Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells

Abstract

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide-alkyne cycloaddition reaction ("click chemistry"), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine-palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.

Figure 1

Selective PEGylation of HPG-Ub with an mPEG-linked phenyl iodide 1n via copper-free Sonogashira cross-coupling. (a) Reaction scheme. (b) Coomassie blue staining of SDS-PAGE gel showing selective formation of the PEGylated Ub (Ub-mPEG): lane 1, control reaction with wild-type ubiquitin; lane 2: control reaction with HPG-Ub in the presence of 50 equiv of 1n but absence of palladium complex; lane 3: reaction with HPG-Ub in the presence of 50 equiv of 1n and palladium catalyst.

Figure 2

Fluorescent labeling of HPG–Ub by fluorescein iodide 1a in E. coli cells via copper-free Sonogashira cross-coupling. (a) Fluorescence image of the cell pellets upon excitation at 365 nm. Pellet 1, cells collected after treatment with 100 µM of fluorescein iodide 1a but not palladium complex; Pellet 2, cells collected after treatment with 100 µM 1a, 1 mM palladium complex, 5 mM sodium ascorbate in Na₂HPO₄ buffer at 37 °C for 4 h; Pellet 3, cells collected after treatment of the pre-activated mixture of palladium complex (1 mM) and 1a (100 µM). See Supporting Information for the treatment details. (b) In-gel fluorescence (left panel) and Coomassie blue staining (right panel) of SDS-PAGE analysis of the proteins captured by Ni-NTA-agarose beads from the lysates of the three cell pellets.