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Azanone ((1)HNO, nitroxyl) is a highly reactive molecule with interesting chemical and biological properties. Like nitric oxide (NO), its main biologically related targets are oxygen, thiols, and metalloproteins, particularly heme proteins. As HNO dimerizes with a rate constant between 10(6) and 10(7) M(-1) s(-1), reactive studies are performed using donors, which are compounds that spontaneously release HNO in solution. In the present work, we studied the reaction mechanism and kinetics of two azanone donors Angelís Salt and toluene sulfohydroxamic acid (TSHA) with eight different Mn porphyrins as trapping agents. These porphyrins differ in their total peripheral charge (positively or negatively charged) and in their Mn(III)/Mn(II) reduction potential, showing for each case positive (oxidizing) and negative (reducing) values. Our results show that the reduction potential determines the azanone donor reaction mechanism. While oxidizing porphyrins accelerate decomposition of the donor, reducing porphyrins react with free HNO. Our results also shed light into the donor decomposition mechanism using ab initio methods and provide a thorough analysis of which MnP are the best candidates for azanone trapping and quantification experiments.

Growing interest in redox-active compounds as therapeutics for oxidative stress-related diseases led to the design of metalloporphyrins as some of the most potent functional SOD-mimics. Herein we report a detailed electrochemical study of the protolytic and redox equilibria of manganese ortho and meta substituted N-ethylpyridyl porphyrins (MnPs), MnTE-2-PyP(5+) and MnTE-3-PyP(5+), in aqueous solutions. The electrochemical parameters of redox processes for all experimentally available species have been determined, as well as their diffusion coefficients and estimated sizes of aqueous cavities. The results indicate that possible changes of the intracellular acidity cannot affect the antioxidant activity of MnPs in vivo, since no change in the E(Mn(III)P/Mn(II)P) values was observed below pH 10. Furthermore, the results confirm that both of these MnPs can be efficient redox scavengers of peroxynitrite (ONOO(-)), another major damaging species in vivo. This can occur by either single-electron reduction or two-electron reduction of ONOO(-), involving either the Mn(IV)P/Mn(III)P redox couple or Mn(IV)P/Mn(II)P redox couple. In addition to kred(ONOO(-)) reported previously, the thermodynamic parameters calculated herein imply a strong and identical driving force for the reaction of both ortho and meta isomeric MnPs with ONOO(-). An enlargement of both Mn(III)P complexes upon an increase of the solution pH was also observed and attributed to the reduction of positive charge on the central ion caused by deprotonation of the axial water molecules. This expansion of aqueous cavities suggests the formation of a solvent cage and the increased lipophilicity of Mn(III)P complexes caused by increased electron density on the Mn ion.

Cationic Mn porphyrins are among the most potent catalytic antioxidants and/or cellular redox modulators. Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl(5)) is the Mn porphyrin most studied in vivo and has successfully rescued animal models of a variety of oxidative stress-related diseases. The stability of an authentic MnTE-2-PyPCl(5) sample was investigated hereon by thermogravimetric, derivative thermogravimetric, and differential thermal analyses (TG/DTG/DTA), under dynamic air, followed by studies at selected temperatures to evaluate the decomposition path and appropriate conditions for storage and handling of these materials. All residues were analyzed by thin-layer chromatography (TLC) and UV-vis spectroscopy. Three thermal processes were observed by TG/DTG. The first event (endothermic) corresponded to dehydration, and did not alter the MnTE-2-PyPCl(5) moiety. The second event (endothermic) corresponded to the loss of EtCl (dealkylation), which was characterized by gas chromatography-mass spectrometry. The residue at 279°C had UV-vis and TLC data consistent with those of the authentic, completely dealkylated analog, MnT-2-PyPCl. The final, multi-step event corresponded to the loss of the remaining organic matter to yield Mn(3)O(4) which was characterized by IR spectroscopy. Isothermal treatment at 188°C under static air for 3h yielded a mixture of partially dealkylated MnPs and traces of the free-base, dealkylated ligand, H(2)T-2-PyP, which reveals that dealkylation is accompanied by thermal demetallation under static air conditions. Dealkylation was not observed if the sample was heated as a solid or in aqueous solution up to ∼100°C. Whereas moderate heating changes sample composition by loss of H(2)O, the dehydrated sample is indistinguishable from the original sample upon dissolution in water, which indicates that catalytic activity (on Mn basis) remains unaltered. Evidently, dealkylation at high temperature compromises sample activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The in vitro autoxidation of N-hydroxyurea (HU) is catalyzed by Mn(III)TTEG-2-PyP(5+), a synthetic water soluble Mn(III) porphyrin which is also a potent mimic of the enzyme superoxide dismutase. The detailed mechanism of the reaction is deduced from kinetic studies under basic conditions mostly based on data measured at pH = 11.7 but also including some pH-dependent observations in the pH range 9-13. The major intermediates were identified by UV-vis spectroscopy and electrospray ionization mass spectrometry. The reaction starts with a fast axial coordination of HU to the metal center of Mn(III)TTEG-2-PyP(5+), which is followed by a ligand-to-metal electron transfer to get Mn(II)TTEG-2-PyP(4+) and the free radical derived from HU (HU˙). Nitric oxide (NO) and nitroxyl (HNO) are minor intermediates. The major pathway for the formation of the most significant intermediate, the {MnNO} complex of Mn(II)TTEG-2-PyP(4+), is the reaction of Mn(II)TTEG-2-PyP(4+) with NO. We have confirmed that the autoxidation of the intermediates opens alternative reaction channels, and the process finally yields NO(2)(-) and the initial Mn(III)TTEG-2-PyP(5+). The photochemical release of NO from the {MnNO} intermediate was also studied. Kinetic simulations were performed to validate the deduced rate constants. The investigated reaction has medical implications: the accelerated production of NO and HNO from HU may be utilized for therapeutic purposes.

