Fluorescent Probe for Carboxypeptidase Detection via ProTide Chemistry
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Solution Overview
Problem
Existing carboxypeptidase activity-detecting fluorescent probes face challenges such as poor sensitivity, difficulty in detecting activity in living cells, and complex synthesis processes.
Innovation Solution
A novel carboxypeptidase activity-detecting fluorescent probe is developed using ProTide chemistry, which features a compound represented by a specific general formula that undergoes a large fluorescence intensity change at one reaction point, enabling visualization of carboxypeptidase activity in living cells with high sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a fluorescent probe with two reaction points is used to detect carboxypeptidase activity, then the probe can detect pancreatic fluid leakage in animal models, but sensitivity and quantification are poor and it is difficult to detect activity in living cells
Solution Approach 1:
The probe is divided into distinct functional modules: a fluorophore unit, a P1 unit with amino acid residue, and a P2 unit with C-terminal amino acid residue connected via ProTide chemistry. This segmentation allows the probe to maintain multiple reaction points while improving overall detection performance through optimized modular structure
Solution Approach 2:
The invention changes the chemical parameters of the probe by introducing ProTide chemistry linkage between the P1 and P2 units, which modifies the probe's reactivity and fluorescence properties to achieve better sensitivity and quantification capability
2Measurement precision
If a fluorescent probe utilizing azoformyl group reactivity is used, then fluorescence increase by 300 times or more is achieved and PSMA activity can be detected, but the synthesis is complicated and the azo group may be reduced by intracellular reductase
Solution Approach 1:
The invention changes the chemical linkage parameter from azoformyl group to ProTide chemistry, which maintains the fluorescence enhancement capability while simplifying the synthesis process and eliminating the vulnerability to intracellular reductase
Solution Approach 2:
The probe uses a stable, non-reducible ProTide linkage that functions as a single-use detection element without requiring complex synthesis or stabilization measures, making it more practical for biological applications
3Measurement precision
If a fluorescent probe utilizing azoformyl group reactivity is used, then fluorescence increase by 300 times or more is achieved, but the azo group may be reduced by intracellular reductase
Solution Approach 1:
The invention avoids the harmful reduction of the azo group by replacing it with ProTide chemistry, which is stable against intracellular reductase while maintaining the desired fluorescence response to carboxypeptidase activity
Solution Approach 2:
The chemical parameter of the linkage group is changed from azoformyl to ProTide, which fundamentally alters the probe's stability profile to resist intracellular reduction while preserving detection sensitivity
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 a significant fluorescence intensity change upon reacting with carboxypeptidase, allowing for sensitive detection of carboxypeptidase activity in living cells and potentially aiding in the visualization of minute cancer sites during surgery and in breast cancer imaging.
Implementation Method 1
the fluorophore is a fluorophore in which an absorption/fluorescence wavelength greatly changes or a quenched state changes to a fluorescent state by elimination of a —P(═O)(—R1)-T-S moiety
Data Source
AI summary
A compound is represented by the following general formula (I):The compound or a salt thereof provides a fluorescent probe for detecting carboxypeptidase activity In the formula, R1 represents an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms; T represents an amino acid residue or a residue of an amino acid derivative; S represents a C-terminal amino acid residue.represents a fluorophore, and the fluorophore is a fluorophore in which an absorption/fluorescence wavelength greatly changes or a quenched state changes to a fluorescent state by elimination of the —P(═O)(—R1)-T-S moiety.