Stereoelectronic Insight Into Heme Carbene Catalysis: Bridging Enzymatic and Synthetic Systems.

Iron porphyrin carbenes (IPCs), pivotal intermediates in enzymatic carbene transfer reactions, adopt diverse bonding modes and electronic configurations that govern their catalytic functions. This review systematically compares three IPC isomers—terminal carbenes, bridged carbenes, and N‐alkyl derivatives—isolated from enzymatic systems with synthetic analogues. X‐ray crystal structure analysis, which reveals protein‐induced structural adaptations and isomer‐specific distortions, also facilitates Mössbauer and electron paramagnetic resonance (EPR) spectroscopic studies. Besides ground state assignments on the bridged carbene [Fe(TPP){C═C(p‐ClPh)2}C1] ((dxy)2(dxz,dyz)2(dz2)1) and the N‐alkyl Mb(N‐CH2COBzyl)(His) ((dxy)2(dxz)1(dyz)1(dz2)1(dx2−y2)1), Mössbauer studies also reveal that alongside carbene fragment migration, the bridged carbene, which show shorter Fe─C(R) distance than the σ type Fe─C(sp2/sp3) bond, retains strong π acceptor capability, in contrast to the stronger σ donor and π acceptor capability of the terminal carbene and the N‐alky derivative which nearly loses the bonding to iron. Accordingly, very large tetragonalities (|Δ/λ| > 16) are observed for the bridged carbene Mb(CHCO2Et)(NMH) ((dxy)2(dxz,dyz)3) which exhibited rhombic EPR pattern under the near parallel ligand orientation. This work establishes critical connections between synthetic porphyrin and enzymatic systems, highlighting the adjustable scaffolds of engineered proteins in stabilizing various intermediates and advancing the mechanistic understanding of carbene transfer reactions.

In the context of homogeneous catalysis, open-shell systems are often quite challenging to characterize. Nuclear magnetic resonance (NMR) spectroscopy is the most frequently applied tool to characterize organometallic compounds, but NMR spectra are usually broad, difficult to interpret and often futile for the study of paramagnetic compounds. As such, electron paramagnetic resonance (EPR) has proven itself as a useful spectroscopic technique to characterize paramagnetic complexes and reactive intermediates. EPR spectroscopy is a particularly useful tool to investigate their electronic structures, which is fundamental to understand their reactivity. This paper describes some selected examples of studies where EPR spectroscopy has been useful for the characterization of open-shell organometallic complexes. The paper concentrates in particular on systems where EPR spectroscopy has proven useful to understand catalytic reaction mechanisms involving paramagnetic organometallic catalysts. The expediency of EPR spectroscopy in the study of organometallic chemistry and homogenous catalysis is contextualized in the introductory Sect. 1. Section 2 of the review focusses on examples of C–C and C–N bond formation reactions, with an emphasis on catalytic reactions where ligand/substrate non-innocence plays an important role. Both carbon and nitrogen centered radicals have been shown to play an important role in these reactions. A few selected examples of catalytic alcohol oxidation proceeding via related N-centered ligand radicals are included in this section as well. Section 3 covers examples of the use of EPR spectroscopy to study important commercial ethylene oligomerization and polymerization processes. In Sect. 4 the use of EPR spectroscopy to understand the mechanisms of Atom Transfer Radical Polymerization is discussed. While this review focusses predominantly on the application of EPR spectroscopy in mechanistic studies of C–C and C–N bond formation reactions mediated by organometallic catalysts, a few selected examples describing the application of EPR spectroscopy in other catalytic reactions such as water splitting, photo-catalysis, photo-redox-catalysis and related reactions in which metal initiated (free) radical formation plays a role are included as well. EPR spectroscopic investigation in this area of research are dominated by EPR spectroscopic studies in isotropic solution, including spin trapping experiments. These reactions are highlighted in Sect. 5. EPR spectroscopic studies have proven useful to discern the correct oxidation states of the active catalysts and also to determine the effective concentrations of the active species. EPR is definitely a spectroscopic technique that is indispensable in understanding the reactivity of paramagnetic complexes and in conjunction with other advanced techniques such as X-ray absorption spectroscopy and pulsed laser polymerization it will continue to be a very practical tool.