The difference in electrostatics and reduction potentials between manganese ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP) and manganese meta-tetrakis(N-ethylpyridinium-3-yl)porphyrin (MnTE-3-PyP) is a challenging topic, particularly because of the high likelihood for their clinical development. Hence, a detailed study of the protolytic and electrochemical speciation of Mn(II-IV)TE-2-PyP and Mn(II-IV)TE-3-PyP in a broad pH range has been performed using the combined spectrophotometric and potentiometric methods. The results reveal that in aqueous solutions within the pH range ∼2-13 the following species exist: (H(2)O)Mn(II)TE-m-PyP(4+), (HO)Mn(II)TE-m-PyP(3+), (H(2)O)(2)Mn(III)TE-m-PyP(5+), (HO)(H(2)O)Mn(III)TE-m-PyP(4+), (O)(H(2)O)Mn(III)TE-m-PyP(3+), (O)(H(2)O)Mn(IV)TE-m-PyP(4+) and (O)(HO)Mn(IV)TE-m-PyP(3+) (m = 2, 3). All the protolytic equilibrium constants that include the accessible species as well as the thermodynamic parameters for each particular protolytic equilibrium have been determined. The corresponding formal reduction potentials related to the reduction of the above species and the thermodynamic parameters describing the accessible reduction couples were calculated as well.

Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO(3)(*-), peroxyl radical, and less efficiently H(2)O(2). By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds.

The activation parameters and the rate constants of the water-exchange reactions of Mn(III)TE-2-PyP(5+) (meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin) as cationic, Mn(III)TnHex-2-PyP(5+) (meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin) as sterically shielded cationic, and Mn(III)TSPP(3-) (meso-tetrakis(4-sulfonatophenyl)porphyrin) as anionic manganese(iii) porphyrins were determined from the temperature dependence of (17)O NMR relaxation rates. The rate constants at 298 K were obtained as 4.12 x 10(6) s(-1), 5.73 x 10(6) s(-1), and 2.74 x 10(7) s(-1), respectively. On the basis of the determined entropies of activation, an interchange-dissociative mechanism (I(d)) was proposed for the cationic complexes (DeltaS(double dagger) = approximately 0 J mol(-1) K(-1)) whereas a limiting dissociative mechanism (D) was proposed for Mn(III)TSPP(3-) complex (DeltaS(double dagger) = +79 J mol(-1) K(-1)). The obtained water exchange rate of Mn(III)TSPP(3-) corresponded well to the previously assumed value used by Koenig et al. (S. H. Koenig, R. D. Brown and M. Spiller, Magn. Reson. Med., 1987, 4, 52-260) to simulate the (1)H NMRD curves, therefore the measured value supports the theory developed for explaining the anomalous relaxivity of Mn(III)TSPP(3-) complex. A magnitude of the obtained water-exchange rate constants further confirms the suggested inner sphere electron transfer mechanism for the reactions of the two positively charged Mn(iii) porphyrins with the various biologically important oxygen and nitrogen reactive species. Due to the high biological and clinical relevance of the reactions that occur at the metal site of the studied Mn(iii) porphyrins, the determination of water exchange rates advanced our insight into their efficacy and mechanism of action, and in turn should impact their further development for both diagnostic (imaging) and therapeutic purposes.

Lipophilicity/bioavailibility of Mn(III) N-alkylpyridylporphyrin-based superoxide dismutase (SOD) mimics has a major impact on their in vivo ability to suppress oxidative stress. Meta isomers are less potent SOD mimics than ortho analogues but are 10-fold more lipophilic and more planar. Enhanced lipophilicity contributes to their higher accumulation in cytosol of SOD-deficient Escherichia coli, compensating for their lower potency; consequently, both isomers exert similar-to-identical protection of SOD-deficient E. coli. Thus meta isomers may be prospective therapeutics as are ortho porphyrins.

The awareness of the beneficial effects of Mn porphyrin-based superoxide dismutase (SOD) mimics and peroxynitrite scavengers on decreasing oxidative stress injuries has increased the use of these compounds as mechanistic probes and potential therapeutics. Simple Mn2+ salts, however, have SOD-like activity in their own right both in vitro and in vivo. Thus, quantification/removal of residual Mn2+ species in Mn-based therapeutics is critical to an unambiguous interpretation of biological data. Herein we report a simple, sensitive, and specific method to determine residual Mn2+ in Mn porphyrin preparations that combines a hydrometallurgical approach for separation/speciation of metal compounds with a spectrophotometric strategy for Mn determination. The method requires only common chemicals and a spectrophotometer and is based on the extraction of residual Mn2+ by bis(2-ethylhexyl)hydrogenphosphate (D2EHPA) into kerosene, re-extraction into acid, and neutralization followed by UV-vis determination of the Mn2+ levels via a Cd2+-catalyzed metallation of the H2TCPP4- porphyrin indicator. The overall procedure is simple, sensitive, specific, and amenable to adaptation. This quantification method has been routinely used by us for a large variety of water-soluble porphyrins.

