Ligand-Free Palladium-Mediated Site-Specific Protein Labeling Inside Gram-Negative Bacterial Pathogens

Abstract

Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

Selective chemical protein modification

“…It was also shown that PEG chains offer new reactivity and a significant enhancement in rate as self-liganding (internally chelating) boronic acids when used with Pd(OAc) 2 to give a high-yielding site-specific PEGylation of proteins 128 . This self-liganded effect has more recently been exploited by Li et al 130 , who found that PEG-conjugated fluorophores with Pd(NO 3 ) 2 could efficiently catalyse the Sonogashira crosscoupling, even intracellularly, in E. coli and Shigella.…”

Palladium-mediated chemistry in living cells

“…Attracted by the compatibility and applications of Pd species and bacteria [11 ,18], Chen reported ligand-free palladium-mediated site-specific protein labelling inside Gram-negative bacteria [28]. Here Pd(NO 3 ) 2 was used as a simple, efficient and biocompatible reagent for fluorescent labelling of target proteins via a copper-free Sonogashira coupling reaction.…”

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

“…Similarly, this Review will not cover the click chemistries catalyzed by palladium for bioconjugation of purified proteins or in cells. 529,530 4.12.3.2.2. Staudinger−Bertozzi Ligation.…”