Cell‐Penetrating Peptides as Delivery Vehicles for a Protein‐Targeted Terbium Complex

Chemical protein labeling strategies leverage orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences to attach fluorophores or other useful functionalities to proteins in live cells.[1] Ideally, such approaches could be used to label intracellular proteins with particularly bright and photostable fluorophores (e.g., AlexaFluors, cyanines),[2] photosensitive dyes that facilitate superresolution imaging,[3] or luminescent lanthanide complexes that enable highly sensitive, time-gated microscopy.[4] In practice, however, these types of labels are often impermeable to cell membranes, and chemical labeling approaches have either been limited to studies of cell-surface proteins or have required more onerous methods of intracellular delivery like microinjection or electroporation.[3–4] For example, our laboratory has recently developed a series of trimethoprim (TMP)-lanthanide complex conjugates that selectively and tightly (~nM KD) bind to Escherichia coli dihydrofolate reductase (eDHFR).[5] With these compounds, the useful properties of lanthanide luminescence (narrow, multi-wavelength emission, long luminescent lifetime) can be easily imparted to recombinant fusion proteins. By selectively labeling eDHFR fusion proteins in live cells with one conjugate, TMP-Lumi4(Tb), we showed that interactions between eDHFR fusions and green fluorescent protein (GFP) fusions could be imaged at high signal-to-background ratio using time-gated, luminescence resonance energy transfer (LRET) microscopy.[5b] However, intracellular delivery of cell-impermeable TMP-Lumi4-Tb required either reversible plasma membrane permeabilization or osmotic lysis of pinocytic vesicles.