Unconventional chemistry to generate enabling reporting tools to be used in mono- and poly-ADP-ribosylation biology

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Biologists working in the field of protein modifications are in need of new tools to interrogate the cellular events and mechanisms which are linked to the transfer of adenosine diphosphate (ADP) ribose units from nicotinamide adenine dinucleotide (NAD) on proteins. Through this transfer, NAD plays a pivotal role in the regulation of DNA repair, stress resistance and cell death, linking metabolism to cell survival. In mammalian cells, mono-ADP ribosylation reactions are catalysed by mono-ADP-ribosyltransferases (MART) which transfer one ADP-ribosyl unit on proteins' side chains as means of regulating the activity of the latter. Whilst having been under intense scrutiny for over a decade because of their importance in cellular homeostasis, cell division, cellular "ageing" and cancer, both the MART enzymes and their biological targets have largely remained elusive. This is mainly due to the lack of effective and specific techniques to study that biology, more particularly the limited access to specific antibodies. A key factor to identify ADP-ribosylated proteins in blot experiments and mass spectroscopy analyses is to have access to specific antibodies which can detect and report the presence of such modified proteins in functional assays. The current challenges in preparing such antibodies come from the fact that mono-ADP ribosylated peptides must remain stable to degrading enzymes for as long as the immune response is actively fighting the foreign entity and creating antibodies. To access modified proteins which look like mono-ADP ribosylated proteins but which are more stable, NAD analogues which are good substrates for MART must be synthesised. Additionally, the modifications thus introduced must be sufficiently minimal to induce the generation of antibodies which will also recognise the "true" ADP-ribosylated protein targets. This latter aspect is critical if these antibodies are to become widely used in MAR biology. The biology of polymeric ADP-ribosylation of proteins, catalysed by ADP-ribosyl polymerases (PARP) has been more readily studied due to the development of a broad range of finely-tuned biological tools, such as highly efficient antibodies and in situ detection assays. In human, five members of the PARP family are DNA binding enzymes activated by breaks to the DNA and are critical to the base excision repair process, with the sixth member (PARP13) being a newly identified mRNA binding protein. Despite extensive research leading to major drug discovery programs, poly(ADP-ribose) (PAR), as a chemical entity, has retained numerous levels of unexplored complexity which are critical to understanding its cellular properties. More specifically, the recent identification of four different poly(ADP-ribose) binding motifs have revolutionised how the decoding of this "appendage" by its partner-proteins can be explained. The limitations now reside in accessing functional and structurally defined short strands of poly-ADP-ribose that would help probe and rationalise the recognition and recruitment events elicited by the binding partners for these structurally different epitopes. Therefore, substantial progress could be achieved in the field of ADP-ribosylation if specific antibodies to MAR-ylation could be generated and stable structure-specific PAR fragments became more widely available. To access this type of tools, novel chemistry must be devised to access the required functionalities in a modular manner. We are the only synthetic laboratory that has specialised in phosphorus and nucleoside chemistry where the use of unconventional solvents and mechanochemistry allows for high synthetic efficacy in the preparation of NAD and ADP-ribose type molecules. Here, we will synthesise NAD derivatives which will be used to raise antibodies specific to protein MAR-ylation and PAR fragments with reporting properties to enable structural and functional analyses of the poly-ADP-ribolome.