Positive Charge on Porphyrin Ligand and Nature of Metal Center Define Basic Physicochemical Properties of Cationic Manganese and Iron Porphyrins in Aqueous Solution.

Recently, comprehensive studies on positively charged manganese porphyrins show that these compounds, known for their superoxide dismutase (SOD) mimetic ability, can be equally reactive toward a broad array of other redox active molecules of biological relevance present in a cellular milieu. In this context, the examination of some fundamental aspects of physicochemical behavior of metalloporphyrins behind their rich aqueous chemistry is believed to provide a valuable basis for the understanding of newly observed biological effects of these compounds in vivo and throw more light on a potential use of common SOD porphyrin mimetics for other redox active cellular targets in order to earn desirable therapeutic effects. Herein, we present versatile characteristics of highly positively charged Mn(P) and Fe(P) porphyrins (with up to +9 and +8 overall charge, respectively) with regard to their acid-base equilibria, metal coordination sphere, water-exchange dynamics, redox properties, and substitution behavior toward selected ligands. For the purpose of these comparative studies, we synthesized for the first time a 9-fold cationic manganese(III) porphyrin. The findings reported in this study enabled highlighting the most important similarities and differences characterizing the aqueous chemistry of positively charged manganese and iron porphyrins and, therefore, outlining the potential factors which can affect the intimate underlying mechanism behind the redox cycling of these metalloporphyrins.

Switching between Inner- and Outer-Sphere PCET Mechanisms of Small-Molecule Activation: Superoxide Dismutation and Oxygen/Superoxide Reduction Reactivity Deriving from the Same Manganese Complex.

Readily exchangeable water molecules are commonly found in the active sites of oxidoreductases, yet the overwhelming majority of studies on small-molecule mimics of these enzymes entirely ignores the contribution of water to the reactivity. Studies of how these enzymes can continue to function in spite of the presence of highly oxidizing species are likewise limited. The mononuclear MnII complex with the potentially hexadentate ligand N-(2-hydroxy-5-methylbenzyl)-N,N',N'-tris(2-pyridinylmethyl)-1,2-ethanediamine (LOH) was previously found to act as both a H2O2-responsive MRI contrast agent and a mimic of superoxide dismutase (SOD). Here, we studied this complex in aqueous solutions at different pH values in order to determine its (i) acid-base equilibria, (ii) coordination equilibria, (iii) substitution lability and operative mechanisms for water exchange, (iv) redox behavior and ability to participate in proton-coupled electron transfer (PCET) reactions, (v) SOD activity and reductive activity toward both oxygen and superoxide, and (vi) mechanism for its transformation into the binuclear MnII complex with (H)OL-LOH and its hydroxylated derivatives. The conclusions drawn from potentiometric titrations, low-temperature mass spectrometry, temperature- and pressure-dependent 17O NMR spectroscopy, electrochemistry, stopped-flow kinetic analyses, and EPR measurements were supported by the structural characterization and quantum chemical analysis of proposed intermediate species. These comprehensive studies enabled us to determine how transiently bound water molecules impact the rate and mechanism of SOD catalysis. Metal-bound water molecules facilitate the PCET necessary for outer-sphere SOD activity. The absence of the water ligand, conversely, enables the inner-sphere reduction of both superoxide and dioxygen. The LOH complex maintains its SOD activity in the presence of •OH and MnIV-oxo species by channeling these oxidants toward the synthesis of a functionally equivalent binuclear MnII species.

Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide Dismutase (SOD) Mimetics: Fluorescently Labeled MnSOD Mimetics, X-ray Absorption Spectroscopy, and X-ray Fluorescence Microscopy 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 are largely unknown. Therefore, we have used X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS) to study the cellular fate of a number of MnPAMs. We have also synthesized and characterized fluorescently labeled (pyrene and rhodamine) manganese(II) pyane [manganese(II) trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[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 manganese(II) pyane type pentaazamacrocycles leads to a 10-100-fold increase in the overall cellular manganese levels compared to the physiological levels of manganese in control cells. In treated cells in general, manganese 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 the relative cellular manganese levels. Analysis of the 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 manganese(II) phosphate. Higher phosphate binding constants, determined from the effect of the phosphate concentration on in vitro SOD activity, were associated with more extensive metabolism of the amine-based MnPAMs to manganese(II) phosphate. In contrast, the imine-based manganese(II) pydiene complex that is prone to hydrolysis was entirely decomposed after uptake and free manganese(II) was oxidized to a manganese(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 the corresponding phosphate binding constants.

Nitrogen Oxide Atom-Transfer Redox Chemistry; Mechanism of NO(g) to Nitrite Conversion Utilizing µ-oxo Heme-Fe(III)-O-Cu(II)(L) Constructs.

