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

Abstract

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.