Organic isocyanates are versatile intermediates that provide access to a wide range of functionalities. In this work, we have developed the first synthetic method for preparing aliphatic isocyanates via direct C–H activation. This method proceeds efficiently at room temperature and can be applied to functionalize secondary, tertiary, and benzylic C–H bonds with good yields and functional group compatibility. Moreover, the isocyanate products can be readily converted to substituted ureas without isolation, demonstrating the synthetic potential of the method. To study the reaction mechanism, we have synthesized and characterized a rare MnIV–NCO intermediate and demonstrated its ability to transfer the isocyanate moiety to alkyl radicals. Using EPR spectroscopy, we have directly observed a MnIV intermediate under catalytic conditions. Isocyanation of celestolide with a chiral manganese salen catalyst followed by trapping with aniline afforded the urea product in 51% enantiomeric excess. This represents the only example of an asymmetric synthesis of an organic urea via C–H activation. When combined with our DFT calculations, these results clearly demonstrate that the C–NCO bond was formed through capture of a substrate radical by a MnIV–NCO intermediate.

A reactive hydroxoferric porphyrazine complex, [(PyPz)FeIII(OH) (OH2)]4+ (1, PyPz = tetramethyl-2,3-pyridino porphyrazine), has been prepared via one-electron oxidation of the corresponding ferrous species [(PyPz)FeII(OH2)2]4+ (2). Electrochemical analysis revealed a pH-dependent and remarkably high FeIII–OH/FeII–OH2 reduction potential of 680 mV vs Ag/AgCl at pH 5.2. Nernstian behavior from pH 2 to pH 8 indicates a one-proton, one-electron interconversion throughout that range. The O–H bond dissociation energy of the FeII–OH2 complex was estimated to be 84 kcal mol–1. Accordingly, 1 reacts rapidly with a panel of substrates via C–H hydrogen atom transfer (HAT), reducing 1 to [(PyPz)FeII(OH2)2]4+ (2). The second-order rate constant for the reaction of [(PyPz)FeIII(OH) (OH2)]4+ with xanthene was 2.22 × 103 M–1 s–1, 5–6 orders of magnitude faster than other reported FeIII–OH complexes and faster than many ferryl complexes.

The oxygen rebound mechanism, proposed four decades ago, is invoked in a wide range of oxygen- and heteroatom-transfer reactions. In this process, a high-valent metal-oxo species abstracts a hydrogen atom from the substrate to generate a carbon-centered radical, which immediately recombines with the hydroxometal intermediate with very fast rate constants that can be in the nanosecond to picosecond regime. In addition to catalyzing C–O bond formation, we found that manganese porphyrins can also directly catalyze C–H halogenations and pseudohalogenations, including chlorination, bromination, and fluorination as well as C–H azidation. For these cases, we showed that long-lived substrate radicals are involved, indicating that radical rebound may involve a barrier in some cases. In this study, we show that axial ligands significantly affect the oxygen rebound rate. Fluoride, hydroxide, and oxo ligands all slow down the oxygen rebound rate by factors of 10–40-fold. The oxidation of norcarane by a manganese porphyrin coordinated with fluoride or hydroxide leads to the formation of significant amounts of radical rearranged products. cis-Decalin oxidation afforded both cis- and trans-decalol. Xanthene afforded dioxygen trapped products and the radical dimer product, bixanthene, under aerobic and anaerobic conditions, respectively. DFT calculations probing the rebound step show that the rebound barrier increases significantly (by 3.3, 5.4, and 6.0 kcal/mol, respectively) with fluoride, hydroxide, and oxo as axial ligands.Keywords: DFT; heterorebound catalysis; iron porphyrin; manganese porphyrin; oxygen rebound;

Reactions that directly transform aliphatic C–H bonds into alkyl azides are noticeably lacking in the repertoire of synthetic reactions, despite the importance of molecules containing C–N3 bonds in organic synthesis, chemical biology, and drug discovery. Harnessing the ubiquity of C–H bonds in organic molecules and the versatility of the azide functional group, such transformations could have broad applications in various disciplines. Radical C–H activation represents an appealing strategy to achieve aliphatic C–H azidation, as it overcomes many drawbacks of conventional organometallic approaches in activating inert aliphatic C–H bonds. Novel C–H azidation methodologies could be realized by combining radical C–H activation via hydrogen atom abstraction with suitable azide-transfer reagents. In this perspective, we survey the history of radical C–H azidation and summarize several significant recent advances in the field. All radical C–H azidations to date follow a general approach comprising an initial radical C–H abstraction step and a subsequent azide transfer to the incipient carbon-centered radicals. A particular focus of this perspective is on the beneficial effects of using transition-metal catalysts in C–H azidation reactions, which have “tamed” azide radicals and led to reactions that proceed efficiently under much milder conditions and provide broader substrate scope and higher regioselectivities and stereoselectivities, compared to previous approaches.Keywords: azidation; catalysis; C−H activation; organic azides; radical

