Imaging kidney inflammation using an oxidatively activated MRI probe.

Imaging tools for kidney inflammation could improve care for patients suffering inflammatory kidney diseases by lessening reliance on percutaneous biopsy or biochemical tests alone. During kidney inflammation, infiltration of myeloid immune cells generates a kidney microenvironment that is oxidizing relative to normal kidney. Here, we evaluated whether magnetic resonance imaging (MRI) using the redox-active iron (Fe) complex Fe-PyC3A as an oxidatively activated probe could serve as a marker of kidney inflammation using mouse models of unilateral ischemia-reperfusion injury (IRI) and lupus nephritis (MRL-lpr mice). We imaged unilateral IRI in gp91phox knockout mice, which are deficient in the nicotinamide oxidase II (NOX2) enzyme required for myeloid oxidative burst, as loss of function control, and imaged MRL/MpJ mice as non-kidney involved lupus control. Gadoterate meglumine was used as a non-oxidatively activated control MRI probe. Fe-PyC3A safety was preliminarily examined following a single acute dose. FePyC3A generated significantly greater MRI signal enhancement in the IRI kidney compared to the contralateral kidney in wild-type mice, but the effect was not observed in the NOX2-deficient control. Fe-PyC3A also generated significantly greater kidney enhancement in MRL-lpr mice compared to MRL/MpJ control. Gadoterate meglumine did not differentially enhance the IRI kidney over the contralateral kidney and did not differentially enhance the kidneys of MRL-lpr over MRL/MpJ mice. Fe-PyC3A was well tolerated at the highest dose evaluated, which was a 40-fold greater than required for imaging. Thus, our data indicate that MRI using Fe-PyC3A is specific to an oxidizing kidney environment shaped by activity of myeloid immune cells and support further evaluation of Fe-PyC3A for imaging kidney inflammation.

Development of a Suite of Gadolinium-Free OATP1-Targeted Paramagnetic Probes for Liver MRI.

Five amphiphilic, anionic Mn(II) complexes were synthesized as contrast agents targeted to organic anion transporting polypeptide transporters (OATP) for liver magnetic resonance imaging (MRI). The Mn(II) complexes are synthesized in three steps, each from the commercially available trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) chelator, with T1-relaxivity of complexes ranging between 2.3 and 3.0 mM-1 s-1 in phosphate buffered saline at an applied field strength of 3.0 T. Pharmacokinetics were assessed in female BALB/c mice by acquiring T1-weighted images dynamically for 70 min after agent administration and determining contrast enhancement and washout in various organs. Uptake of Mn(II) complexes in human OATPs was investigated through in vitro assays using MDA-MB-231 cells engineered to express either OATP1B1 or OATP1B3 isoforms. Our study introduces a new class of Mn-based OATP-targeted contrast that can be broadly tuned via simple synthetic protocols.

A Rationally Designed Complex Replenishes the Transferrin Iron Pool Directly and with High Specificity.

Many forms of anemia are caused or complicated by pathologic restriction of iron (Fe). Chronic inflammation and certain genetic mutations decrease the activity of ferroportin, the only Fe-exporter protein, so that endogenously recycled or nutritionally absorbed Fe cannot be exported to the extracellular Fe carrier protein transferrin for delivery to the bone marrow. Diminished ferroportin activity renders anemia correction challenging as Fe administered intravenously or through nutritional supplementation is trafficked through the ferroportin-transferrin axis. Utilizing judicious application of coordination chemistry principles, we designed an Fe complex (Fe-BBG) with solution thermodynamics and Fe dissociation kinetics optimized to replenish the transferrin-Fe pool rapidly, directly, and with precision. Fe-BBG is unreactive under conditions designed to force redox cycling and production of reactive oxygen species. The BBG ligand has a low affinity for divalent metal ions and does not compete for binding of other endogenously present ions including Cu and Zn. Treatment with Fe-BBG confers anemia correction in a mouse model of iron-refractory iron-deficiency anemia. Repeated exposure to Fe-BBG did not cause adverse clinical chemistry changes or trigger the expression of genes related to oxidative stress or inflammation. Fe-BBG represents the first entry in a promising new class of transferrin-targeted Fe replacement drugs.

Molecular Magnetic Resonance Imaging of Aneurysmal Inflammation Using a Redox Active Iron Complex.

