Design of protein-based “turn on” molecular probes for intracellular bond cleavage
The clinical success of antibody-drug conjugates and numerous other stimuli-responsive drug delivery systems motivates the need to develop molecular tools to quantitatively study their intracellular processing. To this end, researchers have developed fluorescent-based probes utilizing Förster resonance energy transfer (FRET). Quenched probes have the potential to eliminate fluorescence bleed through, which is common in FRET-based systems. However, the hydrophobicity of many broad-spectrum fluorescence quenchers can complicate the design of protein-based molecular probes. In this work, we investigate the potential for 2,4-dinitroaniline (2,4-DNA) to serve as a hydrophilic fluorescence quencher. A support-free synthesis of oligothioetheramide (oligoTEA) linkers was developed and applied to the design of quenched fluorescence probes. These quenched probes were based on intramolecular static quenching of boron dipyrromethene (BODIPY)-FL by 2,4-DNA. Probes containing a reduction-sensitive disulfide bond and a protease-sensitive valine–citrulline-PABC linker were synthesized using a model protein – human transferrin. Within HeLa cells, the apparent degradation rate of the disulfide bond was greater than the valine–citrulline-PABC linker. This work establishes a versatile method for synthesizing multifunctional crosslinkers and identifies 2,4-DNA as an effective fluorescence quencher for protein-based bioconjuates.
- This article is part of the themed collection: MSDE Emerging Investigators 2020