Cholesteryl-Tagged Fluorescent NAD+: A Membrane-Permeable Esterase-Sensitive Probe for ADP-ribosylation Studies

ADP-ribosylation is a key protein modification involved in cell signaling pathways, where ADP-ribose units are incorporated into the macromolecules by PARP enzymes, using NAD+ as a substrate. Over years many NAD+ tools have been developed for studies on the complex role of the ADP-ribosylation, although its inability to penetrate the cell membrane poses limitations. In this work, we present a straightforward synthesis of a cell-permeable, fluorescent NAD+ analog with an esterase-sensitive linker. The NAD+ double-conjugate contained a Texas Red fluorophore and a cholesterol ligand, incorporated via a [4-(acetyloxy)phenyl]methyl N-carbamate bond. This bond improved the probe’s stability in buffer compared to our previous design and allowed for efficient cleavage by human carboxylesterase. The probe was successfully accepted as an ADP-ribosylation substrate by PARP1 and PARP10, both before and after cleavage. We demonstrated the probe’s ability to penetrate the cell membrane and used it to visualize ADP-ribosylated proteins and monitor fluorescence changes in HeLa cells under oxidative stress.