Mn(III) N-alkylpyridylporphyrins are among the most potent known SOD mimics and catalytic peroxynitrite scavengers and modulators of redox-based cellular transcriptional activity. In addition to their intrinsic antioxidant capacity, bioavailability plays a major role in their in vivo efficacy. Although of identical antioxidant capacity, lipophilic MnTnHex-2-PyP is up to 120-fold more efficient in reducing oxidative stress injuries than hydrophilic MnTE-2-PyP. Owing to limitations of an analytical nature, porphyrin lipophilicity has been often estimated by the thin-layer chromatographic R(f) parameter, instead of the standard n-octanol/water partition coefficient, P(OW). Herein we used a new methodological approach to finally describe the MnP lipophilicity, using the conventional log P(OW) means, for a series of biologically active ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins. Three new porphyrins (MnTnBu-3-PyP, MnTnHex-3-PyP, and MnTnHep-2-PyP) were synthesized to strengthen the conclusions. The log P(OW) was linearly related to R(f) and to the number of carbons in the alkyl chain (n(C)) for both isomer series, the meta isomers being 10-fold more lipophilic than the analogous ortho porphyrins. Increasing the length of the alkyl chain by one carbon atom increases the log P(OW) value approximately 1 log unit with both isomers. Dramatic approximately 4 and approximately 5 orders of magnitude increases in the lipophilicity of the ortho isomers, by extending the pyridyl alkyl chains from two (MnTE-2-PyP, log P(OW)=-6.89) to six (MnTnHex-2-PyP, log P(OW)=-2.76) and eight carbon atoms (MnTnOct-2-PyP, log P(OW)=-1.24), parallels the increased efficacy in several oxidative-stress injury models, particularly those of the central nervous system, in which transport across the blood-brain barrier is critical. Although meta isomers are only slightly less potent SOD mimics and antioxidants than their ortho analogues, their higher lipophilicity and smaller bulkiness may lead to a higher cellular uptake and overall similar effectiveness in vivo.

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O2.- dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O2.- related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO2 adduct. The log kcat (O2.-) value for MnTBAP is estimated to be about 3.16, which is approximately 5 and approximately 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only approximately 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k red (ONOO-) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O2.-, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO-/CO3.- from O2.- pathways.

Severe pain syndromes reduce the quality of life of patients with inflammatory and neoplastic diseases, partly because reduced analgesic effectiveness with chronic opiate therapy (i.e., tolerance) leads to escalating doses and distressing side effects. Peroxynitrite-mediated nitroxidative stress in the dorsal horn of the spinal cord plays a critical role in the induction and development of antinociceptive tolerance to morphine. This provides a valid pharmacological basis for developing peroxynitrite scavengers as potent adjuncts to opiates in the management of pain. The cationic Mn(III) ortho-N-alkylpyridylporphyrins MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+) are among the most potent peroxynitrite scavengers, with nearly identical scavenging rate constants (approximately 10(7) M(-1) s(-1)). Yet, MnTnHex-2-PyP(5+) is significantly more lipophilic and more bioavailable and, in turn, was 30-fold more effective in blocking the development of morphine antinociceptive tolerance than MnTE-2-PyP(5+) using the hot-plate test in a well-characterized murine model. The hydrophilic MnTE-2-PyP(5+) and the lipophilic MnTnHex-2-PyP(5+) were 10- and 300-fold, respectively, more effective in inhibiting morphine tolerance than the hydrophilic Fe(III) porphyrin FeTM-4-PyP(5+). Both Mn porphyrins decreased levels of TNF-alpha, IL-1 beta, and IL-6 to normal values. Neither of them affected acute morphine antinociceptive effects nor caused motor function impairment. Also neither was able to reverse already established morphine tolerance. We have recently shown that the anionic porphyrin Mn(III) tetrakis(4-carboxylatophenyl)porphyrin is selective in removing ONOO(-) over O(2)(-), but at approximately 2 orders of magnitude lower efficacy than MnTE-2-PyP(5+) and MnTnHex-2-PyP(5+), which in turn parallels up to 100-fold lower ability to reverse morphine tolerance. These data (1) support the role of peroxynitrite rather than superoxide as a major mechanism in blocking the development of morphine tolerance and (2) show that lipophilicity is a critical parameter in enhancing the potency of such novel peroxynitrite scavengers.