The remarkable aliphatic C–H hydroxylations catalyzed by the heme-containing enzyme, cytochrome P450, have attracted sustained attention for more than four decades. The effectiveness of P450 enzymes as highly selective biocatalysts for a wide range of oxygenation reactions of complex substrates has driven chemists to develop synthetic metalloporphyrin model compounds that mimic P450 reactivity. Among various known metalloporphyrins, manganese derivatives have received considerable attention since they have been shown to be versatile and powerful mediators for alkane hydroxylation and olefin epoxidation. Mechanistic studies have shown that the key intermediates of the manganese porphyrin-catalyzed oxygenation reactions include oxo- and dioxomanganese(V) species that transfer an oxygen atom to the substrate through a hydrogen abstraction/oxygen recombination pathway known as the oxygen rebound mechanism. Application of manganese porphyrins has been largely restricted to catalysis of oxygenation reactions until recently, however, due to ultrafast oxygen transfer rates.In this Account, we discuss recently developed carbon–halogen bond formation, including fluorination reactions catalyzed by manganese porphyrins and related salen species. We found that biphasic sodium hypochlorite/manganese porphyrin systems can efficiently and selectively convert even unactivated aliphatic C–H bonds to C–Cl bonds. An understanding of this novel reactivity derived from results obtained for the oxidation of the mechanistically diagnostic substrate and radical clock, norcarane. Significantly, the oxygen rebound rate in Mn-mediated hydroxylation is highly correlated with the nature of the trans-axial ligands bound to the manganese center (L–MnV═O). Based on the ability of fluoride ion to decelerate the oxygen rebound step, we envisaged that a relatively long-lived substrate radical could be trapped by a Mn–F fluorine source, effecting carbon–fluorine bond formation. Indeed, this idea led to the discovery of the first Mn-catalyzed direct aliphatic C–H fluorination reactions utilizing simple, nucleophilic fluoride salts. Mechanistic studies and DFT calculations have revealed a trans-difluoromanganese(IV) species as the key fluorine transfer intermediate. In addition to catalyzing normal 19F-fluorination reactions, manganese salen complexes were found to enable the incorporation of radioactive 18F fluorine via C–H activation. This advance represented the first direct Csp3–H bond 18F labeling with no-carrier-added [18F]fluoride and facilitated the late-stage labeling of drug molecules for PET imaging. Given the high reactivity and enzymatic-like selectively of metalloporphyrins, we envision that this new Heteroatom-Rebound Catalysis (HRC) strategy will find widespread application in the C–H functionalization arena and serve as an effective tool for forming new carbon–heteroatom bonds at otherwise inaccessible sites in target molecules.

Ferryl porphyrins, P–FeIV═O, are central reactive intermediates in the catalytic cycles of numerous heme proteins and a variety of model systems. There has been considerable interest in elucidating factors, such as terminal oxo basicity, that may control ferryl reactivity. Here, the sulfonated, water-soluble ferryl porphyrin complexes tetramesitylporphyrin, oxoFeIVTMPS (FeTMPS-II), its 2,6-dichlorophenyl analogue, oxoFeIVTDClPS (FeTDClPS-II), and two other analogues are shown to be protonated under turnover conditions to produce the corresponding bis-aqua-iron(III) porphyrin cation radicals. The results reveal a novel internal electromeric equilibrium, P–FeIV═O ⇆ P+–FeIII(OH2)2. Reversible pKa values in the range of 4–6.3 have been measured for this process by pH-jump, UV–vis spectroscopy. Ferryl protonation has important ramifications for C–H bond cleavage reactions mediated by oxoiron(IV) porphyrin cation radicals in protic media. Both solvent O–H and substrate C–H deuterium kinetic isotope effects are observed for these reactions, indicating that hydrocarbon oxidation by these oxoiron(IV) porphyrin cation radicals occurs via a solvent proton-coupled hydrogen atom transfer from the substrate that has not been previously described. The effective FeO–H bond dissociation energies for FeTMPS-II and FeTDClPS-II were estimated from similar kinetic reactivities of the corresponding oxoFeIVTMPS+ and oxoFeIVTDClPS+ species to be ∼92–94 kcal/mol. Similar values were calculated from the two-proton P+–FeIII(OH2)2 pKaobs and the porphyrin oxidation potentials, despite a 230 mV range for the iron porphyrins examined. Thus, the iron porphyrin with the lower ring oxidation potential has a compensating higher basicity of the ferryl oxygen. The solvent-derived proton adds significantly to the driving force for C–H bond scission.

We report a manganese-catalyzed aliphatic C–H azidation reaction that can efficiently convert secondary, tertiary, and benzylic C–H bonds to the corresponding azides. The method utilizes aqueous sodium azide solution as the azide source and can be performed under air. Besides its operational simplicity, the potential of this method for late-stage functionalization has been demonstrated by successful azidation of various bioactive molecules with yields up to 74%, including the important drugs pregabalin, memantine, and the antimalarial artemisinin. Azidation of celestolide with a chiral manganese salen catalyst afforded the azide product in 70% ee, representing a Mn-catalyzed enantioselective aliphatic C–H azidation reaction. Considering the versatile roles of organic azides in modern chemistry and the ubiquity of aliphatic C–H bonds in organic molecules, we envision that this Mn-azidation method will find wide application in organic synthesis, drug discovery, and chemical biology.

