The Ortho Effect Makes Manganese(III)Meso-Tetrakis(N-Methylpyridinium-2-yl)Porphyrin a Powerful and Potentially Useful Superoxide Dismutase Mimic*

The ortho, meta, andpara isomers of manganese(III) 5,10,15,20-tetrakis(N-methylpyridyl)porphyrin, MnTM-2-PyP5+, MnTM-3-PyP5+, and MnTM-4-PyP5+, respectively, were analyzed in terms of their superoxide dismutase (SOD) activity in vitro and in vivo. The impact of their interaction with DNA and RNA on the SOD activity in vivo and in vitro has also been analyzed. Differences in their behavior are due to the combined steric and electrostatic factors. In vitro catalytic activities are closely related to their redox potentials. The half-wave potentials (E½) are +0.220 mV, +0.052 mV, and +0.060 Vversus normal hydrogen electrode, whereas the rates of dismutation (k cat) are 6.0 × 107, 4.1 × 106, and 3.8 × 106 m −1 s−1 for theortho, meta, and para isomers, respectively. However, the in vitro activity is not a sufficient predictor of in vivo efficacy. The ortho andmeta isomers, although of significantly different in vitro SOD activities, have fairly close in vivo SOD efficacy due to their similarly weak interactions with DNA. In contrast, due to a higher degree of interaction with DNA, thepara isomer inhibited growth of SOD-deficientEscherichia coli.


Manganese Porphyrins
The manganese complexes were prepared by metallation of the porphyrin ligands in either water or in methanol at porphyrin:manganese ratios of 1:5, 1:13, and 1:100. In both solvents and at all metal to porphyrin ratios, the same compound, as evidenced by the Soret band, was obtained. Metallation in methanol was accomplished under refluxing conditions, whereas in water it was achieved at room temperature. When the pH of the water was raised to ϳ12.3 (20,25), 3 metallation was accomplished in ϳ15 min. Routinely, a 1:20 ratio of porphyrin to metal in water at room temperature was applied. The reaction was monitored by UV-visible spectroscopy. Upon completion of metallation (pH brought to ϳ7), the solution was filtered to remove manganese hydroxo species, and the PF 6 Ϫ salt of manganese complex was precipitated by the addition of a concentrated aqueous solution of NH 4 PF 6 (11,13). The product was thoroughly washed with 2-propanol:diethyl ether ϭ 1:1 and dried in vacuo at room temperature. The compound was then dissolved in acetone, the solution was filtered, and a concentrated acetone solution of tetrabutylammonium chloride was added, until the majority of the porphyrin had precipitated in the form of its chloride salt. The precipitate was washed thoroughly with acetone and dried in vacuo at room temperature. Usually the chloride salt was again dissolved in water and precipitated as PF 6 Ϫ salt. The latter was again dissolved in acetone and reisolated as chloride salt. This procedure ensured elimination of excess of metal. The chloride salts of metalloporphyrins were analyzed in terms of elemental analysis, UV-visible spectroscopy (in the range of 0.5-50 M), and thin-layer chromatography.  Table I, and the related spectra are given in Fig. 2. We obtained ⑀(MnTM-4-PyP 5ϩ ) ϭ 130,000 cm Ϫ1 M Ϫ1 , whereas the literature value is 93,000 cm Ϫ1 M Ϫ1 (26). Also, our molar absorptivity for MnTM-2-PyPCl 5 is 129,000 cm Ϫ1 M Ϫ1 , whereas the literature value is 190,000 cm Ϫ1 M Ϫ1 (27). One explanation for this discrepancy is the inadequate procedures used previously, usually aggressive ones that afford undesired substitution of the ring or even cause its reduction (27) and eventual degradation. The efficient yet nonaggressive metallation in water should be the most appropriate choice. Exposure of porphyrin to the N,N-dimethylformamide (DMF), especially when DMF is either a metallation medium or a solvent for modification of the porphyrin under refluxing conditions, during which it undergoes decomposition (28,29), should be avoided whenever possible. We have found that both metalloporphyrin (MnTMPyP 5ϩ ) and its parent ligand (H 2 TMPyP 4ϩ ) suffer significant changes when exposed to either anhydrous DMF or DMF/H 2 O (9:1) at ϳ90°C for prolonged periods. Thus, in 48 h, the molar absorptivity of a metal-free porphyrin falls to 50% of its initial value. The same happened when metalloporphyrin was left for 48 h in DMF/H 2 O (9:1) at ϳ90°C. Under anhydrous conditions, the metalloporphyrin is more resistant. The same changes were previously observed in the case of manganese(III) 5,10,15,20-tetrakis-(4-carboxyphenyl)porphyrin (MnTBAP ϩ 5 ). The modification of the porphyrin ring, hydrogen bonding interaction of the pyrrole nitrogen hydrogen with DMF (30), dimerization, and metal-centered reduction are among possible routes that can lead to the complete destruction of the porphyrin ring. 4

Stability of the Metalloporphyrins
The stability of manganese porphyrins was studied under strongly acidic and alkaline conditions and in the presence of up to 1000-fold excess EDTA. All three porphyrins were resistant to protonation as well as to ligand exchange. Even after ϳ1 h in 36% hydrochloric acid, no demetallation was observed at ϳ6 M porphyrin concentration. A very slow demetallation was observed in 98% sulfuric acid in the case of meta compound, where ϳ50% of demetallation occurred at 6 M porphyrin in a 24-h period at room temperature. Under the same conditions, only negligible demetallation was observed in the case of para, whereas the ortho isomer appeared to be the most resistant, showing no observable demetallation in 24 h. Their stability can be ascribed to the combination of steric hindrance and electronic effects (31,32).
The stability toward H 2 O 2 was measured at 25°C with 5 M porphyrin, 5 mM H 2 O 2 in 0.05 M phosphate buffer at pH 7.8. The half-times for the oxidative degradation of the porphyrin ring were 105, 28, and 30 s for the ortho, meta, and para isomers, respectively. This observation is consistent with their redox properties, i.e. resistance toward oxidation (anodic shift of both reduction and oxidation potentials) (1-7), due to the closer position and therefore greater inductive effect of the ortho positive charges on the porphyrin ring.

SOD Activity in Vitro
Xanthine oxidase was the source of O 2 . , and ferricytochrome c was its indicating scavenger (33). Reduction of cytochrome c was followed at 550 nm. Assays were conducted in the presence and absence of 0.

The Interaction of Metalloporphyrins with Nucleic Acids
These interactions were followed by UV-visible spectroscopy, by cyclic voltammetry, through inhibition of their SOD-like activity, and by ultrafiltration.
UV-visible Spectroscopy-The interaction of metalloporphyrins with nucleic acids was performed both in the presence (27 mM) and absence of ascorbic acid in 0.05 M phosphate buffer, pH 7.8, at 6 M porphyrin, 4.7 mM DNA and RNA. The RNA stock solutions, ranging between 18 and 76 mM, were prepared in water, whereas DNA stock solutions ranging between 18 and 38 mM were prepared in buffer due to the lower DNA solubility. The concentration of nucleic acids was calculated on the basis of mononucleotide. All the experiments with ascorbate were performed anaerobically in a specially designed cuvette (35) purged with argon.
Ultrafiltration-The retention of the porphyrins by a molecular weight 3000 cut-off filter Ϯ the nucleic acids (11 and 22 mM) was determined filtering the 0.5 mM solutions of porphyrin in 0.05 M phosphate buffer, pH 7.8, 0.1 M NaCl.
SOD Assay-The assay was performed at the porphyrin concentration that caused 50% inhibition of the cytochrome c reduction (IC 50 ). At the IC 50 porphyrin concentration, its SOD activity was titrated with nucleic acid in the concentration range of 0.4 -21 M, depending upon the isomer investigated.