Cationic Mn porphyrins are among the most potent SOD mimics and peroxynitrite scavengers. They have been widely and successfully used in different models of oxidative stress and are either progressing towards or are in phase I of clinical trials. The most frequently used compounds are Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+) or AEOL10113), its methyl analogue (MnTM-2-PyP(5+) or AEOL10112), and Mn(III) meso-tetrakis(4-benzoic acid)porphyrin (MnTBAP). A great discrepancy between the in vivo data obtained with Calbiochem preparations and those of authentic MnTE-2-PyP(5+) and MnTM-2-PyP(5+) samples were recently observed. Surprisingly, the commercial samples were invariably of poor identity and consisted of mixtures of nearly equal contributions of non-alkylated, mono-, di-, tri- and tetraalkylated porphyrins, lacking thus the major structural entity that determines their antioxidant potency, i.e., the four positively charged orthoN-alkylpyridyl groups that afford thermodynamic tuning of the active site and electrostatic guidance of anionic superoxide and peroxynitrite species toward the metal center. The MnTE-2-PyP(5+) and MnTM-2-PyP(5+) compounds were not even the major species in the commercial samples sold as "MnTE-2-PyP" and "MnTM-2-PyP", respectively. While we have already reported the insufficient impurity of the MnTBAP samples from Alexis and other suppliers, in one more recent lot the situation is dramatic, as 25% of the sample was not MnTBAP, but metal-free ligand, H(2)TBAP. The (unintentional) use of the Mn porphyrins of low quality compromises therapeutic and/or mechanistic conclusions. Simple techniques, which include thin-layer chromatography, electrospray-mass spectrometry, UV-vis spectroscopy, and electrochemistry described here could be used routinely to check the overall quality of Mn porphyrins in order to avoid misleading conclusions and waste of valuable resources (animals, compounds, time, manpower).

Mn(III) tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), a potent catalytic superoxide and peroxynitrite scavenger, has been beneficial in several oxidative stress-related diseases thus far examined. Pharmacokinetic studies are essential for the better assessment of the therapeutic potential of MnTE-2-PyP(5+) and similar compounds, as well as for the modulation of their bioavailability and toxicity. Despite high hydrophilicity, this drug entered mitochondria after a single 10 mg/kg intraperitoneal injection at levels high enough (5.1 muM; 2.95 ng/mg protein) to protect against superoxide/peroxynitrite damage. Utilizing the same analytical approach, which involves the reduction of MnTE-2-PyP(5+) followed by the exchange of Mn(2+) with Zn(2+) and HPLC/fluorescence detection of ZnTE-2-PyP(4+), we measured levels of MnTE-2-PyP(5+) in mouse plasma, liver, kidney, lung, heart, spleen, and brain over a period of 7 days after a single intraperitoneal injection of 10 mg/kg. Two B6C3F1 female mice per time point were used. The pharmacokinetic profile in plasma and organs was complex; thus a noncompartmental approach was utilized to calculate the area under the curve, c(max), t(max), and drug elimination half-time (t(1/2)). In terms of levels of MnTE-2-PyP(5+) found, the organs can be classified into three distinct groups: (1) high levels (kidney, liver, and spleen), (2) moderate levels (lung and heart), and (3) low levels (brain). The maximal levels in plasma, kidney, spleen, lung, and heart are reached within 45 min, whereas in the case of liver a prolonged absorption phase was observed, with the maximal concentration reached at 8 h. Moreover, accumulation of the drug in brain continued beyond the time of the experiment (7 days) and is likely to be driven by the presence of negatively charged phospholipids. For tissues other than brain, a slow elimination phase (single exponential decay, t(1/2)=60 to 135 h) was observed. The calculated pharmacokinetic parameters will be used to design optimal dosing regimens in future preclinical studies utilizing this and similar compounds.

Mn porphyrins are among the most efficient SOD mimics with potency approaching that of SOD enzymes. The most potent ones, Mn(III) N-alkylpyridylporphyrins bear positive charges in a close proximity to the metal site, affording thermodynamic and kinetic facilitation for the reaction with negatively charged superoxide. The addition of electron-withdrawing bromines onto beta-pyrrolic positions dramatically improves thermodynamic facilitation for the O2*- dismutation. We have previously characterized the para isomer, Mn(II)Br(8)TM-4-PyP(4+) [Mn(II) beta-octabromo-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin]. Herein we fully characterized its meta analogue, Mn(II)Br(8)TM-3-PyP(4+) with respect to UV/vis spectroscopy, electron spray mass spectrometry, electrochemistry, O2*- dismutation, metal-ligand stability, and the ability to protect SOD-deficient Escherichia coli in comparison with its para analogue. The increased electron-deficiency of the metal center stabilizes Mn in its +2 oxidation state. The metal-centered Mn(III)/Mn(II) reduction potential, E((1/2))=+468 mV vs NHE, is increased by 416 mV with respect to non-brominated analogue, Mn(III)TM-3-PyP(5+) and is only 12 mV less positive than for para isomer. Yet, the complex is significantly more stable towards the loss of metal than its para analogue. As expected, based on the structure-activity relationships, an increase in E((1/2)) results in a higher catalytic rate constant for the O2*- dismutation, log k(cat)> or =8.85; 1.5-fold increase with respect to the para isomer. The IC(50) was calculated to be < or =3.7 nM. Manipulation of the electron-deficiency of a cationic porphyrin resulted, therefore, in the highest k(cat) ever reported for a metalloporphyrin, being essentially identical to the k(cat) of superoxide dismutases (log k(cat)=8.84-9.30). The positive kinetic salt effect points to the unexpected, unique and first time recorded behavior of Mn beta-octabrominated porphyrins when compared to other Mn porphyrins studied thus far. When species of opposing charges react, the increase in ionic strength invariably results in the decreased rate constant; with brominated porphyrins the opposite was found to be true. The effect is 3.5-fold greater with meta than with para isomer, which is discussed with respect to the closer proximity of the quaternary nitrogens of the meta isomer to the metal center than that of the para isomer. The potency of Mn(II)Br(8)TM-3-PyP(4+) was corroborated by in vivo studies, where 500 nM allows SOD-deficient E. coli to grow >60% of the growth of wild type; at concentrations > or =5 microM it exhibits toxicity. Our work shows that exceptionally high k(cat) for the O2*- disproportionation can be achieved not only with an N(5)-type coordination motif, as rationalized previously for aza crown ether (cyclic polyamines) complexes, but also with a N(4)-type motif as in the Mn porphyrin case; both motifs sharing "up-down-up-down" steric arrangement.