The efficient and selective partial oxidation of light alkanes using potassium periodate and potassium chloride is reported. Yields of methane functionalization in trifluoroacetic acid reach >40% with high selectivity for methyl trifluoroacetate. Periodate and chloride also functionalize ethane and propane in good yields (>20%).

A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapid-mixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pH-jump spectrophotometry revealed a basic pKa of 10.0 for the FeIV−O−H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C−H and phenolic substrates, with second-order rate constants for C−H and O−H bond scission in the range of 10–107 M−1⋅s−1. Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C−H and O−H bond scission.

We describe an efficient system for the direct partial oxidation of methane, ethane, and propane using iodate salts with catalytic amounts of chloride in protic solvents. In HTFA (TFA = trifluoroacetate), >20% methane conversion with >85% selectivity for MeTFA have been achieved. The addition of substoichiometric amounts of chloride is essential, and for methane the conversion increases from <1% in the absence of chloride to >20%. The reaction also proceeds in aqueous HTFA as well as acetic acid to afford methyl acetate. 13C labeling experiments showed that less than 2% of methane is overoxidized to 13CO2 at 15% conversion of 13CH4. The system is selective for higher alkanes: 30% ethane conversion with 98% selectivity for EtTFA and 19% propane conversion that is selective for mixtures of the mono- and difunctionalized TFA esters. Studies of methane conversion using a series of iodine-based reagents [I2, ICl, ICl3, I(TFA)3, I2O4, I2O5, (IO2)2S2O7, (IO)2SO4] indicated that the chloride enhancement is not limited to iodate.

We describe the first late-stage 18F labeling chemistry for aliphatic C–H bonds with no-carrier-added [18F]fluoride. The method uses Mn(salen)OTs as an F-transfer catalyst and enables the facile labeling of a variety of bioactive molecules and building blocks with radiochemical yields (RCY) ranging from 20% to 72% within 10 min without the need for preactivation of the labeling precursor. Notably, the catalyst itself can directly elute [18F]fluoride from an ion exchange cartridge with over 90% efficiency. Using this feature, the conventional and laborious dry-down step prior to reaction is circumvented, greatly simplifying the mechanics of this protocol and shortening the time for automated synthesis. Eight drug molecules, including COX, ACE, MAO, and PDE inhibitors, have been successfully [18F]-labeled in this way.

Net reductive elimination (RE) of MeX (X = halide or pseudo-halide: Cl−, CF3CO2−, HSO4−, OH−) is an important step during Pt-catalyzed hydrocarbon functionalization. Developing Rh(I/III)-based catalysts for alkane functionalization is an attractive alternative to Pt-based systems, but very few examples of RE of alkyl halides and/or pseudo-halides from RhIII complexes have been reported. Here, we compare the influence of the ligand donor strength on the thermodynamic potentials for oxidative addition and reductive functionalization using [tBu3terpy]RhCl (1) {tBu3terpy = 4,4′,4′′-tri-tert-butylpyridine} and [(NO2)3terpy]RhCl (2) {(NO2)3terpy = 4,4′,4′′-trinitroterpyridine}. Complex 1 oxidatively adds MeX {X = I−, Cl−, CF3CO2− (TFA−)} to afford [tBu3terpy]RhMe(Cl)(X) {X = I− (3), Cl− (4), TFA− (5)}. By having three electron-withdrawing NO2 groups, complex 2 does not react with MeCl or MeTFA, but reacts with MeI to yield [(NO2)3terpy]RhMe(Cl)(I) (6). Heating 6 expels MeCl along with a small quantity of MeI. Repeating this experiment but with excess [Bu4N]Cl exclusively yields MeCl, while adding [Bu4N]TFA yields a mixture of MeTFA and MeCl. In contrast, 3 does not reductively eliminate MeX under similar conditions. DFT calculations successfully predict the reaction outcome by complexes 1 and 2. Calorimetric measurements of [tBu3terpy]RhI (7) and [tBu3terpy]RhMe(I)2 (8) were used to corroborate computational models. Finally, the mechanism of MeCl RE from 6 was investigated via DFT calculations, which supports a nucleophilic attack by either I− or Cl− on the Rh–CH3 bond of a five-coordinate Rh complex.

A series of cationic cobalt porphyrins was found to catalyze electrochemical water oxidation to O2 efficiently at room temperature in neutral aqueous solution. Co–5,10,15,20-tetrakis-(1,3-dimethylimidazolium-2-yl)porphyrin, with a highly electron-deficient meso-dimethylimidazolium porphyrin, was the most effective catalyst. The O2 formation rate was 170 nmol⋅cm−2⋅min−1 (kobs = 1.4 × 103 s−1) with a Faradaic efficiency near 90%. Mechanistic investigations indicate the generation of a CoIV-O porphyrin cation radical as the reactive oxidant, which has accumulated two oxidizing equivalents above the CoIII resting state of the catalyst. The buffer base in solution was shown to play several critical roles during the catalysis by facilitating both redox-coupled proton transfer processes leading to the reactive oxidant and subsequent O–O bond formation. More basic buffer anions led to lower catalytic onset potentials, extending below 1 V. This homogeneous cobalt-porphyrin system was shown to be robust under active catalytic conditions, showing negligible decomposition over hours of operation. Added EDTA or ion exchange resin caused no catalyst poisoning, indicating that cobalt ions were not released from the porphyrin macrocycle during catalysis. Likewise, surface analysis by energy dispersive X-ray spectroscopy of the working electrodes showed no deposition of heterogeneous cobalt films. Taken together, the results indicate that Co–5,10,15,20-tetrakis-(1,3-dimethylimidazolium-2-yl)porphyrin is an efficient, homogeneous, single-site water oxidation catalyst.