Electrochemical Characterization
Measurements were performed using CH Instruments (computer supported) model 600 Voltammetric Analyzer. A three-electrode setup system in a small volume cell (0.5-3 ml, with a 3 mm-diameter button glassy carbon working electrode (Bioanalytical Systems) was used. Prior to each experiment, the electrode was cleaned with 0.3 m alumina, sonicated in deionized water for 1 min, rinsed with stream of distilled water, wiped with a paper tissue, and allowed to air dry for 5 min. The reference electrode was a standard Ag/AgCl electrode (Bioanalytical Systems, 3 M NaCl gel filling solution), and the auxilliary electrode was a 0.5-mm platinum wire. Solutions containing 0.05 M phosphate buffer, pH 7.8, 0.1 M NaCl and 0.5 mM metalloporphyrin were used. The effect of nucleic acids (0.05-45 mM) on the redox properties of the metalloporphyrins was studied. Ultrapure argon (less than 1 ppm oxygen), humidified, was purged through all the solutions for 30 min. Scan rates were 10 -500 mV/s, typically 100 mV/s.

SOD Activity in Vivo
Escherichia coli strains wild type AB1157 (control strain) and SODdeficient JI132 (sodAsodB) were obtained from J. A. Imlay (36). Growth was followed in casamino acids medium and in minimal (five amino acids) medium (37). The cultures were diluted 1:200 from overnight cultures into minimal salts, 0.2% glucose, and 0.2% casamino acids and grown as described previously (37). Deionized water was used throughout. The porphyrins were added after inoculation in the range of 5-150 M. Growth was followed turbidimetrically at 700 nm. Anaerobic conditions were achieved in a Coy chamber.

RESULTS
The isomers of manganese(III) meso-tetrakis(N-methyl-pyridyl)porphyrin were prepared from their parent metal-free ligands and were characterized. The metal-free ligands were characterized as well, and their UV-visible data, given under "Materials and Methods," agree well with literature data (15). The metalloporphyrins resist strongly acidic (36% HCl) and basic conditions (1 M NaOH). The ortho isomer appeared thrice as resistant toward oxidative degradation with H 2 O 2 than the para one, which is consistent with the anodic shift of the redox potential due to the ortho effect (15)(16)(17)(18)(19)(20)(21). The spectral characteristics of all isomers are given in Table I, and their UV-visible spectra in Fig. 2, A and B. Consistent with the same ortho effect, a blue shift of the Soret band was observed in the case of the ortho isomer. The molar absorptivities of the ortho and para isomers of manganese complexes were almost equal, as are the absorptivities of their diprotonated parent ligands as reported here and elsewhere (20).
SOD Activity in Vitro-Inhibition of cytochrome c reduction by the porphyrins, when plotted as (v o /v i ) -1 versus concentration (38) yielded a straight line as shown for the ortho isomer in Fig. 3. The concentration that causes 50% of the inhibition of cytochrome c reduction by O 2 . , IC 50 (1 unit of activity) was found The activity of the compounds tested was also expressed as the percentage of the Cu,Zn-SOD activity, assuming k SOD ϭ 2 ϫ 10 9 M Ϫ1 s Ϫ1 (34,39,40) and in terms of specific activity, i.e., units of the activity per milligram of the compound. No SOD activity was observed for all three isomers of metal-free porphyrins. None of these compounds interfered with the xanthine oxidase reaction. The addition of catalase at 15 g/ml did not affect the SOD-like activity of the compounds. These data are given in Table II.
The Interaction of Metalloporphyrins with Nucleic Acids-Crude cell extract of the SOD-deficient (sodAsodB) strain, added to the assay mixture, inhibited the SOD-like activity of these compounds. The presence of nucleic acids in the cell extract is responsible for that effect. Thus removal of nucleic acids by precipitation with protamine sulfate (41) eliminated this effect of crude extract. Neither the cell extract nor the protamine sulfate interfered with xanthine oxidase reaction.