OBJECTIVES: Inflammation plays a key role in driving brain aneurysmal instability and rupture, but clinical tools to noninvasively differentiate between inflamed and stable aneurysms are lacking. We hypothesize that imaging oxidative changes in the aneurysmal microenvironment driven by myeloid inflammatory cells may represent a noninvasive biomarker to evaluate rupture risk. In this study, we performed initial evaluation of the oxidatively activated probe Fe-PyC3A as a tool for magnetic resonance imaging (MRI) of inflammation in a rabbit model of saccular aneurysm. MATERIALS AND METHODS: The difference in longitudinal relaxivity ( r1 ) in reduced and oxidized states of Fe-PyC3A was measured in water and blood plasma phantoms at 3 T. A rabbit saccular aneurysm model was created by endovascular intervention/elastinolysis with subsequent decellularization in situ. Rabbits were imaged at 4 weeks (n = 4) or 12 weeks (n = 4) after aneurysmal induction, when luminal levels of inflammation reflected by the presence of myeloperoxidase positive cells are relatively high and low, respectively, using a 3 T clinical scanner. Both groups were imaged dynamically using a 2-dimensional T1-weighted fast field echo pulse MRI sequence before and up to 4 minutes postinjection of Fe-PyC3A. Dynamic imaging was then repeated after an injection of gadobutrol (0.1 mmol/kg) as negative control probe. Rabbits from the 12-week aneurysm group were also imaged before and 20 minutes and 3 hours after injection of Fe-PyC3A using an axial respiratory gated turbo-spin echo (TSE) pulse sequence with motion-sensitized driven equilibrium (MSDE) preparation. The MSDE/TSE imaging was repeated before, immediately after dynamic acquisition (20 minutes postinjection), and 3 hours after injection of gadobutrol. Aneurysmal enhancement ratios (ERs) were calculated by dividing the postinjection aneurysm versus skeletal muscle contrast ratio by the preinjection contrast ratio. After imaging, the aneurysms were excised and inflammatory infiltrate was characterized by fluorometric detection of myeloperoxidase activity and calprotectin immunostaining, respectively. RESULTS: In vitro relaxometry showed that oxidation of Fe-PyC3A by hydrogen peroxide resulted in a 15-fold increase of r1 at 3 T. Relaxometry in the presence of blood plasma showed no more than a 10% increase of r1 , indicating the absence of strong interaction of Fe-PyC3A with plasma proteins. Dynamic imaging with Fe-PyC3A generated little signal enhancement within the blood pool or adjacent muscle but did generate a transient increase in aneurysmal ER that was significantly greater 4 weeks versus 12 weeks after aneurysm induction (1.6 ± 0.30 vs 1.2 ± 0.03, P < 0.05). Dynamic imaging with gadobutrol generated strong aneurysmal enhancement, but also strong enhancement of the blood and muscle resulting in smaller relative ER change. In the 12-week group of rabbits, MSDE/TSE imaging showed that ER values measured immediately after dynamic MRI (20 minutes postinjection) were significantly higher ( P < 0.05) in the case of Fe-PyC3A (1.25 ± 0.06) than for gadobutrol injection (1.03 ± 0.03). Immunohistochemical corroboration using anticalprotectin antibody showed that leukocyte infiltration into the vessel walls and luminal thrombi was significantly higher in the 4-week group versus 12-week aneurysms (123 ± 37 vs 18 ± 7 cells/mm 2 , P < 0.05). CONCLUSIONS: Magnetic resonance imaging using Fe-PyC3A injection in dynamic or delayed acquisition modes was shown to generate a higher magnetic resonance signal enhancement in aneurysms that exhibit higher degree of inflammation. The results of our pilot experiments support further evaluation of MRI using Fe-PyC3A as a noninvasive marker of aneurysmal inflammation.

Peroxidasin Deficiency Re-programs Macrophages Toward Pro-fibrolysis Function and Promotes Collagen Resolution in Liver.

BACKGROUND & AIMS: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix proteins. Extracellular matrix stabilization influences the speed of tissue recovery. Here, we studied the expression and function of peroxidasin (PXDN), a peroxidase that uses hydrogen peroxide to cross-link collagen IV during liver fibrosis progression and regression. METHODS: Mouse models of liver fibrosis and cirrhosis patients were analyzed for the expression of PXDN in liver and serum. Pxdn-/- and Pxdn+/+ mice were either treated with carbon tetrachloride for 6 weeks to generate toxin-induced fibrosis or fed with a choline-deficient L-amino acid-defined high-fat diet for 16 weeks to create nonalcoholic fatty liver disease fibrosis. Liver histology, quantitative real-time polymerase chain reaction, collagen content, flowcytometry and immunostaining of immune cells, RNA-sequencing, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A. RESULTS: In human and mouse cirrhotic tissue, PXDN is expressed by stellate cells and is secreted into fibrotic areas. In patients with nonalcoholic fatty liver disease, serum levels of PXDN increased significantly. In both mouse models of liver fibrosis, PXDN deficiency resulted in elevated monocyte and pro-fibrolysis macrophage recruitment into fibrotic bands and caused decreased accumulation of cross-linked collagens. In Pxdn-/- mice, collagen fibers were loosely organized, an atypical phenotype that is reversible upon macrophage depletion. Elevated ROS in Pxdn-/- livers was observed, which can result in activation of hypoxic signaling cascades and may affect signaling pathways involved in macrophage polarization such as TNF-a via NF-kB. Fibrosis resolution in Pxdn-/- mice was associated with significant decrease in collagen content and improved liver function. CONCLUSION: PXDN deficiency is associated with increased ROS levels and a hypoxic liver microenvironment that can regulate recruitment and programming of pro-resolution macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest a novel pathway that is involved in the resolution of scar tissue.