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.

We evaluate herein the impact of positive charge distribution on the in vitro and in vivo properties of Mn porphyrins as redox modulators possessing the same overall 5+ charge and of minimal stericity demand: Mn(III) meso-tetrakis(trimethylanilinium-4-yl)porphyrin (MnTTriMAP(5+)), Mn(III) meso-tetrakis(N,N'-dimethylpyrazolium-4-yl)porphyrin (MnTDM-4-PzP(5+)), Mn(III) meso-tetrakis(N,N'-dimethylimidazolium-2-yl)porphyrin (MnTDM-2-ImP(5+)), and the ortho and para methylpyridinium complexes Mn(III) meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (MnTM-4-PyP(5+)) and Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnTM-2-PyP(5+)). Both Mn(III)/Mn(II) reduction potential and SOD activity within the series follow the order: MnTTriMAP(5+)

Superoxide is involved in a plethora of pathological and physiological processes via oxidative stress and/or signal transduction pathways. Superoxide dismutase (SOD) mimics have, thus, been actively sought for clinical and mechanistic purposes. Manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP) is one of the most intensely explored "SOD mimics" in biology and medicine. However, we show here that this claimed SOD activity of MnTBAP in aqueous media is not corroborated by comprehensive structure-activity relationship studies for a wide set of Mn porphyrins and that MnTBAP from usual commercial sources contains different amounts of noninnocent trace impurities (Mn clusters), which inhibited xanthine oxidase and had SOD activity in their own right. In addition, the preparation and thorough characterization of a high-purity MnTBAP is presented for the first time and confirmed that pure MnTBAP has no SOD activity in aqueous medium. These findings call for an assessment of the relevance and suitability of using MnTBAP (or its impurities) as a mechanistic probe and antioxidant therapeutic; conclusions on the physiological and pathological role of superoxide derived from studies using MnTBAP of uncertain purity should be examined judiciously. An unequivocal distinction between the biological effects due to MnTBAP and that of its impurities can only be unambiguously made if a pure sample is/was used. This work also illustrates the contribution of fundamental structure-activity relationship studies not only for drug design and optimization, but also as a "watchdog" mechanism for checking/spotting eventual incongruence of drug activity in chemical and biological settings.

Thirteen new manganese porphyrins and two porphodimethenes bearing one to three different substituents at the meso positions in a variety of architectures have been synthesized. The substituents employed generally are (i) electron-withdrawing to tune the reduction potential to the desirable range (near +0.3V vs NHE), and/or (ii) lipophilic to target the interior of lipid bilayer membranes and/or the blood-brain barrier. The influence of the substituents on the Mn(III)/Mn(II) reduction potentials has been characterized, and the superoxide dismutase activity of the compounds has been examined.

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnIIITE-2-PyP5+ (AEOL-10113) has proven effective in treating oxidative stress-induced conditions including cancer, radiation damage, diabetes, and central nervous system trauma. The ortho cationic pyridyl nitrogens of MnTE-2-PyP5+ are essential for its high antioxidant potency. The exceptional ability of MnIIITE-2-PyP5+ to dismute O2.- parallels its ability to reduce ONOO- and CO3-. Decreasing levels of these species are considered its predominant mode of action, which may also involve redox regulation of signaling pathways. Recently, Ferrer-Sueta at al. (Free Radic. Biol. Med. 41:503-512; 2006) showed, with submitochondrial particles, that>or=3 microM MnIIITE-2-PyP5+ was able to protect components of the mitochondrial electron transport chain from peroxynitrite-mediated damage. Our study complements their data in showing, for the first time that micromolar mitochondrial concentrations of MnIIITE-2-PyP5+ are obtainable in vivo. For this study we have developed a new and sensitive method for MnIIITE-2-PyP5+ determination in tissues. The method is based on the exchange of porphyrin Mn2+ with Zn2+, followed by the HPLC/fluorescence detection of ZnIITE-2-PyP4+. At 4 and 7 h after a single 10 mg/kg intraperitoneal administration of MnIIITE-2-PyP5+, the mice (8 in total) were anesthetized and perfused with saline. Mitochondria were then isolated by the method of Mela and Seitz (Methods Enzymol.55:39-46; 1979). We found MnIIITE-2-PyP5+ localized in heart mitochondria to 2.95 ng/mg protein. Given the average value of mitochondrial volume of 0.6 microL/mg protein, the calculated MnIIITE-2-PyP5+ concentration is 5.1 microM, which is sufficient to protect mitochondria from oxidative damage. This study establishes, for the first time, that MnIIITE-2-PyP5+, a highly charged metalloporphyrin, is capable of entering mitochondria in vivo at levels sufficient to exert there its antioxidant action; such a result encourages its development as a prospective therapeutic agent.

