Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Redox regulation of protein tyrosine phosphatase activity by hydroxyl radical

Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H₂O₂ include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H₂O₂ abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. Unfortunately, H₂O₂ is poorly reactive in chemical terms and the second order rate constants for the H₂O₂-mediated PTP inactivation are ~ 10 M^− 1 s^− 1, which is too slow to be compatible with the transient signaling events occurring at the physiological concentrations of H₂O₂. We find that hydroxyl radical is produced from H₂O₂ solutions in the absence of metal chelating agent by the Fenton reaction. We show that the hydroxyl radical is capable of inactivating the PTPs and the inactivation is active site directed, through oxidation of the catalytic Cys to sulfenic acid, which can be reduced by low molecular weight thiols. We also show that hydroxyl radical is a kinetically more efficient oxidant than H₂O₂ for inactivating the PTPs. The second-order rate constants for the hydroxyl radical-mediated PTP inactivation are at least 2–3 orders of magnitude higher than those mediated by H₂O₂ under the same conditions. Thus, hydroxyl radical generated in vivo may serve as a more physiologically relevant oxidizing agent for PTP inactivation. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.     

Prooxidant action of carazolol in the Fenton‐like reaction

Carazolol [4‐(2‐hydroxy‐3‐isopropyl‐amino‐propoxy)‐carbazole], a β₃‐adrenoceptor agonist, is clinically used in the treatment of hypertension, cardiac arrhythmias and angina pectoris. Despite the beneficial effect of the drug, its high dose may contribute to cardiotoxicity. This study was conducted to examine whether carazolol can influence hydroxyl radical formation by a Fenton‐like reaction [Co(II) + H₂O₂ + HO^‐] in the presence of ethylenediaminetetraacetic acid. The oxygen free radicals and singlet oxygen (¹O₂) formation was traced by three different assay methods: chemiluminescence (CL), an electron spin resonance (ESR) spin trapping with 2,2,6,6‐tetramethyl‐4‐piperidine and 5,5‐dimethyl‐1‐pyrroline‐1‐oxide, and spectrophotometric determination of ¹O₂ based on bleaching of p‐nitrosodimethylaniline. The effect of hydroxyl radical inhibitors and ¹O₂ quenchers on peroxidation of carazolol was also examined. The results indicated that carazolol enhanced the HO radical and ¹O₂ formation in a Fenton‐like reaction. Copyright © 2010 John Wiley & Sons, Ltd.     

Bicarbonate-catalyzed hydrogen peroxide oxidation of cysteine and related thiols

The effect of bicarbonate on the rates of the H₂O₂ oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pK_a values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M⁻¹ s⁻¹ for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO₂). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M⁻¹ s⁻¹. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations.     

Chlorinated and brominated phosphatidylcholines are generated under the influence of the Fenton reagent at low pH-a MALDI-TOF MS study

Lipid (phospholipid) oxidation is an increasingly important research topic due to the significant physiological relevance. The Fenton reaction, i.e. the transition metal catalyzed decomposition of H₂O₂ is frequently used to generate hydroxyl radicals (HO). Lipids with unsaturated fatty acyl residues are primarily converted by HO radicals into peroxides.In contrast, chloro- and bromohydrins as well as dihalogenides are formed by the addition of HOCl or HOBr to the olefinic groups of the fatty acyl residues of lipids or under the influence of the enzyme myeloperoxidase (MPO) from Cl⁻ and H₂O₂. We will show here by using MALDI-TOF MS for product analysis that halogenated products may also be generated in the presence of the Fenton reagent, if either FeCl₂ or FeBr₂ is used. In the presence of FeSO₄, however, peroxides are exclusively generated. It will also be shown that the generation of halogen-containing products is a competing reaction with the cleavage of the double bond under generation of the corresponding aldehyde or carboxylic acid that is favored at prolonged incubation times and at elevated pH.     

