Nicotinamide-appended fluorophores as fluorescent redox sensors

Abstract

Fluorescent sensors have proved invaluable in elucidating the regulation and dysregulation of redox processes in biology, but understanding of the breadth of biological redox reactions requires development of new sensors based on a range of sensing groups with varied reduction potentials. The aim of this work was to investigate the use of nicotinamide as a redox switch when conjugated to two classes of amino-fluorophores. We prepared four fluorophore conjugates based on 7-aminocoumarins and 4-amino-1,8-naphthalimides via the nicotinamide Zincke salt. These conjugates all showed clear fluorescence changes in response to chemical reduction, but this reduction was irreversible both chemically and electrochemically. The reduction behaviour of the 1,8-naphthalimides was investigated further by spectroelectrochemistry, revealing that conjugate NNpR1 showed the clearest spectral changes upon both chemical and electrochemical reduction. Cells dosed with NNpR1 and maintained under hypoxic conditions exhibited a significantly higher green:blue fluorescence ratio than cells cultivated under normoxia, confirming the potential of this molecule as a sensor for reductive biological environments.