The low-molecular weight water-soluble Fe(III) and Mn(III) porphyrins--in biologically relevant phosphate-buffered saline medium with ascorbic acid as a source of electrons, under aerobic conditions but without co-oxidant - catalyze the hydroxylation of anti-cancer drug cyclophosphamide to active metabolite 4-hydroxycyclophosphamide in yields similar or higher than those typically obtained by the action of liver enzymes in vivo. The Fe(III) meso tetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin, highly electron-deficient at the metal site, was the most effective catalyst. If proven viable in vivo, this methodology could be expanded to localized or systemic activation of the entire family of oxazaphosphorine-based (and many other) anti-cancer drugs and become a powerful tool for an aggressive treatment of tumors with less toxic side effects to the patient.

Closed head injury induces cerebral oxidative stress. The efficacy of a Mn (III) porphyrin catalytic antioxidant was assessed in a mouse closed head injury model. Mice were subjected to closed head injury and treated 15 min later with an i.v. bolus of vehicle or 3 mg/kg MnTE-2-PyP5+. Aconitase activity, Fluoro-Jade staining, glial fibrillary acidic protein immunoreactivity, and rotarod falling latencies were measured. Closed head injury altered all variables. MnTE-2-PyP5+ had no effect on any variable with the exception of attenuation of aconitase inactivation at 2 h post-closed head injury. In a second experiment, mice received 3 mg/kg or 6 mg/kg MnTE-2-PyP5+ or vehicle i.v. 15 min post-closed head injury. Rotarod and Morris water maze latencies were measured. Closed head injury altered performance in both tests. No statistically significant effect of MnTE-2-PyP5+ was observed. We conclude that single dose MnTE-2-PyP5+ does not alter outcome in this mouse closed head injury model.

Two new tri(ethyleneglycol)-derivatized Mn(III) porphyrins were synthesized with the aim of increasing their bioavailability, and blood-circulating half-life. These are Mn(III) tetrakis(N-(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)pyridinium-2-yl)porphyrin, MnTTEG-2-PyP5+ and Mn(III) tetrakis(N,N'-di(1-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)imidazolium-2-yl)porphyrin, MnTDTEG-2-ImP5+. Both porphyrins have ortho pyridyl or di-ortho imidazolyl electron-withdrawing substituents at meso positions of the porphyrin ring that assure highly positive metal centered redox potentials, E1/2 = +250 mV vs. NHE for MnTTEG-2-PyP5+ and E1/2 = + 412 mV vs. NHE for MnTDTEG-2-ImP5+. As expected, from established E1/2 vs. log kcat(O2 *-) structure-activity relationships for metalloporphyrins (Batinic-Haberle et al., Inorg. Chem., 1999, 38, 4011), both compounds exhibit higher SOD-like activity than any meso-substituted Mn(III) porphyrins-based SOD mimic thus far, log kcat = 8.11 (MnTTEG-2-PyP5+) and log kcat = 8.55 (MnTDTEG-2-ImP5+), the former being only a few-fold less potent in disproportionating O2*- than the SOD enzyme itself. The new porphyrins are stable to both acid and EDTA, and non toxic to E. coli. Despite elongated substituents, which could potentially lower their ability to cross the cell wall, MnTTEG-2-PyP5+ and MnTDTEG-2-ImP5+ exhibit similar protection of SOD-deficient E. coli as their much smaller ethyl analogues MnTE-2-PyP5+ and MnTDE-2-ImP5+, respectively. Consequently, with anticipated increased blood-circulating half-life, these new Mn(III) porphyrins may be more effective in ameliorating oxidative stress injuries than ethyl analogues that have been already successfully explored in vivo.

Pro-inflammatory cytokines have been shown to depress myocardial mechanical function by enhancing peroxynitrite generation in the heart. The contribution of NO synthesized by different NOS isoforms, as well as the contribution of superoxide to this mechanism are still not clear. Isolated working hearts of iNOS(-/-) and wildtype mice were perfused for 120 min in the presence or absence of a mixture of pro-inflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma). iNOS mRNA was detected only in cytokine-treated wildtype hearts. In wildtype hearts, cytokine treatment significantly decreased cardiac work, calculated as cardiac output times peak systolic pressure, to 31+/-9% of original values by the end of perfusion (P <0.05). The decline of cardiac work induced by cytokine treatment was significantly reduced in iNOS(-/-) hearts (63+/-5% of original value). Only cytokine-treated wildtype hearts showed decreased aconitase activity, indicating a higher level of oxidative stress in these hearts. Cytokines increased NADPH oxidase activity in both wildtype and iNOS(-/-) hearts, whereas NADH oxidase and xanthine oxidase/xanthine dehydrogenase activities were unaffected. The SOD mimetic MnTE2PyP prevented the cytokine-induced decline of cardiac work in both wildtype and iNOS(-/-) hearts. Cardiac p38 MAPK activation was unaltered in all experimental groups. Although genetic disruption of the iNOS gene provides partial protection against cytokine-induced cardiac dysfunction, iNOS-independent mechanisms, including contribution of NO from other NOS enzymes and the generation of superoxide, are also important contributors.

