Synthesis of Dimeric ADP-Ribose and Its Structure with Human Poly(ADP-ribose) Glycohydrolase

Abstract

Poly(ADP-ribosyl)ation is a common post-translational modification that mediates a wide variety of cellular processes including DNA damage repair, chromatin regulation, transcription, and apoptosis. The difficulty associated with accessing poly(ADP-ribose) (PAR) in a homogeneous form has been an impediment to understanding the interactions of PAR with poly(ADP-ribose) glycohydrolase (PARG) and other binding proteins. Here we describe the chemical synthesis of the ADP-ribose dimer, and we use this compound to obtain the first human PARG substrate-enzyme co-crystal structure. Chemical synthesis of PAR is an attractive alternative to traditional enzymatic synthesis and fractionation, allowing access to products such as dimeric ADP-ribose, which has been detected but never isolated from natural sources. Additionally, we describe the synthesis of an alkynylated dimer and demonstrate that this compound can be used to synthesize PAR probes including biotin and fluorophore-labeled compounds. The fluorescently labeled ADP-ribose dimer was then utilized in a general fluorescence polarization-based PAR-protein binding assay. Finally, we use intermediates of our synthesis to access various PAR fragments and evaluation of these compounds as substrates for PARG reveals the minimal features for substrate recognition and enzymatic cleavage. Homogeneous PAR oligomers and unnatural variants produced from chemical synthesis will allow for further detailed structural and biochemical studies on the interaction of PAR with its many protein binding partners.

Solid‐Phase Synthesis of Oligo‐ADP‐Ribose

“…Studies on the synthesis of native fragments of ADP-ribosylated proteins have been scarce up to now (Moyle and Muir, 2010;van der Heden van Noort et al, 2010b). The synthesis of fragments of poly-ADPr has been reported only very recently by just two laboratories (Lambrecht et al, 2015;Kistemaker et al, 2015a). …”

Poly(ADP-ribose): From chemical synthesis to drug design

“…[46][47][48] However, as combination of these approaches was not fruitful, sophisticated protection strategy and new methods for the formation of pyrophosphoric bond were developed, resulting in new strategies for the chemical synthesis of PAR oligomers. 41,49 The ADP-ribose dimer represents the simplest form of PAR and can be considered as a model compound. Using H-phosphonate coupling method for the reaction of protected ribose phosphates and protected adenosine monophosphate with protected derivative of 2 0 -O-a-D-ribofuranosyladenosine, containing phosphate group at 5 0 -terminus of adenosine and H-phosphonate group at 5 0 -terminus of the additional ribose moiety, ADP-ribose dimer and propargyl ADP-ribose dimer have been synthesized.…”

“…Using H-phosphonate coupling method for the reaction of protected ribose phosphates and protected adenosine monophosphate with protected derivative of 2 0 -O-a-D-ribofuranosyladenosine, containing phosphate group at 5 0 -terminus of adenosine and H-phosphonate group at 5 0 -terminus of the additional ribose moiety, ADP-ribose dimer and propargyl ADP-ribose dimer have been synthesized. 41 The later compound can be further functionalized by biotin or fluorescent tags via «click chemistry» to evaluate PAR binding with specific proteins (Scheme 4). 41 Another synthetic methodology has been proposed that uses divergently protected amidophosphite 16 for solid-phase synthesis of PAR oligomers (Scheme 5).…”

“…41 The later compound can be further functionalized by biotin or fluorescent tags via «click chemistry» to evaluate PAR binding with specific proteins (Scheme 4). 41 Another synthetic methodology has been proposed that uses divergently protected amidophosphite 16 for solid-phase synthesis of PAR oligomers (Scheme 5). 49 The building block 16 was obtained at the preparative scale (4 mmol) by multistep procedure, involving regioselective glycosylation step, and then was used in solidphase synthesis of PAR oligomers.…”

“…61 Additionally, chemical synthesis could allow facile modification of oligo(ADP-ribose), providing access to unnatural compounds such as the propargyl derivatives, which can be used in combination with click chemistry to study PAR interactions with proteins. 41 The chemical synthesis of PAR will facilitate the study of PAR influence on various cellular processes. For these purposes, the designed and well-documented methods of oligonucleotide delivery into the cell will be used.…”