Review: 104 refs.

Five-coordinate Zn(II), Ni(II), and Cu(II) complexes containing pentadentate N(3)O(2) Schiff base ligands [1A](2-) and [1B](2-) have been synthesized and characterized. X-ray crystallographic studies reveal five coordinate structures in which each metal ion is bound by two imine N-donors, two phenolate O-donors, and a single amine N-donor. Electron paramagnetic resonance (EPR) spectroscopic studies suggest that the N(3)O(2) coordination spheres of [Cu(1A)] and [Cu(1B)] are retained in CH(2)Cl(2) solution and solid-state superconducting quantum interference device (SQUID) magnetometric studies confirm that [Ni(1A)] and [Ni(1B)] adopt high spin (S = 1) configurations. Each complex exhibits two reversible oxidation processes between +0.05 and +0.64 V vs [Fc](+)/[Fc]. The products of one- and two-electron oxidations have been studied by UV/vis spectroelectrochemistry and by EPR spectroscopy which confirm that each oxidation process for the Zn(II) and Cu(II) complexes is ligand-based with sequential formation of mono- and bis-phenoxyl radical species. In contrast, the one-electron oxidation of the Ni(II) complexes generates Ni(III) products. This assignment is supported by spectroelectrochemical and EPR spectroscopic studies, density functional theory (DFT) calculations, and the single crystal X-ray structure of [Ni(1A)][BF(4)] which contains Ni in a five-coordinate distorted trigonal bipyramidal geometry.

Electron spin resonance (ESR) spectroscopy studies on the reduction process of nitroxyl radicals were carried out for 1mM concentration of 14N-labeled nitroxyl radicals in 1 mM concentration of ascorbic acid as a function of time. The half life time and decay rate were estimated for 1mM concentration of 14N labeled nitroxyl radicals in 1 mM concentration of ascorbic acid. From the results, the increase in half life time and decrease in decay rate were calculated for TEMPONE compared with TEMPO and TEMPOL radicals, which indicates the higher stability of TEMPONE radical. The observed radical scavenging activity is also higher for TEMPONE radical. The ESR spectrum was also recorded for 1mM concentration of 14N-labeled nitroxyl radicals in pure water and the ESR parameters, line width, hyperfine coupling constant, g-factor, signal intensity ratio and rotational correlation time were obtained. These results indicate that the TEMPONE radical has narrowest line width and fast tumbling motion compared with TEMPO and TEMPOL. Therefore, this study reveals that the TEMPONE radical can act as a good redox sensitive spin probe for molecular imaging.

Free radical scavenging and antioxidant activity of betanin: Electron spin resonance spectroscopy studies and studies in cultured cells

Iron porphyrin carbenes (IPCs) have been extensively recognized as the reactive intermediates in various iron porphyrin-catalyzed carbene transfer reactions. While donor-acceptor diazo compounds have been frequently used for such transformations, the structures and reactivities of donor-acceptor IPCs are less explored. To date, no crystal structures of donor-acceptor IPC complexes have been reported, and therefore, the involvement of IPC intermediacy for such transformations lacks direct evidence. Here we report the synthesis and NMR characterization of several donor-acceptor IPC complexes from iron porphyrin and corresponding donor-acceptor diazo compounds. The X-ray crystal structure of an IPC complex derived from a morpholine-substituted diazo amide was obtained. The carbene transfer reactivities of those IPCs were tested by the N-H insertion reactions with aniline or morpholine as well as the three-component reaction with aniline and γ,δ-unsaturated α-keto ester based on electrophilic trapping of an ammonium ylide intermediate. Based on these results, IPCs were identified as the real intermediates for iron porphyrin-catalyzed carbene transfer reactions from donor-acceptor diazo compounds.