The release of cytochrome c from mitochondria is a key signaling mechanism in apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanisms remain unclear. Cytochrome c (cyt c) binds to and penetrates lipid structures containing the inner mitochondrial membrane lipid cardiolipin (CL), leading to protein conformational changes and increased peroxidase activity. We describe here a direct visualization of a fluorescent cyt c crossing synthetic, CL-containing membranes in the absence of other proteins. We observed strong binding of cyt c to CL in phospholipid vesicles and bursts of cyt c leakage across the membrane. Passive fluorescent markers such as carboxyfluorescein and a 10-kDa dextran polymer crossed the membrane simultaneously with cyt c, although larger dextrans did not. The data show that these bursts result from the opening of lipid pores formed by the cyt c–CL conjugate. Pore formation and cyt c leakage were significantly reduced in the presence of ATP. We suggest a model, consistent with these findings, in which the formation of toroidal lipid pores is driven by initial cyt c-induced negative spontaneous membrane curvature and subsequent protein unfolding interactions. Our results suggest that the CL–cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis.

Flavins and related molecules catalyze organic Baeyer–Villiger reactions. Combined experimental and DFT studies indicate that these molecules also catalyze the insertion of oxygen into metal–carbon bonds through a Baeyer–Villiger-like transition state.

The extracellular heme-thiolate peroxygenase from Agrocybe aegerita (AaeAPO) has been shown to hydroxylate alkanes and numerous other substrates using hydrogen peroxide as the terminal oxidant. We describe the kinetics of formation and decomposition of AaeAPO compound I upon its reaction with mCPBA. The UV–vis spectral features of AaeAPO-I (361, 694 nm) are similar to those of chloroperoxidase-I and the recently described cytochrome P450-I. The second-order rate constant for AaeAPO-I formation was 1.0 (±0.4) × 107 M–1 s–1 at pH 5.0, 4 °C. The relatively slow decomposition rate, 1.4 (±0.03) s–1, allowed the measurement of its reactivity toward a panel of substrates. The observed rate constants, k2′, spanned 5 orders of magnitude and correlated linearly with bond dissociation enthalpies (BDEs) of strong C–H bond substrates with a log k2′ vs BDE slope of ∼0.4. However, the hydroxylation rate was insensitive to a C–H BDE below 90 kcal/mol, similar to the behavior of the tert-butoxyl radical. The shape and slope of the Brønsted–Evans–Polanyi plot indicate a symmetrical transition state for the stronger C–H bonds and suggest entropy control of the rate in an early transition state for weaker C–H bonds. The AaeAPO-II FeIVO–H BDE was estimated to be ∼103 kcal/mol. All results support the formation of a highly reactive AaeAPO oxoiron(IV) porphyrin radical cation intermediate that is the active oxygen species in these hydroxylation reactions.

A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C–H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both the C–H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C–H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C–H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

Chlorine dioxide, an industrially important biocide and bleach, is produced rapidly and efficiently from chlorite ion in the presence of water-soluble, manganese porphyrins and porphyrazines at neutral pH under mild conditions. The electron-deficient manganese(III) tetra-(N,N-dimethyl)imidazolium porphyrin (MnTDMImP), tetra-(N,N-dimethyl)benzimidazolium (MnTDMBImP) porphyrin, and manganese(III) tetra-N-methyl-2,3-pyridinoporphyrazine (MnTM23PyPz) were found to be the most efficient catalysts for this process. The more typical manganese tetra-4-N-methylpyridiumporphyrin (Mn-4-TMPyP) was much less effective. Rates for the best catalysts were in the range of 0.24–32 TO/s with MnTM23PyPz being the fastest. The kinetics of reactions of the various ClOx species (e.g., chlorite ion, hypochlorous acid, and chlorine dioxide) with authentic oxomanganese(IV) and dioxomanganese(V)MnTDMImP intermediates were studied by stopped-flow spectroscopy. Rate-limiting oxidation of the manganese(III) catalyst by chlorite ion via oxygen atom transfer is proposed to afford a trans-dioxomanganese(V) intermediate. Both trans-dioxomanganese(V)TDMImP and oxoaqua-manganese(IV)TDMImP oxidize chlorite ion by 1-electron, generating the product chlorine dioxide with bimolecular rate constants of 6.30 × 103 M–1 s–1 and 3.13 × 103 M–1 s–1, respectively, at pH 6.8. Chlorine dioxide was able to oxidize manganese(III)TDMImP to oxomanganese(IV) at a similar rate, establishing a redox steady-state equilibrium under turnover conditions. Hypochlorous acid (HOCl) produced during turnover was found to rapidly and reversibly react with manganese(III)TDMImP to give dioxoMn(V)TDMImP and chloride ion. The measured equilibrium constant for this reaction (Keq = 2.2 at pH 5.1) afforded a value for the oxoMn(V)/Mn(III) redox couple under catalytic conditions (E′ = 1.35 V vs NHE). In subsequent processes, chlorine dioxide reacts with both oxomanganese(V) and oxomanganese(IV)TDMImP to afford chlorate ion. Kinetic simulations of the proposed mechanism using experimentally measured rate constants were in agreement with observed chlorine dioxide growth and decay curves, measured chlorate yields, and the oxoMn(IV)/Mn(III) redox potential (1.03 V vs NHE). This acid-free catalysis could form the basis for a new process to make ClO2.

