Recent advances in chemical protein synthesis: method developments and biological applications

Efficient Chemical Synthesis of Multi-Monoubiquitylated and Diubiquitylated Histones by the α-Halogen Ketone-Mediated Strategy

“…The conjugation of three ubiquitin monomers to H3 was performed by mixing proteins 3 and 14 in boric acid buffer at a molar ratio of 6:1. To our pleasure, although a small amount of byproducts was observed, H3 mutant 14 was smoothly converted to the desired H3Ub3 (15) with a conversion rate of ∼82%, which was comparable to the synthetic efficiency of single-monoubiquitylated histones (Figure 2b). Finally, H3Ub3 was obtained with high homogeneity in a 50% yield (Figure 2c).…”

“…Histone ubiquitylation, in which ubiquitin (Ub) is installed onto the lysine residue of histones via an isopeptide bond, is a critical post-translational modification (PTM) for various chromatin-related processes, such as DNA replication, transcription, and repair. − These very different outcomes may result from the diversity of histone ubiquitylation in structures, such as monoubiquitylation bearing a Ub monomer on one lysine, multi-monoubiquitylation in which Ub monomers are conjugated to histones on multiple residues, and polyubiquitylation in which Ub chains are installed onto histones. − The decoding of ubiquitylation-dependent regulation requires the use of precisely ubiquitylated histones to allocate their functions and reveal the underlying molecular mechanisms. − Therefore, remarkable endeavors have been focused on the chemical preparation of those histones. − Total chemical synthesis − and semisynthesis − enable the generation of ubiquitylated histones with a native linkage, while site-specific conjugation chemistries − allow us to produce ubiquitylated histone analogues from recombinant materials. Among these methods, the α-halogen ketone-mediated strategy (Scheme a) has gained attractive attention and been broadly applied by the biology community due to its practicality in preparing ubiquitylated histones and the resistance of the resulting linkages to deubiquitinases.…”

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

“…Chemical protein synthesis (CPS), enabled by chemo-selective peptide ligation reactions like native chemical ligation (NCL), has contributed enormously to the generation of proteins with specific structural modifications and facilitated the selective introduction of unnatural amino acids, biophysical probes, and post-translational modifications. 1 Since its inception by Kent and co-workers, 2 and with great contributions from many research groups, NCL has become the most powerful and dominant method for CPS. 3 With the increasing demand for large and complex proteins, the reactivity of peptidyl thioesters ( e.g.…”

Employing unnatural promiscuity of sortase to construct peptide macrocycle libraries for ligand discovery

“…Additionally, peptide macrocycles have a larger surface than small molecules and are perceived to be more suitable for targeting protein–protein interactions. 4–6 Currently, techniques for generating large-scale peptide macrocycle libraries include creating synthetic combinatorial chemical libraries and genetically encoded peptide libraries. 7,8 Genetically encoded peptide libraries, such as phage display and mRNA display, can easily provide a diversity of 10 9 –10 12 and they are an invaluable avenue for discovering high-affinity peptide ligands.…”