Ultrafiltration data indicated the association of manga-nese(III) porphyrins with DNA and RNA. The association constants of the manganese(III) porphyrins with nucleic acids were calculated according to Equation 1. The data are given in Table III.
Data obtained from the SOD assay, under the conditions where manganese cycles between 2ϩ and 3ϩ state, correspond to porphyrins both oxidized and reduced at the metal center. Each addition of nucleic acids decreased SOD activity by an amount that is linearly related to the concentration of bound porphyrin. This is shown in Fig. 4. The association constants (K a ) for all three isomers are presented in Table III. The K a values for the interactions of the meta isomer with DNA and RNA are essentially equal, whereas they are greater for the interaction of the ortho and para isomers with DNA than with RNA. The association constants are on average 2 orders of magnitude higher than those calculated from ultrafiltration data when manganese was in its 3ϩ state.
Spectrophotometric monitoring of the interaction of metalloporphyrin with nucleic acids was performed anaerobically in the absence and presence of ascorbic acid. (When no ascorbic acid was present, the same interaction of porphyrins with     nucleic acids aerobically and anaerobically was observed). The max and the corresponding absorbances, as well as their changes, when compared with the spectra of metalloporphyrins themselves, are given in Table IV. The optical spectra of the ortho and para isomers in the presence of ascorbic acid and nucleic acids are presented in Fig. 5, A and B, respectively. In the presence of nucleic acids, the Soret bands of the reduced porphyrins were red-shifted, the shift being 2-3-fold larger in the case of RNA. The red shifts were accompanied with hyperchromicity, which was much larger in the case of DNA (Table  IV). In the absence of ascorbic acid, only small shifts in the Soret bands were observed (Fig. 6, A and B).
Electrochemical Characterization-The metal-centered redox behavior for all three isomers was reversible. The corresponding half-wave potentials, E1 ⁄2 were calculated as the average of cathodic and anodic peaks and are given as V versus NHE (Table V) along with the corresponding currents in A. The redox potential of the para isomer agrees well with literature data (37), whereas the E1 ⁄2 of the ortho isomer is ϳ40 mV more positive than reported (21). Cyclic voltammetry was also performed in the presence of RNA and DNA. Essentially no shift in potential was seen when cyclic voltammograms of the ortho isomer in the absence and presence of RNA were compared. In all other cases, anodic shifts were observed in the presence of nucleic acids accompanied with the decrease in the currents (Table V). The magnitude of the shift and of the current decrease reflects a different type and degree of porphyrin interaction with nucleic acids. Representative voltammograms of all three isomers with and without RNA and DNA are presented in Fig. 7, A and B, respectively. SOD-like Activity in Vivo-The metal-free ligands did not show any SOD-like activity in growth experiments at the concentrations at which metalloporphyrins were potent. Moreover, slight toxicity, which was equal in the case of the meta and para isomers and less in the case of the ortho one, was detected. These toxicities may be attributed to their metal-chelating ability as well as to their interaction with nucleic acids.
The effect of the metalloporphyrins on the growth of SODdeficient and wild type strains in the casamino acids medium are shown in Fig. 8. The ortho isomer was clearly the most effective. In the minimal, five-amino acid-containing medium, the ortho isomer again appeared to be the most effective compound, meta expressed a fairly good protection, whereas the para isomer was toxic, as shown in Fig. 9. Special care must be taken to ensure that the metalloporphyrin preparations are   No effect on the growth of wild type was detected at a 25 M concentration of each isomer, but some toxicity was introduced at higher concentrations.
