Toward Selective Synthesis of Protein Olympiadanes via Orthogonal Active Templates in One Step

Unlike small molecules, the topological complexity of macromolecules remains largely unexplored due to the huge synthetic challenge. Herein, we report the development of orthogonal active templates for con-cise and selective synthesis of protein [n]heteroca-tenanes toward protein olympiadanes. An active template (AT-Snoop) was first developed based on the isopeptide-bond-forming RrgA domain with comparable efficiency and excellent orthogonality to the previously reported active template (AT-Spy) based on the CnaB2 domain. Their combination fa-cilitated the selective synthesis of protein [n]cate-nanes from multiple components in one step and the resulting structures were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, liquid chromatography-mass spectrometry, and proteolytic digestion experi-ments. The results offered a promising solution to tackling the daunting challenge of precision synthesis of protein olympiadane with five distinct ring components. Not only did the success provide new tools for protein topology engineering but also spurred and fueled the future exploitation of topology-related functional benefits in protein science.