Abstract
This research project aimed to develop fluorescent analogues of the oCOm molecules reported by Larsen and co-workers.1 It was proposed that the attachment of phenanthrene-based fluorescent reporter groups to these CO prodrugs would allow their distribution to be monitored in biological experiments and therefore, provide insight into their cellular location and mode of action. This was achieved by the synthesis of various phenanthrene-based CORMs, oCOm-57, -58 and -62.
oCOm-62 was synthesised via a Diels-Alder cycloaddition of bromomaleimide derivative 51 and diene 37. This compound was unable to be purified due to low solubility in water and organic solvents, and therefore oCOm-62 was not investigated further. oCOm-57 and -58 were synthesised in good yields from a CuAAC reaction of alkyne 55, copper(I) catalyst 64 and appropriately functionalised mPEG-750 or TEG chains.
oCOm-57, -58 and -62 were fully characterised and their water solubility profiles were investigated by HPLC analysis. oCOm-58 demonstrated an improved water solubility (≥ 440 mM), in comparison to that of oCOm-57 (14.5 mM), and hence the CO release profile of this compound was investigated by HPLC analysis. Unfortunately, the half-life of oCOm-58 was found to be much longer (225 minutes) than those previously reported for similar oCOm molecules.1, 2 The UV-Vis and fluorescence profiles of oCOm-58 and its spent by-product, BP-oCOm-58, were investigated by spectrophotometry. BP-oCOm-58 demonstrated strong fluorescence at 470 nm when excited with 400 nm light, which was almost 25 times as intense as that of oCOm-58.
oCOm-57 and -58 were sent to our collaborators in the Department of Pharmacology at the University of Otago for biological screening. Confocal microscopy was carried out on AC-16 cardiomyocyte cells treated with either oCOm-57 or -58, MitoTracker and the CO selective probe, COP-1. The spent by-product of oCOm-57, BP-oCOm-57, was observed to be predominantly associated with the cell membrane, while BP-oCOm-58 was detected inside the cellular matrix. This made it apparent that a terminal protonated amine group, such as in oCOm-58, provided the driving force for transportation across cell membranes, hence providing valuable information regarding the mode of action of these oCOm analogues.