The SOD activity of the porphyrin-containing cell extract was studied as well. The SOD-deficient strain of E. coli JI132 was grown aerobically in the presence of a 25 M concentration of all three isomers for ϳ8 h. The crude cell extract was prepared, and the porphyrin concentration in the cell extract determined spectrally, being higher for the para isomer (10 M) and lower for the ortho (3 M) and meta (4 M) isomers. At IC 50 of all three isomers, 42% of the SOD-like activity was detected in the case of the ortho isomer, 20% in the case of the meta isomer, and only 6% in the case of the para isomer.  Table V.
a Anodic shift in the potential in the presence of nucleic acids. b The percentage of current obtained in the presence of nucleic acids as compared to the current in the absence of nucleic acids.
resulting in the changes known as the ortho effect (15)(16)(17)(18)(19)(20)(21), was advantageous for several reasons. When the center of positive charge is closer to the porphyrin ring, the redox potential at the manganese is shifted from ϩ60 mV to ϩ220 mV versus NHE ( Fig. 7 and Table V). Because this brings it closer to the value that would provide equal driving force to both half reactions of the catalytic cycle (ϳϩ300 mV) (8), it increases the catalytic effect by ϳ16-fold. Another factor that may contribute to the catalytic efficiency of the ortho isomer is more electrostatic facilitation. Furthermore, in the ortho isomer the N-methyl group is fixed in an axial position relative to the plane of the porphyrin whereas in the para it rotates freely (42)(43)(44)(45). Thus, when metal is in its 2ϩ state (no axially coordinated ligand present), the para isomer is more likely to intercalate into nucleic acids than is the ortho. In fact, several lines of evidence show that the para does interact with both DNA and RNA to a greater extent than does the ortho isomer (Tables III-V and Figs. 4, 6, and 7). The evidence is a current drop of more than 80% when cyclic voltammograms of the para isomer and para isomer in the presence of DNA were compared ( Fig. 7 and Table   V). The current drop suggests that the porphyrin is hidden inside DNA and cannot be approached by the reductant. In the case of the ortho isomer, RNA and DNA had minimal effects on E1 ⁄2 and current ( Fig. 7 and Table V). A lower level of interaction with nucleic acids should allow the ortho isomer to express more of its SOD-like activity within cells and at the same time to diminish toxicity due to interference with the functions of nucleic acids. A striking feature of these compounds is their extreme stability, which makes it likely that they will persist within cells. We have also observed that the meta isomer interacts with DNA similar to the ortho (Tables III and V and Fig.  7), as a consequence of the restricted rotation of N-methylpyridyls (42)(43)(44)(45). Consequently, it behaved as a good SOD mimic in vivo despite the ϳ16-fold less in vitro SOD-like activity (Fig. 9). Due to the reasons discussed above, the para isomer exhibits in vivo toxicity (Fig. 9). The interaction of metalloporphyrins with nucleic acids has been studied (46 -54). Those capable of axial ligation, as well as those possessing other means of steric hindrance, such as the ortho and meta isomers of both H 2 TMPyP 4ϩ and MnTMPyP 5ϩ , are restricted to external association. In contrast, the absence of axial ligation and coplanarity (metal-reduced para isomer) allows intercalation. Our data support this view and indicate that in vivo utility, as a replacement for SOD, is fostered by inability to intercalate into nucleic acids.
The results reported herein for the MnTM-2-PyP 5ϩ are for the mixture of atropoisomers, because we could not resolve them. However, the atropoisomers of ZnTM-2-PyP 4ϩ have been resolved (55). Future work will be directed at resolving the zinc compound and then replacing Zn(II) with Mn(III) in the separated atropoisomers. We are anxious to see whether the atropoisomers will differ in catalytic activity. FIG. 8. The magnitude of growth of SOD-deficient E. coli JI132 in casamino acids medium after a 4-h period, expressed as a percentage of wild type AB1157 growth versus concentrations of the ortho (circles), meta (triangles), and para (squares) isomers of MnTMPyP 5؉ . When no porphyrin was added, the SOD-deficient strain grew at 18% of the growth of wild type (diamond).
FIG. 9. The growth curves of SOD-deficient E. coli JI132 in minimal (five amino acids) medium as affected by the presence of 25 M concentrations of the ortho (circles), meta (inverted triangles), and para (squares) isomer. The growth of JI132 in the absence of porphyrin (diamonds) and the growth of wild type AB1157 (upright triangles) were followed as well.