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

在蛋白质修饰领域工作的生物学家需要新的工具来询问与二磷酸腺苷(ADP)核糖单元从烟酰胺腺嘌呤二核苷酸(NAD)转移到蛋白质上的细胞事件和机制。通过这种转移，NAD在DNA修复、抗逆性和细胞死亡的调节中发挥关键作用，将新陈代谢与细胞生存联系起来。在哺乳动物细胞中，单-ADP核糖化反应是由单-ADP-核糖基转移酶(MART)催化的，MART通过转移蛋白质侧链上的一个ADP-核糖基单位来调节后者的活性。由于它们在细胞内稳态、细胞分裂、细胞“老化”和癌症中的重要性，十多年来一直受到严格的审查，但MART酶及其生物学靶点在很大程度上仍然难以捉摸。这主要是由于缺乏有效和具体的技术来研究这一生物学，尤其是获得特定抗体的机会有限。在印迹实验和质谱分析中识别ADP核糖化蛋白的一个关键因素是获得特定的抗体，这些抗体可以在功能分析中检测和报告这种修饰蛋白的存在。目前在制备这种抗体方面的挑战来自这样一个事实，即只要免疫反应积极地对抗异物并产生抗体，单-ADP核糖化肽就必须对降解酶保持稳定。为了获得看起来像单一ADP核糖化蛋白但更稳定的修饰蛋白，必须合成作为MART良好底物的NAD类似物。此外，由此引入的修饰必须足够小，以诱导抗体的产生，抗体也将识别“真正的”ADP核糖化蛋白靶标。如果这些抗体要广泛应用于MAR生物学，后一个方面是至关重要的。由于一系列微调的生物学工具的发展，如高效抗体和原位检测分析，由ADP-核糖聚合酶(PARP)催化的蛋白质的聚合ADP-核糖化生物学已经得到了更容易的研究。在人类中，PARP家族的五个成员是DNA结合酶，由DNA断裂激活，在碱基切除修复过程中起关键作用，第六个成员(PARP13)是新发现的mRNA结合蛋白。尽管广泛的研究导致了重大的药物发现计划，但作为一个化学实体，聚(ADP-核糖)(PAR)保持了大量未被探索的复杂性，这对了解其细胞特性至关重要。更具体地说，最近发现了四个不同的聚(ADP-核糖)结合基序，这彻底改变了如何解释这种“附属物”是由其伙伴蛋白质解码的。目前的限制在于获得功能和结构上定义的聚-ADP-核糖短链，这将有助于探索和合理化结合伙伴对这些结构不同的表位引发的识别和招募事件。因此，如果能够产生针对MAR的特异性抗体，并使稳定的结构特异性PAR片段得到更广泛的获得，那么在ADP-核糖化领域将取得实质性的进展。要获得这种类型的工具，必须设计新的化学物质以模块化方式获得所需的功能。我们是唯一一家专门从事磷和核苷化学的合成实验室，在制备NAD和ADP-核糖型分子时，使用非传统溶剂和机械力化学可以实现高合成效率。在这里，我们将合成NAD衍生物，这些衍生物将用于产生针对蛋白质MAR-基化的特异性抗体和具有报告特性的PAR片段，以便能够对聚ADP-Ribolome进行结构和功能分析。

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.