Cu(II)/THPTA-Mediated Thiazolidine Deprotection for Living Phages and Cell Surfaces Labeling

Incorporating unnatural bioorthogonal groups into peptides and proteins offers an excellent opportunity to endow them with new properties in a precise and controlled manner. Among these, the α-oxo aldehyde group is particularly suitable for the post-functionalization of peptides and proteins due to its versatility and stability in aqueous buffers. However, the facile and site-specific incorporation of α-oxo aldehyde into proteins, especially in living systems, remains a long-lasting challenge. Here, we describe a novel Cu(II)/THPTA-Mediated Thiazolidine Deprotection (CUT-METHOD) strategy for post-installation of a highly-active α-oxo aldehyde moiety, which is released from a thiazolidine ring borne by a genetically encoded unnatural amino acid ThzK. This reaction is performed under physiological conditions, thereby enabling the chemoselective and site-specific modification of proteins via oxime ligation without compromising their integrity and function. To validate its versatility, we successfully performed site-specific incorporation of α-oxo aldehyde into recombinant proteins and those displayed on M13 filamentous bacteriophage particles and bacterial cell surfaces. In addition, by leveraging Spycatcher/Spytag chemistry and oxime ligation, the bacterial cells bearing aldehyde generated via the CUT-METHOD could be simultaneously decorated with two distinct functional molecules, providing a novel one-pot dual labeling platform for the construction of living bacterial cell-based cancer targeting systems. Put together, we have demonstrated that the CUT-METHOD strategy is a significant addition to the current bioorthogonal chemistry toolbox with broad applications anticipated in the near future.