Enzyme Control Over Ferric Iron Magnetostructural Properties.

Fe3+ complexes in aqueous solution can exist as discrete mononuclear species or multinuclear magnetically coupled species. Stimuli-driven change to Fe3+ speciation represents a powerful mechanistic basis for magnetic resonance sensor technology, but ligand design strategies to exert precision control of aqueous Fe3+ magnetostructural properties are entirely underexplored. In pursuit of this objective, we rationally designed a ligand to strongly favor a dinuclear µ-oxo-bridged and antiferromagnetically coupled complex, but which undergoes carboxylesterase mediated transformation to a mononuclear high-spin Fe3+ chelate resulting in substantial T1 -relaxivity increase. The data communicated demonstrate proof of concept for a novel and effective strategy to exert biochemical control over aqueous Fe3+ magnetic, structural, and relaxometric properties.

Rational Ligand Design Enables pH Control over Aqueous Iron Magnetostructural Dynamics and Relaxometric Properties.

Complexes of Fe3+ engage in rich aqueous solution speciation chemistry in which discrete molecules can react with solvent water to form multinuclear µ-oxo and µ-hydroxide bridged species. Here we demonstrate how pH- and concentration-dependent equilibration between monomeric and µ-oxo-bridged dimeric Fe3+ complexes can be controlled through judicious ligand design. We purposed this chemistry to develop a first-in-class Fe3+-based MR imaging probe, Fe-PyCy2AI, that undergoes relaxivity change via pH-mediated control of monomer vs dimer speciation. The monomeric complex exists in a S = 5/2 configuration capable of inducing efficient T1-relaxation, whereas the antiferromagnetically coupled dimeric complex is a much weaker relaxation agent. The mechanisms underpinning the pH dependence on relaxivity were interrogated by using a combination of pH potentiometry, 1H and 17O relaxometry, electronic absorption spectroscopy, bulk magnetic susceptibility, electron paramagnetic resonance spectroscopy, and X-ray crystallography measurements. Taken together, the data demonstrate that PyCy2AI forms a ternary complex with high-spin Fe3+ and a rapidly exchanging water coligand, [Fe(PyCy2AI)(H2O)]+ (ML), which can deprotonate to form the high-spin complex [Fe(PyCy2AI)(OH)] (ML(OH)). Under titration conditions of 7 mM Fe complex, water coligand deprotonation occurs with an apparent pKa 6.46. Complex ML(OH) dimerizes to form the antiferromagnetically coupled dimeric complex [(Fe(PyCy2AI))2O] ((ML)2O) with an association constant (Ka) of 5.3 ± 2.2 mM-1. The relaxivity of the monomeric complexes are between 7- and 18-fold greater than the antiferromagnetically coupled dimer at applied field strengths ranging between 1.4 and 11.7 T. ML(OH) and (ML)2O interconvert rapidly within the pH 6.0-7.4 range that is relevant to human pathophysiology, resulting in substantial observed relaxivity change. Controlling Fe3+ µ-oxo bridging interactions through rational ligand design and in response to local chemical environment offers a robust mechanism for biochemically responsive MR signal modulation.

Tumor Contrast Enhancement and Whole-Body Elimination of the Manganese-Based Magnetic Resonance Imaging Contrast Agent Mn-PyC3A.