We present a novel platform for investigating the composition-specific interactions of proteins (or other biologically relevant molecules) with model membranes composed of compositionally distinct domains. We focus on the interaction between a mitochondrial-specific lipid, cardiolipin (CL), and a peripheral membrane protein, cytochrome c (cyt c). We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and the lipid of interest, CL, preferentially localizes into one of the domains (the liquid disordered (Ld) phase). The presence of CL-rich and CL-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to CL-rich domains and observe fascinating morphological transitions within these regions of membrane. CL-rich domains start to form small buds and eventually fold up into a collapsed state. We also observe that cyt c can induce a strong attraction between the CL-rich domains of adjacent vesicles as demonstrated by the development of large osculating regions between these domains. Qualitatively similar behavior is observed when other polycationic proteins or polymers of a similar size and net charge are used instead of cyt c. We argue that these striking phenomena can be simply understood by consideration of colloidal forces between the protein and the membrane. We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria.

Protein tyrosine nitration has been observed in a variety of human diseases associated with oxidative stress, such as inflammatory, neurodegenerative, and cardiovascular conditions. However, the pathways leading to nitration of tyrosine residues are still unclear. Recent studies have shown that peroxynitrite (PN), produced by the reaction of superoxide and nitric oxide, can lead to protein nitration and inactivation. Tyrosine nitration may also be mediated by nitrogen dioxide produced by the oxidation of nitrite by peroxidases. Manganese superoxide dismutase (MnSOD), which plays a critical role in cellular defense against oxidative stress by decomposing superoxide within mitochondria, is nitrated and inactivated under pathological conditions. In this study, MnSOD is shown to catalyze PN-mediated self-nitration. Direct, spectroscopic observation of the kinetics of PN decay and nitrotyrosine formation (kcat = 9.3 × 102 M−1 s−1) indicates that the mechanism involves redox cycling between Mn2+ and Mn3+, similar to that observed with superoxide. Distinctive patterns of tyrosine nitration within MnSOD by various reagents were revealed and quantified by MS/MS analysis of MnSOD trypsin digest peptides. These analyses showed that three of the seven tyrosine residues of MnSOD (Tyr34, Tyr9, and Tyr11) were the most susceptible to nitration and that the relative amounts of nitration of these residues varied widely depending upon the nature of the nitrating agent. Notably, nitration mediated by PN, in both the presence and absence of CO2, resulted in nitration of the active site tyrosine, Tyr34, while nitration by freely diffusing nitrogen dioxide led to surface nitration at Tyr9 and Tyr11. Flux analysis of the nitration of Tyr34 by PN−CO2 showed that the nitration rate coincided with the kinetics of the reaction of PN with CO2. These kinetics and the 20-fold increase in the efficiency of tyrosine nitration in the presence of CO2 suggest a specific role for the carbonate radical anion (•CO3−) in MnSOD nitration by PN. We also observed that the nitration of Tyr34 caused inactivation of the enzyme, while nitration of Tyr9 and Tyr11 did not interfere with the superoxide dismutase activity. The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3−.

We report a manganese porphyrin mediated aliphatic C−H bond chlorination using sodium hypochlorite as the chlorine source. In the presence of catalytic amounts of phase transfer catalyst and manganese porphyrin Mn(TPP)Cl 1, reaction of sodium hypochlorite with different unactivated alkanes afforded alkyl chlorides as the major products with only trace amounts of oxygenation products. Substrates with strong C−H bonds, such as neopentane (BDE =∼100 kcal/mol) can be also chlorinated with moderate yield. Chlorination of a diagnostic substrate, norcarane, afforded rearranged products indicating a long-lived carbon radical intermediate. Moreover, regioselective chlorination was achieved by using a hindered catalyst, Mn(TMP)Cl, 2. Chlorination of trans-decalin with 2 provided 95% selectivity for methylene-chlorinated products as well as a preference for the C2 position. This novel chlorination system was also applied to complex substrates. With 5α-cholestane as the substrate, we observed chlorination only at the C2 and C3 positions in a net 55% yield, corresponding to the least sterically hindered methylene positions in the A-ring. Similarly, chlorination of sclareolide afforded the equatorial C2 chloride in a 42% isolated yield. Regarding the mechanism, reaction of sodium hypochlorite with the MnIII porphyrin is expected to afford a reactive MnV═O complex that abstracts a hydrogen atom from the substrate, resulting in a free alkyl radical and a MnIV—OH complex. We suggest that this carbon radical then reacts with a MnIV—OCl species, providing the alkyl chloride and regenerating the reactive MnV═O complex. The regioselectivity and the preference for CH2 groups can be attributed to nonbonded interactions between the alkyl groups on the substrates and the aryl groups of the manganese porphyrin. The results are indicative of a bent [Mnv═O---H---C] geometry due to the C—H approach to the Mnv═O (dπ−pπ)* frontier orbital.

