancer Using a PSMA Inhibitor as a Homing Ligand;Sumith A. Kularatne等;MOLECULAR PHARMACEUTICS;第6卷(第3期);第780–789页 *

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