OBJECTIVES: The goals of this study were to compare the efficacy of the new manganese-based magnetic resonance imaging (MRI) contrast agent Mn-PyC3A to the commercial gadolinium-based agents Gd-DOTA and to Gd-EOB-DTPA to detect tumors in murine models of breast cancer and metastatic liver disease, respectively, and to quantify the fractional excretion and elimination of Mn-PyC3A in rats. METHODS: T1-weighted contrast-enhanced MRI with 0.1 mmol/kg Mn-PyC3A was compared with 0.1 mmol/kg Gd-DOTA in a breast cancer mouse model (n = 8) and to 0.025 mmol/kg Gd-EOB-DTPA in a liver metastasis mouse model (n = 6). The fractional excretion, 1-day biodistribution, and 7-day biodistribution in rats after injection of 2.0 mmol/kg [Mn]Mn-PyC3A or Gd-DOTA were quantified by Mn gamma counting or Gd elemental analysis. Imaging data were compared with a paired t test; biodistribution data were compared with an unpaired t test. RESULTS: The postinjection-preinjection increases in tumor-to-muscle contrast-to-noise ratio (ΔCNR) 3 minutes after injection of Mn-PyC3A and Gd-DOTA (mean ± standard deviation) were 17 ± 3.8 and 20 ± 4.4, respectively (P = 0.34). Liver-to-tumor ΔCNR values at 8 minutes postinjection of Mn-PyC3A and Gd-EOB-DTPA were 28 ± 9.0 and 48 ± 23, respectively (P = 0.11). Mn-PyC3A is eliminated with 85% into the urine and 15% into the feces after administration to rats. The percentage of the injected doses (%ID) of Mn and Gd recovered in tissues after 1 day were 0.32 ± 0.12 and 0.57 ± 0.12, respectively (P = 0.0030), and after 7 days were 0.058 ± 0.051 and 0.19 ± 0.052, respectively (P < 0.0001). CONCLUSIONS: Mn-PyC3A provides comparable tumor contrast enhancement to Gd-DOTA in a mouse breast cancer model and is more completely eliminated than Gd-DOTA; partial hepatobiliary elimination of Mn-PyC3A enables conspicuous delayed phase visualization of liver metastases.

Molecular Magnetic Resonance Imaging Using a Redox-Active Iron Complex.

We introduce a redox-active iron complex, Fe-PyC3A, as a biochemically responsive MRI contrast agent. Switching between Fe3+-PyC3A and Fe2+-PyC3A yields a full order of magnitude relaxivity change that is field-independent between 1.4 and 11.7 T. The oxidation of Fe2+-PyC3A to Fe3+-PyC3A by hydrogen peroxide is very rapid, and we capitalized on this behavior for the molecular imaging of acute inflammation, which is characterized by elevated levels of reactive oxygen species.  Injection of Fe2+-PyC3A generates strong, selective contrast enhancement of inflamed pancreatic tissue in a mouse model (caerulein/LPS model). No significant signal enhancement is observed in normal pancreatic tissue (saline-treated mice). Importantly, signal enhancement of the inflamed pancreas correlates strongly and significantly with ex vivo quantitation of the pro-inflammatory biomarker myeloperoxidase. This is the first example of using metal ion redox for the MR imaging of pathologic change in vivo. Redox-active Fe3+/2+ complexes represent a new design paradigm for biochemically responsive MRI contrast agents.

Manganese-Based Contrast Agents for Magnetic Resonance Imaging of Liver Tumors: Structure-Activity Relationships and Lead Candidate Evaluation.

Gd-based MRI contrast agents (GBCAs) have come under intense regulatory scrutiny due to concerns of Gd retention and delayed toxicity. Three GBCAs comprising acyclic Gd chelates, the class of GBCA most prone to Gd release, are no longer marketed in Europe. Of particular concern are the acyclic chelates that remain available for liver scans, where there is an unmet diagnostic need and no replacement technology. To address this concern, we evaluated our previously reported Mn-based MRI contrast agent, Mn-PyC3A, and nine newly synthesized derivatives as liver specific MRI contrast agents. Within this focused library the transient liver uptake and rate of blood clearance are directly correlated with log P. The complex Mn-PyC3A-3-OBn emerged as the lead candidate due to a combination of high relaxivity, rapid blood clearance, and avid hepatocellular uptake. Mn-PyC3A-3-OBn rendered liver tumors conspicuously hypo-intense in a murine model and is wholly eliminated within 24 h of injection.

A Janus Chelator Enables Biochemically Responsive MRI Contrast with Exceptional Dynamic Range.

We introduce a new biochemically responsive Mn-based MRI contrast agent that provides a 9-fold change in relaxivity via switching between the Mn3+ and Mn2+ oxidation states. Interchange between oxidation states is promoted by a "Janus" ligand that isomerizes between binding modes that favor Mn3+ or Mn2+. It is the only ligand that supports stable complexes of Mn3+ and Mn2+ in biological milieu. Rapid interconversion between oxidation states is mediated by peroxidase activity (oxidation) and l-cysteine (reduction). This Janus system provides a new paradigm for the design of biochemically responsive MRI contrast agents.