A water-soluble manganese porphyrin, 5,10,15,20-tetrakis-(1,3-dimethylimidazolium-2-yl)porphyrinatomanganese(III) (MnIIITDMImP) is shown to react with H2O2 to generate a relatively stable dioxomanganese(V) porphyrin complex (a compound I analog). Stopped-flow kinetic studies revealed Michaelis Menton-type saturation kinetics for H2O2. The visible spectrum of a compound 0 type intermediate, assigned as MnIII(OH)(OOH)TDMImP, can be directly observed under saturating H2O2 conditions (Soret band at 428 nm and Q bands at 545 and 578 nm). The rate-determining O−O heterolysis step was found to have a very small activation enthalpy (ΔH≠ = 4.2 ± 0.2 kcal mol−1) and a large, negative activation entropy (ΔS≠ = −36 ± 1 cal mol−1 K−1). The O−O bond cleavage reaction was pH independent at 8.8 < pH < 10.4 with a first-order rate constant of 66 ± 12 s−1. These observations indicate that the O−O bond in MnIII(OH)(OOH)TDMImP is cleaved via a concerted “push−pull” mechanism. In the transition state, the axial (proximal) −OH is partially deprotonated (“push”), while the terminal oxygen in −OOH is partially protonated (“pull”) as a water molecule is released to the medium. This mechanism is reminiscent of O−O bond cleavage in heme enzymes, such as peroxidases and cytochrome P450, and similar to the fast, reversible O−Br bond breaking and forming reaction mediated by similar manganese porphyrins. The small enthalpy of activation suggests that this O−O bond cleavage could also be made reversible.

Oxygenated heme proteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. PN is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN interactions with heme proteins. In this Forum, we survey the range of reactions of PN with heme proteins, with particular attention to myoglobin and cytochrome c. While these two proteins are textbook paradigms for oxygen binding and electron transfer, respectively, both have recently been shown to have other important functions that involve NO and PN. We have recently described direct evidence that ferrylmyolgobin (ferrylMb) and nitrogen dioxide (NO2) are both produced during the reaction of PN and metmyolgobin (metMb) (Su, J.; Groves, J. T. J. Am. Chem. Soc. 2009, 131, 12979−12988). Kinetic evidence indicates that these products evolve from the initial formation of a caged radical intermediate [FeIV═O·NO2]. This caged pair reacts mainly via internal return with a rate constant kr to form metMb and nitrate in an oxygen-rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, kobs, of heme-mediated PN decomposition. Freely diffusing NO2, which is liberated concomitantly from the radical pair (ke), preferentially nitrates myoglobin Tyr103 and added fluorescein. For cytochrome c, Raman spectroscopy has revealed that a substantial fraction of cytochrome c converts to a β-sheet structure, at the expense of turns and helices at low pH (Balakrishnan, G.; Hu, Y.; Oyerinde, O. F.; Su, J.; Groves, J. T.; Spiro, T. G. J. Am. Chem. Soc., 2007, 129, 504−505). It is proposed that a short β-sheet segment, comprising residues 37−39 and 58−61, extends itself into the large 37−61 loop when the latter is destabilized by protonation of H26, which forms an anchoring hydrogen bond to loop residue P44. This conformation change ruptures the Met80−Fe bond, as revealed by changes in ligation-sensitive Raman bands. It also induces peroxidase activity with the same temperature profile. This process is suggested to model the apoptotic peroxidation of cardiolipin by cytochrome c.