While nitric oxide (NO, nitrogen monoxide) is a critically important signaling agent, its cellular concentrations must be tightly controlled, generally through its oxidative conversion to nitrite (NO2(-)) where it is held in reserve to be reconverted as needed. In part, this reaction is mediated by the binuclear heme a3/CuB active site of cytochrome c oxidase. In this report, the oxidation of NO(g) to nitrite is shown to occur efficiently in new synthetic µ-oxo heme-Fe(III)-O-Cu(II)(L) constructs (L being a tridentate or tetradentate pyridyl/alkylamino ligand), and spectroscopic and kinetic investigations provide detailed mechanistic insights. Two new X-ray structures of µ-oxo complexes have been determined and compared to literature analogs. All µ-oxo complexes react with 2 mol equiv NO(g) to give 1:1 mixtures of discrete [(L)Cu(II)(NO2(-))](+) plus ferrous heme-nitrosyl compounds; when the first NO(g) equiv reduces the heme center and itself is oxidized to nitrite, the second equiv of NO(g) traps the ferrous heme thus formed. For one µ-oxo heme-Fe(III)-O-Cu(II)(L) compound, the reaction with NO(g) reveals an intermediate species ("intermediate"), formally a bis-NO adduct, [(NO)(porphyrinate)Fe(II)-(NO2(-))-Cu(II)(L)](+) (λmax = 433 nm), confirmed by cryo-spray ionization mass spectrometry and EPR spectroscopy, along with the observation that cooling a 1:1 mixture of [(L)Cu(II)(NO2(-))](+) and heme-Fe(II)(NO) to -125 °C leads to association and generation of the key 433 nm UV-vis feature. Kinetic-thermodynamic parameters obtained from low-temperature stopped-flow measurements are in excellent agreement with DFT calculations carried out which describe the sequential addition of NO(g) to the µ-oxo complex.

Structure- and concentration-specific assessment of the physiological reactivity of α-dicarbonyl glucose degradation products in peritoneal dialysis fluids.

In peritoneal dialysis (PD), glucose degradation products (GDPs), which are formed during heat sterilization of dialysis fluids, lead to structural and functional changes in the peritoneal membrane, which eventually result in the loss of its ultrafiltration capacity. To determine the molecular mechanisms behind these processes, the present study tested the influence of the six major α-dicarbonyl GDPs in PD fluids, namely, glyoxal, methylglyoxal, 3-deoxyglucosone (3-DG), 3-deoxygalactosone (3-DGal), 3,4-dideoxyglucosone-3-ene (3,4-DGE), and glucosone with respect to their potential to impair the enzymatic activity of RNase A as well as their effects on cell viability. For comprehensive risk assessment, the α-dicarbonyl GDPs were applied separately and in concentrations as present in conventional PD fluids. Thus, it was shown that after 5 days, glucosone impaired RNase A activity most distinctly (58% remaining activity, p < 0.001 compared to that of the control), followed by 3,4-DGE (62%, p < 0.001), 3-DGal (66%, p < 0.001), and 3-DG (76%, p < 0.01). Methylglyoxal and glyoxal caused weaker inactivation with significant effects only after 10 days of incubation (79%, 81%, p < 0.001). Profiling of the advanced glycation end products formed during the incubation of RNase A with methylglyoxal revealed predominant formation of the arginine modifications imidazolinone, CEA/dihydroxyimidazoline, and tetrahydropyrimidine at Arg10, Arg33, Arg39, and Arg85. Particularly, modification at Arg39 may severely affect the active site of the enzyme. Additionally, structure- and concentration-specific assessment of the cytotoxicity of the α-dicarbonyl GDPs was performed. Although present at very low concentration, the cytotoxic effect of PD fluids after 2 days of incubation was exclusively caused by 3,4-DGE (14% cell viability, p < 0.001). After 4 days of incubation, 3-DGal (13% cell viability, p < 0.001), 3-DG (24%, p < 0.001), and, to a lower extent, glyoxal and methylglyoxal (both 57%, p < 0.01) also reduced cell viability significantly. In conclusion, 3,4-DGE, 3-DGal, and glucosone appear to be the most relevant parameters for the biocompatibility of PD fluids.

Bioinspired superoxide-dismutase mimics: The effects of functionalization with cationic polyarginine peptides.

Continuing a bio-mimetic approach, we have prepared peptide conjugates of a superoxide dismutase (SOD) mimic [MnL](+) (where HL=N-(2-hydroxybenzyl)-N,N'-bis[2-(N-methylimidazolyl)methyl]ethane-1,2-diamine), namely [MnL'-Arg(n-1)](n+) (where n=2, 4, 7 and 10) and [MnL'-Gly1](+). [MnL'-Arg(n-1)](n+) contained cationic residue(s) that emulate the electrostatic channel of the enzyme. Physicochemical methods showed that functionalization at the secondary amine of HL did not impair coordination to Mn(II) with association constants (Kassoc) between 1.6 and 3.3×10(6)M(-1). The Mn(III)/Mn(II) redox potential of the conjugates was between 0.27 and 0.30V vs SCE, slightly higher than [MnL](+) under the same conditions, but remain at a value that facilitates O2(-) dismutation. The catalytic rate constant (kcat) of the dismutation for the series was studied using a direct stopped-flow method, which showed that for compounds with the same overall charge, the alkylation of the secondary amine of [MnL](+) (kcat=5.0±0.1×10(6)M(-1)s(-1)) led to a lower value (i.e. for [MnL'Gly](+), kcat=4.2±0.1×10(6)M(-1)s(-1)). However, under the same conditions, kcat values between 5.0±0.4×10(6)M(-1)s(-1) and 6.6±0.1×10(6)M(-1)s(-1) were determined for [MnL'-Arg(n-1)](n+) conjugates, indicating that the cationic residue(s) compensated for the loss in activity. Analysis of the effect of ionic strength on the kcat strongly suggested that not all the charges were involved, but only the closest ones electrostatically influenced the SOD active metal centre.