The cellular fates of Mn(II) pentaazamacrocyclic SOD mimetics – fluorescently-labelled MnSOD mimetics, XAS and XFM studies

Manganese(II) pentaazamacrocyclic complexes (MnPAMs) can act as small molecule mimics of manganese superoxide dismutase (MnSOD) with potential therapeutic application in conditions linked to oxidative stress. Previously, the in vitro mechanism of action has been determined, their activity has been demonstrated in cells and some representatives of this class of MnSOD mimetics have entered clinical trials. However MnPAM uptake, distribution and metabolism in cells is largely unknown. Therefore, we have used X-ray fluorescence microscopy (XFM) and X-ray absorbance spectroscopy (XAS) to study the cellular fate of a number of MnPAMs. We have also synthesized and characterized fluorescently-labelled (pyrene and rhodamine) Mn(II)pyane ((manganese(II)trans-2,13-dimethyl-3,6,9,12,18pentaazabicyclo[12.3.1]-octadeca-1(18),14,16-triene)) derivatives and investigated their utility for cellular imaging of MnPAMs. Their SOD activity was determined via a direct stopped-flow technique. XFM experiments show that treatment with amine-based Mn(II)pyane-type pentaazamacrocycles leads to a 10to 100-fold increase in overall cellular manganese levels compared to physiological levels of Mn in control cells. In treated cells in general, Mn was distributed throughout the cell body, with a couple of notable exceptions. The lipophilicity of the MnPAMs – examined by partitioning in octanol-buffer system – was a good predictor of relative cellular Mn levels. Analysis of XAS data of treated cells revealed that some fraction of amine-based MnPAMs taken up by the cells remained intact, with the rest transformed into SOD active Mn(II) phosphate. Higher phosphate binding constants – determined from the effect of phosphate concentration on in vitro SOD activity – were associated with more extensive metabolism of the amine-based MnPAMs to Mn(II) phosphate. In contrast, the imine-based Mn(II)pydiene complex that is prone to hydrolysis was entirely decomposed after uptake and free Mn(II) was oxidized to a Mn(III)-oxide type species, in cytosolic compartments – possibly mitochondria. Complex stability constants (determined for some of the MnPAMs) are less indicative of the cellular fate of the complexes than corresponding phosphate binding constants.