The detection and kinetic characterization of a cytochrome P450 model compound I, [OFeIV−4-TMPyP]+ (1), in aqueous solution shows extraordinary reaction rates for C−H hydroxylations. Stopped-flow spectrophotometric monitoring of the oxidation of FeIII−4-TMPyP with mCPBA revealed the intermediate 1, which displays a weak, blue-shifted Soret band at 402 nm and an absorbance at 673 nm, typical of a porphyrin π-radical cation. This intermediate was subsequently transformed into the well-characterized OFeIV−4-TMPyP. Global analysis afforded a second-order rate constant k1 = (1.59 ± 0.06) × 107 M−1 s−1 for the formation of 1 followed by a first-order decay with k2 = 8.8 ± 0.1 s−1. 1H and 13C NMR determined 9-xanthydrol to be the major product (∼90% yield) of xanthene oxidation by 1. Electrospray ionization mass spectrometry carried out in 47.5% 18OH2 indicated 21% 18O incorporation, consistent with an oxygen-rebound reaction scenario. Xanthene/xanthene-d2 revealed a modest kinetic isotope effect, kH/kD = 2.1. Xanthene hydroxylation by 1 occurred with a very large second-order rate constant k3 = (3.6 ± 0.3) × 106 M−1 s−1. Similar reactions of fluorene-4-carboxylic acid and 4-isopropyl- and 4-ethylbenzoic acid also gave high rates for C−H hydroxylation that correlated well with the scissile C−H bond energy, indicating a homolytic hydrogen abstraction transition state. Mapping the observed rate constants for C−H bond cleavage onto the Brønsted−Evans−Polanyi relationship for similar substrates determined the H−OFeIV−4-TMPyP bond dissociation energy to be ∼100 kcal/mol. The high kinetic reactivity observed for 1 is suggested to result from a high porphyrin redox potential and spin-state-crossing phenomena. More generally, subtle charge modulation at the active site may result in high reactivity of a cytochrome P450 compound I.

Mechanistically informative chemical probes are used to characterize the activity of functional alkane hydroxylases in whole cells. Norcarane is a substrate used to reveal the lifetime of radical intermediates formed during alkane oxidation. Results from oxidations of this probe with organisms that contain the two most prevalent medium-chain-length alkane-oxidizing metalloenzymes, alkane ω-monooxygenase (AlkB) and cytochrome P450 (CYP), are reported. The results—radical lifetimes of 1–7 ns for AlkB and less than 100 ps for CYP—indicate that these two classes of enzymes are mechanistically distinguishable and that whole-cell mechanistic assays can identify the active hydroxylase. The oxidation of norcarane by several recently isolated strains (Hydrocarboniphaga effusa AP103, rJ4, and rJ5, whose alkane-oxidizing enzymes have not yet been identified) is also reported. Radical lifetimes of 1–3 ns are observed, consistent with these organisms containing an AlkB-like enzyme and inconsistent with their employing a CYP-like enzyme for growth on hydrocarbons.

The manganese meso-dimethylimidazolium porphyrin complex Mn(III)[TDMImP] reacted with HOBr/OBr− to generate the corresponding oxo-Mn(V)[TDMImP] species. The rate of this process accelerated with increasing pH. A forward rate constant, kfor, of 1.65 × 106 M−1 s−1 was determined at pH 8. Under these conditions, the oxo-Mn(V) species is short-lived and is transformed into the corresponding oxo-Mn(IV) complex. A first-order rate constant, kobs, of 0.66 s−1 was found for this reduction process at pH 8. The mechanism of this reduction process, which was dependent on bromide ion, appeared to proceed via an intermediate Mn(III)–O–Br complex. Thus, both a fast, reversible Mn(III)–O–Br bond heterolysis and a slower homolytic pathway occur in parallel in this system. The reverse oxidation reaction between oxo-Mn(V)[TDMImP] and bromide was investigated as a function of pH. The rate of this oxo-transfer reaction (krev = 1.4 × 103 M−1 s−1 at pH 8) markedly accelerated as the pH was lowered. The observed first-order dependence of the rate on [H+] indicates that the reactive species responsible for bromide oxidation is a protonated oxo-hydroxo complex and the stable species present in solution at high pH is dioxo-Mn(V)[TDMImP], [OMn(V)O]−. The oxo-Mn(V) species retains nearly all of the oxidative driving force of the hypohalite. The equilibrium constant Kequi = kfor/krev for the reversible process was determined at three different pH values (Kequi = 1.15 × 103 at pH 8) allowing the measurement of the redox potentials E of oxo-Mn(V)/Mn(III) (E = 1.01 V at pH 8). The redox potential for this couple was extrapolated over the entire pH scale using the Nernst relationship and compared to those of the manganese 2- and 4-meso-N-methylpyridinium porphyrin couples oxo-Mn(V)[2-TMPyP]/Mn(III)[2-TMPyP], oxo-Mn(V)[4-TMPyP]/Mn(III)[4-TMPyP], OBr−/Br− and H2O2/H2O. Notably, the redox potential of oxo-Mn(V)/Mn(III) for the imidazolium porphyrin approaches that of H2O2/H2O at low pH.

A new and remarkably facile sp3-C–O bond forming reaction of β-hydroxyalkyl Rh porphyrins to form epoxides has been discovered and its mechanism investigated.

The bioinorganic chemistry of iron is central to life processes. Organisms must recruit iron from their environment, control iron storage and trafficking within cells, assemble the complex, iron-containing redox cofactors of metalloproteins, and manage a myriad of biochemical transformations by those enzymes. The coordination chemistry and the variable oxidation states of iron provide the essential mechanistic machinery of this metabolism. Our current understanding of several aspects of the chemistry of iron in biology are discussed with an emphasis on the oxygen activation and transfer reactions mediated by heme and nonheme iron proteins and the interactions of amphiphilic iron siderophores with lipid membranes.

