First-generation species-selective chemical probes for fluorescence imaging of human senescence-associated β-galactosidase

Human senescence-associated β-galactosidase (SA-β-gal), the most widely used biomarker of aging, is a valuable tool for assessing the extent of cell 'healthy aging' and potentially predicting the health life span of an individual. Human SA-β-gal is an endogenous lysosomal enzyme expressed from <i>GLB1</i>, the catalytic domain of which is very different from that of <i>E. coli</i> β-gal, a bacterial enzyme encoded by <i>lacZ</i>. However, existing chemical probes for this marker still lack the ability to distinguish human SA-β-gal from β-gal of other species, such as bacterial β-gal, which can yield false positive signals. Here, we show a molecular design strategy to construct fluorescent probes with the above ability with the aid of structure-based steric hindrance adjustment catering to different enzyme pockets. The resulting probes normally work as traditional SA-β-gal probes, but they are unique in their powerful ability to distinguish human SA-β-gal from <i>E. coli</i> β-gal, thus achieving species-selective visualization of human SA-β-gal for the first time. NIR-emitting fluorescent probe <b>KSL11</b> as their representative further displays excellent species-selective recognition performance in biological systems, which has been herein verified by testing in senescent cells, in <i>lacZ</i>-transfected cells and in <i>E. coli</i>-β-gal-contaminated tissue sections of mice. Because of our probes, it was also discovered that SA-β-gal content in mice increased gradually with age and SA-β-gal accumulated most in the kidneys among the main organs of naturally aging mice, suggesting that the kidneys are the organs with the most severe aging during natural aging.