Cholesterol, not polyunsaturated fatty acids, is target molecule in oxidation induced by reactive oxygen species in membrane of human erythrocytes

The oxidation of polyunsaturated fatty acids (PUFAs) by reactive oxygen species (ROS) is linked to aging and to many diseases. We herein employ initiating peroxyl radical (ROO•) derived from the decomposition of 2,2′-azobis(2-amidinopropane dihydrochloride), hydroxyl radical generated by the Fenton reaction and peroxyl radical (ROO•) and alkoxyl radical (LO•) derived from PUFAs by addition of Cu²⁺ as ROS sources to oxidize glycerides under alkaline conditions in the presence of methanol instead of being treated traditionally by diazomethane (CH₂N₂) under acidic conditions (pH=2.0), to obtain corresponding methyl esters for the combination of gas chromatography with mass spectrometry determination. It was found that all the PUFAs in the membrane are perfectly preserved after oxidation by ROS, even though sufficient time is available for the interaction between human erythrocytes and ROS. This indicates that ROS do not damage PUFAs during reaction time. However, three products (cholesta-4,6-dien-3-ol, cholesta-4,6-dien-3-one, and cholesta-3,5-dien-7-one) are produced from the oxidation of cholesterol within this time frame. This qualitative finding, suggests that the cholesterol in the membrane of human erythrocytes is more susceptible to ROS-induced oxidation than are PUFAs, and compels us to re-evaluate the physiological roles of cholesterol and PUFAs in the human erythrocyte membrane.     

Kinetics of reaction of nitrogen dioxide with dihydrorhodamine and the reaction of the dihydrorhodamine radical with oxygen: implications for quantifying peroxynitrite formation in cells

Dihydrorhodamine 123 (RhH₂) has been used to detect ‘reactive nitrogen species’, including peroxynitrite and its radical decomposition products, peroxynitrite probably oxidizing RhH₂ to rhodamine (Rh) via radical products rather than directly. In this study, the radical intermediate (RhH) was generated by pulse radiolysis, and shown to react with oxygen with a rate constant k ∼ 7 × 10⁸ M^-1 s^-1. This fast reaction was exploited in experiments observing Rh being formed slowly (k ∼ 4-7 × 10⁵ M^-1 s^-1) from oxidation of RhH₂ by nitrogen dioxide in a rate-limiting step, >1000-fold slower than the corresponding oxidation by carbonate radicals. The time-dependent uptake of RhH₂ into mammalian cells was measured, with average intracellular levels reaching only ∼10 μM with the protocol used. The combination of low loading and relatively low reactivity of oxidants towards RhH₂ compared to competing cellular nucleophiles suggests rather a small fraction of peroxynitrite-derived radicals (mainly CO₃⁻) may be scavenged intracellularly by RhH₂.     

The effect of neighboring methionine residue on tyrosine nitration and oxidation in peptides treated with MPO, H2O2, and NO2 or peroxynitrite and bicarbonate: Role of intramolecular electron transfer mechanism?

Recent reports suggest that intramolecular electron transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H₂O₂ and NO₂⁻ and peroxynitrite (ONOO⁻) or ONOO⁻ and bicarbonate (HCO₃⁻) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analyzed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. 280 (2005) 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO⁻ (or ONOOCO₂⁻) or MPO/H₂O₂/NO₂⁻ system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO⁻ (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer in these peptides. Further support for the interpretation was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed.     

Kinetics and mechanism of the reaction of octacarbonyl dicobalt with ethyl diazoacetate

Kinetics of the reaction of octacarbonyl dicobalt with ethyl diazoacetate leading to [μ²-{ethoxycarbonyl(methylene)}-μ²-(carbonyl)-bis(tricarbonyl-cobalt)] (Co-Co) (1), dinitrogen, and carbon monoxide were investigated at 10 °C in heptane solution. The initial rate of the reaction was measured by following both the gas evolution and the decrease of the octacarbonyl dicobalt concentration. The rate is first order with respect to octacarbonyl dicobalt and a complex order with respect to ethyl diazoacetate and carbon monoxide depending on the ratio of their concentrations. This is in accord with the formation of a heptacarbonyl dicobalt reactive intermediate (k₁ (10 °C) = (1.22 ± 0.06) × 10⁻³ s⁻¹) for which carbon monoxide and ethyl diazoacetate compete (k₋₁/k₂ (10 °C) = 1.34 ± 0.07).     

Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non-fluorescent terephthalate (TA²⁻) with hydroxyl radicals or ferryl-oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2-hydroxyterephthalate (OH-TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH-TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o- phthalaldehyde, for amino acid detection, had no effect on OH-TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose-dependent manner. The presence of TA²⁻ in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ' ex vivo' showed TA²⁻ to be non-toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe²⁺ + H₂O₂) indicate a mechanism whereby TA²⁻ forms a primary complex with Fe²⁺ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.     

A study on the reaction of copper complex of dioxotetraamine with superoxide ion by spectrophotometry and pulse radiolysis

The reactions of a dioxotetraamine Cu(II) complex [Cu(H₋₂L)] (L is 6-(9-fluorenyl)-1,4,8,11-tetraazaandencane-5,7-dione)with O₂ ⁻ were investigated by electrochemistry, UV-Vis spectrophotometry and pulse radiolysis, respectively. In DMSO solution, [Cu^II(H₋₂L)] was oxidized into [Cu^III(H₋₂L)]⁺ by O₂ ⁻, a consecutive reaction was observed with [Cu^III(H₋₂L)(O₂ ²⁻)] ⁻ as intermediates (k₁=1.71×10³ M⁻¹ s⁻¹, k₂=1.2×10⁻² s⁻¹). The mechanism of O₂ ⁻ dismutation catalyzed by the complex involved alternate oxidation and reduction of Cu(II) by O₂ ⁻ and the k_cat is 6.07 × 10⁷ M⁻¹ s⁻¹ (pH 7.4).     

Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins

‘Footprinting’ describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions. The hydroxyl radical (·OH) is a particularly valuable footprinting probe by virtue of it being among the most reactive of chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved ·OH footprinting has been developed based on the Fenton reaction, Fe(II) + H₂O₂ → Fe(III) + ·OH + OH⁻. This method can be implemented in laboratories using widely available three-syringe quench flow mixers and inexpensive reagents to study local changes in the solvent accessibility of DNA, RNA and proteins associated with their biological function.     

The oxidation of aminoethylcysteine ketimine dimer by oxygen reactive species

Summary The prominent spontaneous reaction of aminoethylcysteine ketimine in the neutral pH range is the concentration-dependent dimerization (Hermann, 1961). The carboxylated dimer first produced loses the free carboxyl yielding the more stable decarboxylated dimer (named simply the dimer in this note). In the search for a possible biochemical activity of this uncommon tricyclic compound we have assayed whether it could interact with oxygen reactive species (H₂O₂, O₂ ^–,OH) thus exhibiting a scavenging effect of possible biomedical interest. The dimer interacts with H₂O₂ producing compounds detectable by chromatographic procedures. The presence of Fe²⁺ stimulates the oxidative reaction by yielding the hydroxyl radical (the Fenton reaction). Using the system xanthine oxidase-xanthine as superoxide producer, the dimer oxidation by O₂ ^– has also been documented. Among the oxidation products the presence of taurine and cysteic acid has been established. Identification of remaining oxidation products and investigation of the possible function of the dimer as a biological scavenger of oxygen reactive species are now oncoming.Abbreviations HPLC high performance liquid chromatography - AAÅ amino acid analyzer - SOD superoxide dismutase - EDTA ethylenediaminetetraacetic acid     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Evidence for a slow and oxygen-insensitive intra-molecular long range electron transfer from tyrosine residues to the semi-oxidized tryptophan 214 in human serum albumin: its inhibition by bound copper (II)

A slow, long range electron transfer (SLRET) in human serum albumin (HSA) is observed from an intact tyrosine (Tyr) residue to the neutral tryptophan (Trp) radical (Trp·) generated in pulse radiolysis. This radical is formed, at neutral pH, through oxidation with Br₂^·− radical anions of the single Trp 214 present. The SLRET rate constant of ~0.2 s⁻¹ determined is independent of HSA concentration and radiation dose, consistent with an intra-molecular process. This is the slowest rate constant so far reported for an intra-molecular LRET. In sharp contrast with the LRET reported for other proteins, the SLRET observed here is insensitive to oxygen, suggesting that the oxidized Trp is inaccessible to—or do not react with radiolytically generated O₂^·−. In N₂O-saturated solutions, the SLRET is inhibited by Cu²⁺ ions bound to the His 3 residue of the N-terminal group of HSA but it is partially restored in O₂-saturated solutions.     

