Monoacyl Fluorescent Probe for Real-Time Protease Detection
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Solution Overview
Problem
Existing fluorescent probes for measuring proteases require multiple steps for fluorescence activation, resulting in poor real-time response and quantification suitability due to the need for hydrolysis of both acyl groups to change from a closed to an open ring structure.
Innovation Solution
A fluorescent probe utilizing a diaminorhodamine structure with a hydroxy(lower alkyl) group substitution, where the carboxyl group on the benzene ring is acylated and then de-acylated, allowing a structural change from a closed to an open ring structure at one reaction site, enabling prompt fluorescence activation at neutral pH.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a diacyl type fluorescent probe is used for measuring protease, then the probe can detect protease activity, but the fluorescence response requires multiple hydrolysis steps resulting in poor real-time response property
Solution Approach 1:
The invention extracts and removes one acyl group from the traditional diacyl probe structure, creating a monoacyl probe structure. This extraction eliminates the need for dual hydrolysis steps while retaining the essential protease recognition and fluorescence activation functions, thereby achieving real-time response.
Solution Approach 2:
The probe structure is segmented into essential and non-essential components. The invention identifies that only one acyl group is necessary for both protease recognition and fluorescence activation, separating the functional requirements from the traditional dual-acyl structure and eliminating redundant elements.
2Measurement precision
If a diacyl type fluorescent probe is used for measuring protease, then the probe can detect protease activity, but the multiple hydrolysis steps result in poor suitability for quantification
Solution Approach 1:
By extracting one acyl group from the diacyl structure, the invention simplifies the reaction pathway to a single hydrolysis step. This reduction in complexity directly improves quantification suitability while preserving the probe's ability to detect and measure protease activity accurately.
3Stability of the object's composition
If the carboxyl group on the benzene ring is replaced with a hydroxy(lower alkyl) group and the amino group is acylated, then the probe achieves closed ring non-fluorescent structure, but requires acyl group removal to activate fluorescence
Solution Approach 1:
The invention modifies the chemical parameters by replacing the carboxyl group with a hydroxy(lower alkyl) group, which fundamentally changes the ring-closing mechanism. This parameter change enables the ring to close through different chemistry, achieving stable non-fluorescent state that can be activated by a single hydrolysis event rather than requiring complex multi-step processes.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The probe achieves several hundred times higher fluorescence intensity and provides superior real-time response and quantification capabilities for protease measurement, with the open ring form being retained in cells without leakage, enabling long-term sensitive imaging.
Implementation Method 1
hydrolysis of the acyl group at one side by a protease
Implementation Method 2
fluorescent probes are functional molecules which are substantially non-fluorescent in the absence of a target substance, and become fluorescent after a reaction with the target substance
Data Source
Figure 1(a)~1(d)
Figure 2~3
AI summary
A compound represented by the formula (I) or a salt thereof (R1 represents hydrogen atom, or a substituent; R2 to R7 represent hydrogen atom, hydroxyl group, an alkyl group, or a halogen atom; R8 and R9 represent hydrogen atom, or an alkyl group; X represents a C1-C3 alkylene group; and R10 represents an acyl group), which promptly causes a structural change from a non-fluorescent closed ring structure to a strongly fluorescent open ring structure by hydrolysis of an acyl group, and is useful as a fluorescent probe for measuring a protease showing superior suitability for quantification.