A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.

Cellular studies have indicated that some Fe-S proteins, and the aconitases in particular, are targets for nitric oxide. Specifically, NO has been implicated in the intracellular process of the conversion of active cytosolic aconitase containing a [4Fe-4S] cluster, to its apo-form which functions as an iron-regulatory protein. We have undertaken the in vitro study of the reaction of NO with purified forms of both mitochondrial and cytosolic aconitases by following enzyme activity and by observing the formation of EPR signals not shown by the original reactants. Inactivation by either NO solutions or NO-producing NONOates under anaerobic conditions is seen for both enzyme isoforms. This inactivation, which occurs in the presence or absence of substrate, is accompanied by the appearance of the g = 2.02 signals of the [3Fe-4S] clusters and the g approximately 2.04 signal of a protein-bound dinitrosyl-iron-dithiol complex in the d7 state. In addition, in the reaction of cytosolic aconitase, the transient formation of a thiyl radical, g parallel = 2.11 and g perpendicular = 2.03, is observed. Disassembly of the [3Fe-4S] clusters of the inactive forms of the enzymes upon the anaerobic addition of NO is also accompanied by the formation of the g approximately 2.04 species and in the case of mitochondrial aconitase, a transient signal at g approximately 2. 032 appeared. This signal is tentatively assigned to the d9 form of an iron-nitrosyl-histidyl complex of the mitochondrial protein. Inactivation of the [4Fe-4S] forms of both aconitases by either superoxide anion or peroxynitrite produces the g = 2.02 [3Fe-4S] proteins.

We report the high-frequency (139.5 GHz) electron paramagnetic resonance (EPR) spectrum of the tyrosyl radical of photosystem II. A rhombic powder pattern with principal g values g1 = 2.007 82, g2 = 2.004 50, and g3 = 2.002 32 is observed. The well-defined turning points and the value of the largest principal g value are indicative of ordered hydrogen bonding to the tyrosyl phenyl oxygen. Hyperfine structure is resolved on all three turning points. Proton hyperfine couplings obtained from the simulation of the 139.5 GHz EPR spectrum are in good agreement with X-band electron spin echo−electron nuclear double resonance studies. The high-frequency EPR spectrum was acquired under conditions of saturation in which the dispersion signal is detected. Proper replication of the high-frequency EPR spectral features is only achieved in simulations which account for the line shapes characteristic of saturated dispersion signals. Comparison of the spectrum with spectra of non-hydrogen bonded tyrosyl radicals indicate...

