Impact of electrostatics in redox modulation of oxidative stress by Mn porphyrins: Protection of SOD-deficient Escherichia coli via alternative mechanism where Mn porphyrin acts as a Mn carrier

Abstract

Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective SOD mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the beta-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([Mn(III)TCPP](3-) or MnTBAP(3-)) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([Mn(III)TSPP](3-)), to yield the corresponding anionic analogues [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-), respectively, is described along with characterization data, stability studies, and their ability to substitute for SOD in SOD-deficient Escherichia coli. Despite the Mn(III)/Mn(II) reduction potential of [Mn(III)Br(8)TCPP](3-) and [Mn(III)Br(8)TSPP](3-) being close to the SOD-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (Mn(III)TM-2-PyP(5+)), the SOD activity of both anionic brominated porphyrins ([Mn(III)Br(8)TCPP](3-), E(1/2)=+213 mV vs NHE, log k(cat)=5.07; [Mn(III)Br(8)TSPP](3-), E(1/2)=+209 mV, log k(cat)=5.56) is considerably lower than that of Mn(III)TM-2-PyP(5+) (E(1/2)=+220 mV, log k(cat)=7.79). This illustrates the impact of electrostatic guidance of O(2)(-) toward the metal center of the mimic. With low k(cat), the [Mn(III)TCPP](3-), [Mn(III)TSPP](3-), and [Mn(III)Br(8)TCPP](3-) did not rescue SOD-deficient E. coli. The striking ability of [Mn(III)Br(8)TSPP](3-) to substitute for the SOD enzymes in the E. coli model does not correlate with its log k(cat). In fact, the protectiveness of [Mn(III)Br(8)TSPP](3-) is comparable to or better than that of the potent SOD mimic Mn(III)TM-2-PyP(5+), even though the dismutation rate constant of the anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [Mn(III)Br(8)TSPP](3-) system is not the Mn complex, but the free-base porphyrin [H(2)Br(8)TSPP](4-) instead. Control experiments with extracellular MnCl(2) showed the lack of E. coli protection, indicating that "free" Mn(2+) cannot enter the cell to a significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [Mn(III)Br(8)TSPP](3-) is not an SOD mimic per se but carries Mn into the E. coli cell.

SOD-like activity of Mn(II) β-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrin equals that of the enzyme itself

“…Further, along with our mechanistic studies we observed a dramatic impact of the positive charges of the Mn porphyrin on the O 2 •− dismutation; the rate constants are more than two orders of magnitude higher when compared to the neutral or negatively charged porphyrins [3,4]. Thus, Mn III TE-2-PyP 5+ is 130-fold more potent SOD mimic than its singly charged Mn III Br 8 T-2-PyP + [3], and 400-fold more potent than its negatively charged analogue [Mn III Br 8 TSPP] 3− [4]. Charge distribution had also remarkable impact [2].…”

“…Synthesis of β-octabromo-meso-tetrakis(N-methylpyridinium-3-yl)porphyrinato-manganese (II) chloride, Mn II Br 8 TM-3-PyPCl 4 This fully β-brominated complex was prepared from H 2 TM-3-PyP 4+ in four steps. The first three steps were based on the synthetic protocol reported by Richards et al [30] In the third step, the Cu II Br 8 TM-3-PyP 4+ complex (45.6 mg, 0.0301 mmol) was demetallated by the slow addition of a concentrated sulfuric acid (6.0 mL, 111 mmol) while cooled to 10°C…”

“…We have been developing Mn porphyrin-based SOD mimics for over a decade. We have established structure activity relationship (SAR) for both Mn and Fe porphyrins [1] between the metalcentered redox potential and the rate constant for O 2 •− dismutation and refined it recently to account for the differently charged/sized Mn porphyrins [2][3][4][5]. The introduction of both electron-withdrawing groups and positive charges onto the porphyrin core affords thermodynamic and kinetic/electrostatic facilitation for the approach of superoxide to the redox-cycling metal site [1][2][3][4].…”

“…In summary both metal-centered redox potential and charges in close proximity to the metal site have a major impact on the antioxidant potency of the Mn porphyrin. As our knowledge on porphyrins increases it became obvious that another major factor is their in vivo availability [2,4,24,25]. The optimal combination of thermodynamics, kinetics, bioavailability and low toxicity will determine the best drug candidate.…”

“…Para isomer was toxic due to its interactions with nucleic acids [26]. Guided by these collective in vitro, in vivo, and SAR results [1][2][3][4][5]12,[24][25][26], we resumed herein our studies on the meta Mn(III) Nalkylpyridylporphyrins by preparing a brominated derivative of Mn III TM-3-PyP 5+ . This is the first report on the synthesis and characterization of Mn II Br 8 TM-3-PyP 4+ (Fig.…”