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

07 June 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


Chemical modifications
Unnatural amino acid
Phage and bacterial surface display

Supplementary materials

Supporting Information-Cu(II)/THPTA-Mediated Thiazolidine Deprotection for Living Phages and Cell Surfaces Labeling
Detailed methods and experimental procedures could be found in the Supporting Information file, including recombinant protein expression, phage preparation, dual-labeling of bacterial cell surface, HPLC, ESI-MS, SDS-PAGE, Western blot, et al.


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