Chemical Synthesis Of Post-Translational Modified Histone Probes For Functional Studies

Bio-based chemicals can increase the added value of biomass derived compounds.At present,the transformation and application of biomass derived compounds mainly focus on organic compounds such as alcohols,aldehydes,and carboxylic acids,while the utilization of important biomass derived compounds such as peptides and proteins is very scarce.An important reason is that the current research on the structure and function of complex biological macromolecules such as proteins is not thorough enough.As a key component of human hereditary material,histones are also an important class of biomass derived compound.In-depth and systematic study of histones to understand the structure,protein-protein interactions and functions of histones.It is of great significance to realize the bio based chemical transformation of histones.Histone post-translational modifications(HPTMs)directly influence the properties of the histones and mediate critical nuclear functions including DNA replication,gene transcription,chromatin remodelling and hetero-chromatin establishment and maintenance.There are a large number of complex protein-protein interactions in these processes,which constitute a series of precise and diverse regulatory mechanisms.Elucidating the above regulatory mechanisms at the molecular level not only elucidate the relevant underlying biological events,but also further reveals the mechanisms by which these interacting perturbations regulate disease occurrence.The key to achieving this is to explore and discover the protein-protein interaction map of post-translational modified histones.In recent years,posttranslational modification histone chemical probes have played an important role in the study of protein-protein interactions mediated by post-translational modifications.However,these histone probes carrying chemical groups still have cumbersome operations and low yields in the synthesis method.More importantly,in situ screening of post-translationally modified histone interactome in living cells still requires prior genetic manipulations such as gene editing in research samples.In order to solve the above deficiencies,this study carried out the following three aspects of research work around the synthesis of novel strategies and in vivo applications of post-translational modified histone chemical probes.Firstly,we developed new methods of chemical synthesis for novel histone posttranslational modification chemical probes,such as the recently discovered iodination and lactylation.Using the sequential ligation of four fragments based on peptide hydrazides,we obtained the C.elegans HIS-58Y81-iodination by chemical total synthesis for the first time,and constructed the first iodinated HIS-58 nucleosome pulldown probe.In addition,we synthesized the first fully protected lactylated lysine for solid-phase peptide synthesis(SPPS)with a Fmoc-protection strategy and synthesized the histone H3K181a pull-down probe.Using this probe,we successfully analyzed potential lactylated binding proteins in nuclear lysates.Subsequently,we report the efficiently synthesis of a new diazirine-based histone chemical probes through the construction of terminal diazirine via phenyliodonium diacetate(PIDA)mediated transformation.The construction of the key building block containing diazirine photo-crosslinking groups can be completed by one-step reaction with common used amino acids.By using this key building block,we can rapidly synthesize multigram quantities of H3K4me3 terminal-diazirine-based peptide probe.Moreover,we demonstrated that the photoaffinity probe obtained by this method can effectively capture H3K4me3 reader in complicated cell lysate.Finally,in order to achieve direct exploration of the intracellular histone interactome of non-genetic manipulation samples,we developed a class of histone photoaffinity probes carrying photo-crosslinking groups,nuclear localization peptide sequences,and cell penetrating peptide sequences.The new probe can precisely target the nucleus in living cells,enabling in situ screening of histone interactome.By using nucleus-targeted H3K9bhb,H3K9me3,H3K9ac,H3K181a photoaffinity probes,this study achieved the first post-translational modification of histone-protein interactome analysis in non-gene-edited cells and hard-to-transfect living cells.We have identified a range of potential binding proteins that provide new clues to elucidate the fundamental epigenetic regulation mechanisms by the above modifications.We developed cellpenetrating and organelle-targeted histone chemical probes,which may become a powerful toolkit to uncover diverse PTM-mediated protein-protein interactions between histone and its epigenetic regulatory effectors in living cells.