Hydrogen atom abstraction from HOOOCl by chlorine atom and OH radical

Gas-phase reactions of HOOOCl with both Cl atom and OH radical are investigated using ab initio methods. The structures of all reactants, products, intermediates, and transition states have been optimized and characterized with the quadratic configuration interaction (QCISD) method. The overall mechanism for the Cl + HOOOCl and OH + HOOOCl reaction is the formation of HCl + O₂ + ClO and H₂O + O₂ + ClO, respectively. The rate-limiting step in each reaction is the abstraction of hydrogen from HOOOCl by either Cl or OH radicals and the barrier height is predicted to be 1.9 kcal mol⁻¹ and 8.1 kcal mol⁻¹ for abstraction by Cl atom and OH radical, respectively. Since both barriers for hydrogen abstraction are high, the reaction is suggested to be slow. These results also suggest that an atmospheric removal mechanism for HOOOCl may result from reaction with Cl atoms rather than with OH radicals, and that photolysis of HOOOCl may be the major removal mechanism for the intermediate.     

Butane-2,3-dionethiosemicarbazone: an oxime with antioxidant properties

Oximes are compounds generally used to reverse the acetylcholinesterase (AChE) inhibition caused by organophosphates (OPs). The aim of this study was to examine the capacity of the butane-2,3-dionethiosemicarbazone oxime to scavenge different forms of reactive species (RS) in vitro, as well as counteract their formation. The potential antioxidant and toxic activity of the oxime was assayed both in vitro and ex vivo. The obtained results indicate a significant hydrogen peroxide (H₂O₂), nitric oxide (NO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity at 0.275, 0.5 and 5 μM of oxime, respectively (p ≤ 0.05). The oxime exhibited a powerful inhibitory effect on dihydroxybenzoate formation (25 μM) (p ≤ 0.05) and also decreased deoxyribose degradation induced by Fe²⁺ and via Fenton reaction (0.44 and 0.66 mM, respectively) (p ≤ 0.05). The oxime showed a significant inhibitory effect on σ-phenantroline reaction with Fe²⁺ (0.4 mM) suggesting a possible interaction between the oxime and iron. A significant decrease in the basal and pro-oxidant-induced lipid peroxidation in brain, liver, and kidney of mice was observed both in vitro and ex vivo (p ≤ 0.05). In addition, in our ex vivo experiments the oxime did not depict any significant changes in thiol levels of liver, kidney and brain as well as did not modify the δ-aminolevulinate dehydratase (δ-ALA-D) activity in these tissues. Taken together our results indicate an in vitro and ex vivo antioxidant activity of the oxime possibly due to its scavenging activity toward different RS and a significant iron interaction.     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.     

Preparation of phthalocyanine(3−) species as bulk material by reduction of the dichloro(phthalocyaninato)antimony(V) complex cation

By reduction of dichloro(phthalocyaninato)antimony(V) complex, [Sb(pc)Cl₂]⁺ (pc²⁻=phthalocyaninate, C₃₂H₁₆N₈ ²⁻), its pc³⁻ species has been prepared as bulk material using elemental silver as the electron donor. One electron reduction of [Sb(pc)Cl₂]⁺ proceeded without any side reaction. The obtained pc³⁻ species has been characterized by its ESR, electronic absorption, infra-red, and mass spectra. The electronic absorption spectrum of the [Sb(pc³⁻)Cl₂] species was identical with that of the known spectroelectrochemically studied [Sb(pc³⁻)Cl₂]. The first infra-red spectrum of the pc³⁻ species of metallophthalocyanine is reported and indicates that the structural change upon the reduction is insignificant.