Electrochemistry of Redox-active Mn Porphyrin-based SOD Mimic MnTnBuOE-2-PyP 5 +-Study of Redox Species Involved in ROS / RNS Scavenging

Manganese ortho tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP 5+ , is a thirdgeneration redox-active compound currently undergoing preclinical exploration. This work is intended to complement the already extensive research of its chemical and biological properties by a simple electrochemical study. The thermodynamic parameters related to the Mn(IV) porphyrin species of MnTnBuOE-2-PyP 5+ determined in this work support its observed reactivity as an efficient scavenger of peroxynitrite. The corresponding driving forces for the possible single-electron or two-electron reductions of ONOO have been estimated as well.

Comparison of MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ with regards to their redox properties (E 1/2 for Mn III P/Mn II P redox couple), reactivities towards superoxide, log k cat (O 2 • -), and peroxynitrite, log k red (ONOO -), as well as lipophilicities (distribution between n-octanol and water, log P OW ) is given in Table 1.Comprehensive studies on bioavailability of these compounds have been performed recently as well [17][18][19], in order to gain further insight into the biodistribution of MnPs, but also provide guidelines for proper dosing regimens.After assessing preliminary efficacy and toxicity data, the porphyrins were administered to mice intraperitoneally (ip) or intravenously (iv) at following doses: 10 mg/kg of MnTE-2-PyP 5+ (ip) and 2 mg/kg of MnTnBuOE-2-PyP 5+ (iv).Drug levels in plasma and different organs were followed during at least 24 hours after a single dose, thus allowing the calculation of relevant pharmacokinetic (PK) parameters via non-compartmental analysis of PK curves.
These studies showed the highest MnP bioavailability in liver (maximum concentration, C max , values of 8.1 and 13.1 µmol per L of tissue homogenate for MnTE-2-PyP 5+ and MnTnBuOE-2-PyP 5+ , respectively) and kidney, followed by spleen, lung and heart, whereas lower levels were found in plasma and lowest in the brain.Remarkably, MnTnBuOE-2-PyP 5+ has 8-fold higher brain bioavailability (expressed as the area under PK curve, AUC, divided by dose) than the less lipophilic MnTE-2-PyP 5+ (Figure 1), which may be highly significant for its potential application as a brain tumor radio-and chemo-therapeutic [20][21].
Additionally, it has been shown that k cat (O 2 • -) parallels the ability of MnPs to reduce ONOO -[4-5,14], due to the same thermodynamic and kinetic factors facilitating the reaction of electron-deficient cationic MnPs with electron-donating anionic species, such as O 2 •and ONOO - [3][4][5]22].Specifically, the removal of ONOO - can occur either through its binding to the Mn(III) site followed by its one-electron reduction to NO 2 • radical (coupled to the oxidation of Mn III P to Mn IV P), or the two-electron reduction of ONOO -by the Mn II P species (coupled to the oxidation of Mn II P to Mn IV P).The latter reaction produces less toxic nitrite, NO 2 -, and might in fact be dominant in vivo due to (i) the abundance of cellular reductants keeping MnPs in Mn(II) state [1,13,[23][24][25] and (ii) a higher driving force making it thermodynamically favorable [26].).The values for MnTE-2-PyP 5+ determined via iv route are taken as 100 % availability (plasma bioavailability via ip route was found to be approximately 84% of bioavailability via iv route for MnTE-2-PyP 5+ ) [18].MnPs' charges have been omitted from the plot for simplicity.
MnTnBuOE-2-PyP 5+ represents the latest generation of redox-active compounds and is currently undergoing preclinical exploration.This work is intended to complement the already extensive research of its chemical and biological properties by a simple electrochemical study.

Results and Discussion
Cyclic voltammograms of the aqueous solutions of Mn III Ps can show two distinct electron transfers: only one current peak pair attributed to the Mn III P/Mn II P redox couple is observed in a neutral or mildly basic medium (pH = 7.8, Figure 2), whereas in the more basic medium an additional current peak pair attributed to the Mn IV P/Mn III P redox couple appears at more positive potentials (pH = 11, Figure 2).The cathodic and anodic current peak potentials, E pc and E pa , of both redox couples shift toward more negative values upon the increased pH of the solution.The cathodic-anodic peak separation, ΔE p = |E pc -E pa | for both redox processes is larger than 59 mV even at the scan rate of 0.02 V s -1 , indicating quasi-reversible electron transfer processes.previously for methyl, ethyl and butyl analogues [15,26,29].Redox couples shown relate to the Mn III P/Mn II P (right cycle) and the Mn IV P/Mn III P couples (left cycle).
These results are in agreement with the previously established scheme of behavior of MnPs in aqueous solutions [26,30].At pH = 7.8 only a single-electron reduction of Mn III P can be observed, shown by eq. ( 1), whereas at pH = 11 the single-electron reduction of Mn IV P is accompanied by a simultaneous dissociation of two protons, according to eq. ( 2).The latter electron transfer cannot be observed in a neutral medium due to a formal potential more positive than that of water (E 0' (O 2 (g),H + (aq.)/H 2 O(l)) = 816 mV vs. SHE at pH = 7) [30][31].Mn III P/Mn II P Mn III P/Mn II P Mn IV P/Mn III P Mn IV P/Mn As shown previously [11], the E 1/2 of Mn III P/Mn II P redox couple of MnTnBuOE-2-PyP 5+ is close to the value of the Mn SOD enzyme itself, accounting for its high reactivity towards O 2 •-(even higher than that of MnTE-2-PyP 5+ , Table 1).Thermodynamic parameters related to the Mn IV P species of MnTnBuOE-2-PyP 5+ determined in this work (Table 2) support its observed reactivity as an efficient scavenger of peroxynitrite.
Estimating the shift of the E 1/2 (Mn IV P/Mn III P) value for +118 mV per unit decrease of pH from pH = 11, yields the values of E 1/2 (Mn IV P/Mn III P) = + 1049 mV vs. NHE at the physiological pH, according to eq. ( 2).The E 1/2 (Mn IV P/Mn II P) at the physiological pH can then be calculated as E 1/2 (Mn IV P/Mn II P) = [E 1/2 (Mn III P/Mn II P) + E 1/2 (Mn IV P/Mn III P)]/2 = + 663 mV vs. NHE.Finally, the driving forces for the single-electron and two-electron reduction of ONOO -, EMF (1e -) = 551 mV and EMF (2e -) = 637 mV, can then be calculated as EMF = E c -E a , where the values of E c are the formal potentials E 0' (ONOO -/NO 2 • ) = 1.6 V and E 0' (ONOO -/NO 2 -) = 1.3 V [32], respectively, whereas the values of E a are the formal potentials E 1/2 (Mn IV P/Mn III P) and E 1/2 (Mn IV P/Mn II P), respectively.

Conclusions
Considerable interest in the impact of oxidative stress on cellular function prompted an intensive search for natural and synthetic antioxidants.High bioavailability of MnTnBuOE-2-PyP 5+ , coupled with its exceptional physicochemical properties as a redox-active ROS/RNS scavenger, makes it a promising candidate in the ongoing cancer and radioprotection preclinical studies.

Table 2 .
Calculated reduction potentials for the Mn