Synthesis and evaluation of beta-galactosidase cleavable linkers for payload release

Abstract

β-galactosidase (β-gal), a hydrolase enzyme that catalyses the hydrolysis of β-D-galactosidic bonds serves as a widely used biomarker for cellular senescence, where senescence-associated β-gal (SA-β-gal) activity distinguishes senescent cells from proliferative ones. Its overexpression in cancerous and senescent tissues makes it a promising enzymatic trigger in enzyme-responsive drug delivery systems, enabling site-specific drug activation with minimal off-target effects. A key component of enzyme-triggered drug release is the self-immolative linker (SIL) that helps in a controlled payload release. This study explores the impact of EWGs on β-gal-responsive payload release by designing and analysing self-immolative linkers conjugated to 4-nitrophenyl chloroformate as a model payload. Upon enzymatic activation, 4-nitrophenolate, a well-characterized leaving group, is spectroscopically monitored to assess reaction kinetics. These findings provide insights into optimizing enzyme-activated prodrug systems for targeted drug delivery in cancer therapy and senescence-associated diseases. By fine-tuning linker design and substituent effects, this research contributes to the development of precise and efficient enzyme-responsive therapeutic strategies.