Natural antioxidants, like phenolic acids, possess a unique chemical space that can protect cellular components from oxidative stress. However, their polar carboxylic acid chemotype reduces full intracellular antioxidant potential due to limited diffusion through biological membranes. Here, we have designed and developed a new generation of hydrophobic turn-on fluorescent antioxidant precursors that upon penetration of the cell membrane, reveal a more polar and more potent antioxidant core and simultaneously become fluorescent allowing their intracellular tracking. Their activation is stimulated by polarity alteration by sensing intracellular signals and specifically biothiols. In our design, the carboxylic group of phenolic acids that originally restricts cell entrance is derivatized and conjugated through Copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) to a coumarin derivative that its fluorescence properties are quenched with a biothiol activatable element. This more hydrophobic precursor readily penetrates cell membrane and once inside the cell the antioxidant core is revealed upon sensing glutathione, its fluorescence is restored in a turn-on manner and the generation of a more polar character traps the molecule inside the cell. This turn-on fluorescent antioxidant precursor that can be applied to phenolic acids, was developed for rosmarinic acid and the conjugate was named as RCG. The selectivity and responsiveness of RCG towards the most abundant biothiols was monitored through a variety of biophysical techniques including UV-Vis, fluorescence and NMR spectroscopy. The electrochemical behavior and free radical scavenging capacity of the precursor RCG and the active compound (RC) was evaluated and compared with the parent compound (rosmarinic acid) through cyclic voltammetry and EPR spectroscopy, respectively. The stability of the newly synthesized bioactive conjugate RC was found significantly higher than the parent rosmarinic acid when exposed to oxygen. Cell uptake experiments were conducted and revealed the internalization of RCG. The degree of intracellular DNA protection offered by RCG and its active drug (RC) on exposure to H2O2 was also evaluated in Jurkat cells.

Charles University 1st Faculty of Medicine Kateřinská 1660 32 12108 Prague 2 Czech Republic

Department of Chemistry Section of Organic Chemistry and Biochemistry University of Ioannina Ioannina 45110 Greece

Department of Oncology St Luke's Cancer Institute Royal Surrey County Hospital Guildford UK

J Heyrovsky Institute of Physical Chemistry of the Czech Academy of Sciences Dolejskova 3 18223 Prague Czech Republic

John Fulcher Neuro Oncology Laboratory Department of Brain Sciences Hammersmith Hospital Imperial College London

Laboratory of Biological Chemistry University of Ioannina School of Health Sciences Faculty of Medicine 451 10 Ioannina Greece

Laboratory of Biomimetic Catalysis and Hybrid Materials Department of Chemistry University of Ioannina 45110 Ioannina Greece

Laboratory of Physical Chemistry of Materials and Environment Department of Physics University of Ioannina 45110 Ioannina Greece

University Research Center of Ioannina Institute of Materials Science and Computing Ioannina Greece

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