The photochemistry of metalloporphyrins and other tetrapyrrolic systems continues to attract considerable interest.1 There have been numerous studies on the photoactivity of various porphyrin complexes with single-bonded axial ligands, including halogens, oxo-anion species, molecular oxygen,2 and a variety of metals, including chromium, manganese, and iron.3 The photochemistry of metalloporphyrins with multiple-bonded axial ligands has not been explored. We report here the photochemistry of several iron porphyrin carbene and vinylidene complexes. Irradiation of these complexes with visible light cleaves the iron-carbon double bond, resulting in a four-coordinate iron(II) porphyrin and a free carbene. This chemistry is unique in the area of transition metal carbene or alkylidene compounds. Porphyrins with multiple-bonded axial ligands are especially relevant to both biological and catalytic oxidative processes.4 Iron porphyrin carbene and nitrene species are isoelectronic with a terminal iron-oxo complex, the proposed intermediate of the cytochrome P-450 monooxygenases and its synthetic analogs. Most recently, the catalytic cyclopropanation of olefins by iron porphyrins using ethyl diazoacetate was thought to involve a porphyrin carbene complex.5 We prepared Fe(TPP)CCl2, Fe(TPP)CBr2, Fe(TPP)CClF, and Fe(TPP)CC(p-C6H4Cl)2 under anaerobic conditions following Mansuy’s procedure using iron metal as the reductant (TPP ) 5,10,15,20-tetraphenylporphyrinate).6 These compounds were further purified via crystallization from benzene/pentane under argon in an inert atmosphere box and then fully characterized.7 To examine the photochemistry of these complexes, degassed benzene solutions (∼1 × 10-4 M) were irradiated with a 300 W Xe arc lamp, which was filtered to remove both infrared and ultraviolet (<360 nm) light in order to prevent sample heating and porphyrin bleaching, respectively. In addition, the photolysis cells were thermostated to an internal solution temperature of 20 °C. In all cases, irradiation of the parent compounds cleanly produced a new porphyrin identified as Fe(TPP) (λmax ) 419, 442, and 537 nm), as shown in Figure 1 for Fe(TPP)CCl2. Both the Soret and Q-bands of the parent compounds are photoactive.8 The formation of Fe(TPP) was confirmed by addition of pyridine after photolysis of the carbene or vinylidene complexes, yielding the bispyridine complex, Fe(TPP)(py)2. Also as expected, exposure of photolysed solutions to oxygen resulted in the formation of the μ-oxo dimer species, [Fe(TPP)]2O. The production of Fe(TPP) in this reaction is indicative of homolytic cleavage of the metal-carbon double bond and production of a carbene, as in the following reaction:

Owing to their importance, diversity and abundance of generated paramagnetic species, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. In contrast to prototypic single-domain and thus single-[4Fe–4S]-containing rSAM enzymes, there is a large subfamily of rSAM enzymes with multiple domains and one or two additional iron–sulfur cluster(s) called the SPASM/twitch domain-containing rSAM enzymes. EPR spectroscopy is a powerful tool that allows for the observation of the iron–sulfur clusters as well as potentially trappable paramagnetic reaction intermediates. Here, we review continuous-wave and pulse EPR spectroscopic studies of SPASM/twitch domain-containing rSAM enzymes. Among these enzymes, we will review in greater depth four well-studied enzymes, BtrN, MoaA, PqqE, and SuiB. Towards establishing a functional consensus of the additional architecture in these enzymes, we describe the commonalities between these enzymes as observed by EPR spectroscopy.

Heme proteins have recently been demonstrated to catalyze cyclopropanation reactions via a putative transfer mechanism. Carbene transfer reactions are not known to occur in natural biological systems, but are highly useful synthetic reactions. There is growing interest in developing new carbene that bring new chemical reactions into the realm of biology, and growing interest in engineering these enzymes for use in organic synthesis. Additionally, the mechanistic details of iron porphyrin-catalyzed transfer reactions are largely unknown, especially with regards to how the enzyme environment influences the outcome of a transfer reaction. This thesis details both the engineering of transferases with novel catalytic capabilities and investigations into how these enzymes catalyze transfer reactions. Chapter 1 introduces heme protein-catalyzed transfer reactions and describes the directed evolution of new enzymes that allow access to a range of useful cyclopropane products. Chapter 2 describes the evolution of an enzyme that performs transfer to silicon–hydrogen bonds, resulting in a highly efficient and selective carbon–silicon bond-forming enzyme, the first of its kind. Chapter 3 focuses on the characterization of a key reactive intermediate, the iron-porphyrin carbene, in the active site of the evolved carbon–silicon bond-forming enzyme. This study provides an explanation of the remarkable enantioselectivity of the enzyme and provides a foundation from which to investigate the enzyme reaction mechanism. The mechanism of carbon– silicon bond formation is elucidated in Chapter 4, and the is then used to explain how the enzyme achieves chemoselectivity, which in turn guides the evolution of enzyme variants with altered chemoselectivity. Finally, two off-cycle catalytic pathways that cause inactivation of the transferase are characterized, and methods to prevent and/or circumvent inactivation are investigated (Chapter 5). Overall, the work presented here expands the repertoire of enzyme-catalyzed reactions and facilitates the continuing development of new transferases by developing our mechanistic understanding of this novel class of enzymes.

