Modular Design Platform for Activatable Fluorescence Probes Targeting Carboxypeptidases Based on ProTide Chemistry

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Carboxypeptidases (CPs) are hydrolases crucial for physiological and pathological processes.
• Existing fluorescence probes for CPs are limited, necessitating a versatile design platform.

Purpose of the Study:

• To establish a modular design platform for carboxypeptidase-targeting fluorescence probes.
• To enable flexible tuning of probe properties for diverse biological and medical applications.

Main Methods:

• Utilized ProTide chemistry, adapting its prodrug activation mechanism for probe design.
• Developed a four-module system (fluorophore, phosphorus substituent, P1 linker amino acid, P1' substrate amino acid) for customizable probes.
• Optimized probe reactivity by modifying the P1 linker amino acid.

Main Results:

• Successfully constructed and validated fluorescence probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (PSMA).
• Demonstrated the probes' efficacy in imaging CP activity in live cells and patient clinical specimens.
• Showcased the platform's ability to fine-tune probe properties, including reactivity and emission wavelength.

Conclusions:

• The ProTide-based modular platform offers a flexible strategy for developing novel carboxypeptidase-targeting fluorescence probes.
• This approach facilitates the study of CP-related biological functions and pathological conditions.
• The developed probes are valuable tools for in vitro and in vivo imaging applications.