Utilising fluorescent reporters to probe the mode of action of norbornen-7-one CO releasing molecules
Hannah Margaret Johnston, Jui Thiang Brian Kueh, Rachael Hannah Hartley, Abigail R. Bland, Fergus M. Payne, Joanne C. Harrison, Ivan A. Sammut, David S. Larsen
Abstract
The synthesis of the fluorescent organic carbon monoxide releasing molecules oCOm-57, oCOm-58, and oCOm-66 are reported. These oCOms are water soluble and exhibit a "turn-on" fluorescent behaviour when CO is released under physiological conditions. oCOm-66 also contains an additional nitro-naphthalimide moiety that functions as a fluorescent reporter. Delivery of CO released from these oCOms to the mitochondria of AC-16 cardiomyocytes was confirmed using confocal microscopy in conjuction with MitoTracker Red. While the neutral, PEGylated oCOm-57 was found to remain in the extracellular environment releasing CO to diffuse into the cellular compartments, the positively charged oCOm-58 and -66 are targeted to the mitochondria where they release CO. Notably, the use of the fluorescent oCOms in live cellular imaging, allows the intracellular CO delivery and oCOm localisation to be characterised. This cellular confocal study also shows that, subtoxic concentrations of CO released from these molecules preserved mitochondrial energetics as indicated by the membrane potential dependent MitoTracker Red.