Room-temperature, single-crystal W-band (~94 GHz) electron paramagnetic resonance (EPR) spectra of a flux-grown fluorapatite (AP30-0) containing 0.8(1) ppm Gd and 0.9 ppm Mn disclose the presence of two even-isotope Gd 3+ centers (electron spin number S = 7/2 and nuclear spin number I = 0) and a 55 Mn 2+ center (S = I = 5/2). The relative abundance of the two Gd 3+ centers (corresponding to recently established centers “a” and “b” containing Gd 3+ ions at the Ca2 and Ca1 sites, respectively) in AP30-0 has been estimated to be 0.20, indicating that “a” in this sample arises from the presence of ~0.2 ppm Gd at the Ca2 site. In addition, high-resolution W-band spectra of this sample at ~120 and ~77 K disclose the 155 Gd and 157 Gd spectra of “a,” in which these isotopes (I = 3/ 2) are only ~0.02 ppm in abundance. To the best of our knowledge, this is the first-ever demonstration of structural characterization of sub-ppm-level trace elements in minerals and their synthetic analogs. Moreover, the fact that the Gd 3+ and 55 Mn 2+ centers in AP30-0 are detected despite the multiplicity of lines arising from their complex fine structures, hyperfine structures, and magneticsite splittings, suggests that the W-band EPR technique is potentially capable of characterizing trace elements with a single unpaired electron (S = 1/2) and zero nuclear spin (I = 0) at even lower concentrations. The spin-Hamiltonian parameters of the 55 Mn 2+ center, including matrices g, D, A, and P, and high-spin term of type S4, have been determined by optimization using the single-crystal W-band EPR spectra of a Gd-doped fluorapatite containing 3.0(4) ppm Mn. The principal-axis directions of D and the pseudo-symmetry axes calculated from the S4 parameters confirm that this center corresponds to occupancy of 55 Mn 2+ ions at the Ca1 site. Also, the optimized parameters suggest that the Mn 2+ -substituted Ca1 site in the flux-grown fluorapatite has rhombic (i.e., triclinic) local symmetry [e.g., D/g e β e = 436.2(6) G, E/g e β e = 1.1(1) G], slightly different from the trigonal symmetry of the ideal Ca1 site.

In the last few years, the field of artificial hemoproteins has been expanding through two main strategies involving either the incorporation of synthetic metalloporphyrin derivatives into the chiral cavity of a protein or the directed evolution of natural hemoproteins such as myoglobin and cytochromes P450. First, various synthetic water-soluble porphyrins including ions of transition metals such as iron and manganese have been inserted covalently or by supramolecular anchoring into non-specifically designed native proteins or into proteins modified by a minimum number of mutations. The obtained artificial hemoproteins were able to catalyze oxene transfer reactions such as epoxidation of alkenes or sulfoxidation of sulfides and cyclopropanation reactions with good activities and moderate enantioselectivities. Recently, a second approach, based on the design of the active site of already existing native hemoproteins such as myoglobin and cytochromes P450 by directed evolution, has led to new artificial hemoproteins that are able to catalyze oxene transfer reactions with improved activities as well as with abiological reactions. This approach thus provided promising tools for the catalysis of reactions such as intramolecular or intermolecular carbene and nitrene transfer reactions with high efficiencies. In addition, in all cases, after a few rounds of mutagenesis, mutants that were able to catalyze those reactions with a high enantioselectivity could be obtained. Finally, several groups showed that these new artificial metalloenzymes could also be used for the preparative scale-production of compounds with an excellent enantioselectivity, opening new pathways for the industrial synthesis of compounds of pharmaceutical interest.