Cationic Mn(III) porphyrins substituted on the methine bridge carbons (meso positions) with N-alkylpyridinium or N,N'-diethylimidazolium groups have been prepared and characterized, both chemically and as SOD mimics. The ortho tetrakis N-methylpyridinium compound was substantially more active than the corresponding para isomer. This ortho compound also exhibited a more positive redox potential and greater ability to facilitate the aerobic growth of a SOD-deficient Escherichia coli. Analogs with longer alkyl side chains and with methoxyethyl side chains, as well as with N,N'-diethylimidazolium and N,N'-dimethoxyethylimidazolium groups on the meso positions, have been prepared in anticipation of greater penetration of the cells due to greater lipophilicity. We now report that the more lipophilic compounds were effective at complementing the SOD-deficient E. coli at lower concentrations than were needed with the less lipophilic compounds. The greater efficacy of the more lipophilic compounds was achieved at the cost of greater toxicity that became apparent when these compounds were applied at higher concentrations.

Mn(III) ortho-tetrakis(N-ethylpyridinium-2-yl)porphyrin (Mn(III)TE-2-PyP(5+)) effectively scavenges reactive oxygen and nitrogen species in vitro, and protects in vivo, in different rodent models of oxidative stress injuries. Further, Mn(III)TE-2-PyP(5+) was shown to be readily reduced by cellular reductants such as ascorbic acid and glutathione. We now show that tetrahydrobiopterin (BH(4)) is also able to reduce the metal center. Under anaerobic conditions, in phosphate-buffered saline (pH 7.4) at 25 +/- 0.1 degrees C, reduction of Mn(III)TE-2-PyP(5+) occurs through two reaction steps with rate constants k(1) = 1.0 x 10(4) M(-1) s(-1) and k(2) = 1.5 x 10(3) M(-1) s(-1). We ascribe these steps to the formation of tetrahydrobiopterin radical (BH(4)(.+)) (k(1)) that then undergoes oxidation to 6,7-dihydro-8H-biopterin (k(2)), which upon rearrangement gives rise to 7,8-dihydrobiopterin (7,8-BH(2)). Under aerobic conditions, Mn(III)TE-2-PyP(5+) catalytically oxidizes BH(4). This is also true for its longer chain alkyl analog, Mn(III) ortho-tetrakis(N-n-octylpyridinium-2-yl)porphyrin. The reduced Mn(II) porphyrin cannot be oxidized by 7,8-BH(2) or by l-sepiapterin. The data are discussed with regard to the possible impact of the interaction of Mn(III)TE-2-PyP(5+) with BH(4) on endothelial cell proliferation and hence on tumor antiangiogenesis via inhibition of nitric oxide synthase.

Despite numerous defenses, the brain is vulnerable to oxidative stress resulting from ischemia/reperfusion. Excitotoxic stimulation of superoxide and nitric oxide production leads to formation of highly reactive products, including peroxynitrite and hydroxyl radical, which are capable of damaging lipids, proteins and DNA. Use of transgenic mutants and selective pharmacological antioxidants has greatly increased understanding of the complex interplay between substrate deprivation and ischemic outcome. Recent evidence that reactive oxygen/nitrogen species play a critical role in initiation of apoptosis, mitochondrial permeability transition and poly(ADP-ribose) polymerase activation provides additional mechanisms for oxidative damage and new targets for post-ischemic therapeutic intervention. Because oxidative stress involves multiple post-ischemic cascades leading to cell death, effective prevention/treatment of ischemic brain injury is likely to require intervention at multiple effect sites.

Three new Mn(III) porphyrin catalysts of O2.-dismutation (superoxide dismutase mimics), bearing ether oxygen atoms within their side chains, were synthesized and characterized: Mn(III) 5,10,15,20-tetrakis[N-(2-methoxyethyl)pyridinium-2-yl]porphyrin (MnTMOE-2-PyP(5+)), Mn(III)5,10,15,20-tetrakis[N-methyl-N'-(2-methoxyethyl)imidazolium-2-yl]porphyrin (MnTM,MOE-2-ImP(5+)) and Mn(III) 5,10,15,20-tetrakis[N,N'-di(2-methoxyethyl)imidazolium-2-yl]porphyrin (MnTDMOE-2-ImP(5+)). Their catalytic rate constants for O2.-dismutation (disproportionation) and the related metal-centered redox potentials vs. NHE are: log k(cat)= 8.04 (E(1/2)=+251 mV) for MnTMOE-2-PyP(5+), log k(cat)= 7.98 (E(1/2)=+356 mV) for MnTM,MOE-2-ImP(5+) and log k(cat)= 7.59 (E(1/2)=+365 mV) for MnTDMOE-2-ImP(5+). The new porphyrins were compared to the previously described SOD mimics Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP(5+)), Mn(III) 5,10,15,20-tetrakis(N-n-butylpyridinium-2-yl)porphyrin (MnTnBu-2-PyP(5+)) and Mn(III) 5,10,15,20-tetrakis(N,N'-diethylimidazolium-2-yl)porphyrin (MnTDE-2-ImP(5+)). MnTMOE-2-PyP(5+) has side chains of the same length and the same E(1/2), as MnTnBu-2-PyP(5+)(k(cat)= 7.25, E(1/2)=+ 254 mV), yet it is 6-fold more potent a catalyst of O2.-dismutation , presumably due to the presence of the ether oxygen. The log k(cat)vs. E(1/2) relationship for all Mn porphyrin-based SOD mimics thus far studied is discussed. None of the new compounds were toxic to Escherichia coli in the concentration range studied (up to 30 microM), and protected SOD-deficient E. coli in a concentration-dependent manner. At 3 microM levels, the MnTDMOE-2-ImP(5+), bearing an oxygen atom within each of the eight side chains, was the most effective and offered much higher protection than MnTE-2-PyP(5+), while MnTDE-2-ImP(5+) was of very low efficacy.

