A ribose-functionalized NAD+ with unexpected high activity and selectivity for protein poly-ADP-ribosylation

Nicotinamide adenine dinucleotide (NAD + )-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD + analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3′-OH of nicotinamide riboside enables enzymatic synthesis of an NAD + analogue with high efficiency and yields. Notably, the generated 3′-azido NAD + exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3′-azido NAD + in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3′-azido NAD + provides an important tool for studying cellular PARylation. The study of NAD + dependent ADP-ribosylation can be challenging. Here the authors report on the development of NAD + analogues, using chemo-enzymatic methods, which can be used as probes to label the substrate proteins of poly-ADP-ribose polymerase.