A variety of naturally occurring biomaterials owe their unusual structural and mechanical properties to layers of β-sheet proteins laminated between layers of inorganic mineral. To explore the possibility of fabricating novel two-dimensional protein layers, we studied the self-assembly properties of de novo proteins from a designed combinatorial library. Each protein in the library has a distinct 63 amino acid sequence, yet they all share an identical binary pattern of polar and nonpolar residues, which was designed to favor the formation of six-stranded amphiphilic β-sheets. Characterization of proteins isolated from the library demonstrates that (i) they self assemble into monolayers at an air/water interface; (ii) the monolayers are dominated by β-sheet secondary structure, as shown by both circular dichroism and infrared spectroscopies; and (iii) the measured areas (500- 600 Å2) of individual protein molecules in the monolayers match those expected for proteins folded into amphiphilic β-sheets. The finding that similar structures are formed by distinctly different protein sequences suggests that assembly into β-sheet monolayers can be encoded by binary patterning of polar and nonpolar amino acids. Moreover, because the designed binary pattern is compatible with a wide variety of different sequences, it may be possible to fabricate β-sheet monolayers by using combinations of side chains that are explicitly designed to favor particular applications of novel biomaterials.

The diagnostic substrate tetramethylcyclopropane (TMCP) has been reexamined as a substrate with three drug- and xenobiotic-metabolizing cytochrome P450 enzymes, human CYP2E1, CYP3A4 and rat CYP2B1. The major hydroxylation product in all cases was the unrearranged primary alcohol along with smaller amounts of a rearranged tertiary alcohol. Significantly, another ring-opened product, diacetone alcohol, was also observed. With CYP2E1 this product accounted for 20% of the total turnover. Diacetone alcohol also was detected as a product from TMCP with a biomimetic model catalyst, FeTMPyP, but not with a ruthenium porphyrin catalyst. Lifetimes of the intermediate radicals were determined from the ratios of rearranged and unrearranged products to be 120, 13 and 1 ps for CYP2E1, CYP3A4 and CYP2B1, respectively, corresponding to rebound rates of 0.9 × 1010 s−1, 7.2 × 1010 s−1 and 1.0 × 1012 s−1. For the model iron porphyrin, FeTMPyP, a radical lifetime of 81 ps and a rebound rate of 1.2 × 1010 s−1 were determined. These apparent radical lifetimes are consistent with earlier reports with a variety of CYP enzymes and radical clock substrates, however, the large amounts of diacetone alcohol with CYP2E1 and the iron porphyrin suggest that for these systems a considerable amount of the intermediate carbon radical is trapped by molecular oxygen. These results add to the view that cage escape of the intermediate carbon radical in [FeIV–OH R] can compete with cage collapse to form a C–O bond. The results could be significant with regard to our understanding of iron-catalyzed C–H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1.Graphical abstractDownload high-res image (119KB)Download full-size imageResearch highlights► Tetramethylcyclopropane reveals a range of radical lifetimes for three CYP enzymes. ► A model iron porphyrin/Ar–IO system gave similar results. ► Diacetone alcohol is a significant product for CYP2E1 and the model system.

Net reductive elimination (RE) of MeX (X = halide or pseudo-halide: Cl−, CF3CO2−, HSO4−, OH−) is an important step during Pt-catalyzed hydrocarbon functionalization. Developing Rh(I/III)-based catalysts for alkane functionalization is an attractive alternative to Pt-based systems, but very few examples of RE of alkyl halides and/or pseudo-halides from RhIII complexes have been reported. Here, we compare the influence of the ligand donor strength on the thermodynamic potentials for oxidative addition and reductive functionalization using [tBu3terpy]RhCl (1) {tBu3terpy = 4,4′,4′′-tri-tert-butylpyridine} and [(NO2)3terpy]RhCl (2) {(NO2)3terpy = 4,4′,4′′-trinitroterpyridine}. Complex 1 oxidatively adds MeX {X = I−, Cl−, CF3CO2− (TFA−)} to afford [tBu3terpy]RhMe(Cl)(X) {X = I− (3), Cl− (4), TFA− (5)}. By having three electron-withdrawing NO2 groups, complex 2 does not react with MeCl or MeTFA, but reacts with MeI to yield [(NO2)3terpy]RhMe(Cl)(I) (6). Heating 6 expels MeCl along with a small quantity of MeI. Repeating this experiment but with excess [Bu4N]Cl exclusively yields MeCl, while adding [Bu4N]TFA yields a mixture of MeTFA and MeCl. In contrast, 3 does not reductively eliminate MeX under similar conditions. DFT calculations successfully predict the reaction outcome by complexes 1 and 2. Calorimetric measurements of [tBu3terpy]RhI (7) and [tBu3terpy]RhMe(I)2 (8) were used to corroborate computational models. Finally, the mechanism of MeCl RE from 6 was investigated via DFT calculations, which supports a nucleophilic attack by either I− or Cl− on the Rh–CH3 bond of a five-coordinate Rh complex.

The efficient and selective partial oxidation of light alkanes using potassium periodate and potassium chloride is reported. Yields of methane functionalization in trifluoroacetic acid reach >40% with high selectivity for methyl trifluoroacetate. Periodate and chloride also functionalize ethane and propane in good yields (>20%).