We have studied the role of three Mn(III)porphyrins differing in charge, alkyl substituent length and reactivity, on LDL exposed to low fluxes of peroxynitrite (PN) in the presence of uric acid. Mn(III)porphyrins (5 microM, MnTE-2-PyP(5+), MnTnOct-2-PyP(5+), and MnTCPP(3-)) plus uric acid (300 microM) inhibited cholesteryl ester hydroperoxide formation, changes in REM as well as spared alpha- and gamma-tocopherol. MnTnOct-2-PyP(5+), the more lipophilic compound, was the most effective in protecting LDL lipids, while MnTCPP(3-) exerted the lesser protection. Mn(III)porphyrins react fast with PN ( approximately 10(5)-10(7) M(-1) s(-1)) to yield a O=Mn(IV) complex. The stoichiometry of uric acid consumption was approximately 1.7 moles per mol of PN, in agreement with reactions with both the O=Mn(IV) complex and nitrogen dioxide. A shift from an anti- to a pro-oxidant action of the Mn(III)porphyrin was observed after uric acid was significantly consumed, supporting competition reactions between LDL targets and uric acid for the O=Mn(IV) complex. Overall, the data is consistent with the catalytic reduction of PN in a cycle that involves a one electron oxidation of Mn(III) to Mn(IV) by PN followed by the reduction back to Mn(III) by uric acid. These antioxidant effects should predominate under in vivo conditions having plasma uric acid concentration range between 150 and 500 microM.

Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t(1/2) was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O(2)/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.

The ortho, meta, and para isomers of cationic N-alkylpyridylporphyrins and their Zn(II) complexes were compared in terms of their photodynamic properties. The ortho Zn(II) complex was found to be the most efficient in causing photooxidation of NADH in vitro. In Escherichia coli, however, the para and meta isomers were better photosensitizers than their ortho analogs. The lower potency of the ortho compound in vivo seems to be due to its lower intracellular concentration. All porphyrins tested were more efficient in killing E. coli and in photooxidizing NADH than the hematoporphyrin derivative. Antibiotic resistance did not affect the photokill, which implies that the cationic N-alkylpyridylporphyrins, as their Zn(II) complexes, can be used as bactericidal agents against antibiotic-resistant strains of gram-negative bacteria.

pK(a) values for the hydroxamic acid, alpha-NH(3)(+), and epsilon-NH(3)(+) groups of L-lysinehydroxamic acid (LyHA, H(3)L(2+)) were found to be 6.87, 8.89, and 10.76, respectively, in aqueous solution (I = 0.1 M, NaClO(4)) at 25 degrees C. O,O coordination to Fe(III) by LyHA is supported by H(+) stoichiometry, UV-vis spectral shifts, and a shift in nu(CO) from 1648 to 1592 cm(-1) upon formation of mono(L-lysinehydroxamato)tetra(aquo)iron(III) (Fe(H(2)L)(H(2)O)(4)(4+)). The stepwise formation of tris(L-lysinehydroxamato)iron(III) from Fe(H(2)O)(6)(3+) and H(3)L(2+) was characterized by spectrophotometric titration, and the values for log beta(1), log beta(2), and log beta(3) are 6.80(9), 12.4(2), and 16.1(2), respectively, at 25 degrees C and I = 2.0 M (NaClO(4)). Stopped-flow spectrophotometry was used to study the proton-driven stepwise ligand dissociation kinetics of tris(L-lysinehydroxamato)iron(III) at 25 degrees C and I = 2.0 M (HClO(4)/NaClO(4)). Defining k(n) and k(-n) as the stepwise ligand dissociation and association rate constants and n as the number of bound LyHA ligands, k(3), k(-3), k(2), k(-2), k(1), and k(-1) are 3.0 x 10(4), 2.4 x 10(1), 3.9 x 10(2), 1.9 x 10(1), 1.4 x 10(-1), and 1.2 x 10(-1) M(-1) s(-1), respectively. These rate and equilibrium constants are compared with corresponding constants for Fe(III) complexes of acetohydroxamic acid (AHA) and N-methylacetohydroxamic acid (NMAHA) in the form of a linear free energy relationship. The role of electrostatics in these complexation reactions to form the highly charged Fe(LyHA)(3)(6+) species is discussed, and an interchange mechanism mediated by charge repulsion is presented. The reduction potential for tris(L-lysinehydroxamato)iron(III) is -214 mV (vs. NHE), and a comparison to other hydroxamic acid complexes of Fe(III) is made through a correlation between E(1/2) and pFe.