Accessing Ni(III)-thiolate versus Ni(II)-thiyl bonding in a family of Ni-N2S2 synthetic models of NiSOD.

Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide (O2(• -)) into H2O2 and O2(g) by toggling through different oxidation states of a first-row transition metal ion at its active site. Ni-containing SODs (NiSODs) are a distinct class of this family of metalloenzymes due to the unusual coordination sphere that is comprised of mixed N/S-ligands from peptide-N and cysteine-S donor atoms. A central goal of our research is to understand the factors that govern reactive oxygen species (ROS) stability of the Ni-S(Cys) bond in NiSOD utilizing a synthetic model approach. In light of the reactivity of metal-coordinated thiolates to ROS, several hypotheses have been proffered and include the coordination of His1-Nδ to the Ni(II) and Ni(III) forms of NiSOD, as well as hydrogen bonding or full protonation of a coordinated S(Cys). In this work, we present NiSOD analogues of the general formula [Ni(N2S)(SR')](-), providing a variable location (SR' = aryl thiolate) in the N2S2 basal plane coordination sphere where we have introduced o-amino and/or electron-withdrawing groups to intercept an oxidized Ni species. The synthesis, structure, and properties of the NiSOD model complexes (Et4N)[Ni(nmp)(SPh-o-NH2)] (2), (Et4N)[Ni(nmp)(SPh-o-NH2-p-CF3)] (3), (Et4N)[Ni(nmp)(SPh-p-NH2)] (4), and (Et4N)[Ni(nmp)(SPh-p-CF3)] (5) (nmp(2-) = dianion of N-(2-mercaptoethyl)picolinamide) are reported. NiSOD model complexes with amino groups positioned ortho to the aryl-S in SR' (2 and 3) afford oxidized species (2(ox) and 3(ox)) that are best described as a resonance hybrid between Ni(III)-SR and Ni(II)-(•)SR based on ultraviolet-visible (UV-vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectroscopies, as well as density functional theory (DFT) calculations. The results presented here, demonstrating the high percentage of S(3p) character in the highest occupied molecular orbital (HOMO) of the four-coordinate reduced form of NiSOD (NiSODred), suggest that the transition from NiSODred to the five-coordinate oxidized form of NiSOD (NiSODox) may go through a four-coordinate Ni-(•)S(Cys) (NiSODox-Hisoff) that is stabilized by coordination to Ni(II).

Synthetic analogues of nickel superoxide dismutase: a new role for nickel in biology.

Nickel-containing superoxide dismutases (NiSODs) represent a novel approach to the detoxification of superoxide in biology and thus contribute to the biodiversity of mechanisms for the removal of reactive oxygen species (ROS). While Ni ions play critical roles in anaerobic microbial redox (hydrogenases and CO dehydrogenase/acetyl coenzyme A synthase), they have never been associated with oxygen metabolism. Several SODs have been characterized from numerous sources and are classified by their catalytic metal as Cu/ZnSOD, MnSOD, or FeSOD. Whereas aqueous solutions of Cu(II), Mn(II), and Fe(II) ions are capable of catalyzing the dismutation of superoxide, solutions of Ni(II) are not. Nonetheless, NiSOD catalyzes the reaction at the diffusion-controlled limit (~10(9) M(-1) s(-1)). To do this, nature has created a Ni coordination unit with the appropriate Ni(III/II) redox potential (~0.090 V vs Ag/AgCl). This potential is achieved by a unique ligand set comprised of residues from the N-terminus of the protein: Cys2 and Cys6 thiolates, the amino terminus and imidazole side chain of His1, and a peptide N-donor from Cys2. Over the past several years, synthetic modeling efforts by several groups have provided insight into understanding the intrinsic properties of this unusual Ni coordination site. Such analogues have revealed information regarding the (i) electrochemical properties that support Ni-based redox, (ii) oxidative protection and/or stability of the coordinated CysS ligands, (iii) probable H(+) sources for H(2)O(2) formation, and (iv) nature of the Ni coordination geometry throughout catalysis. This review includes the results and implications of such biomimetic work as it pertains to the structure and function of NiSOD.

Dipeptide-based models of nickel superoxide dismutase: solvent effects highlight a critical role to Ni-S bonding and active site stabilization.

Nickel superoxide dismutase (Ni-SOD) catalyzes the disproportionation of the superoxide radical to O(2) and H(2)O(2) utilizing the Ni(III/II) redox couple. The Ni center in Ni-SOD resides in an unusual coordination environment that is distinct from other SODs. In the reduced state (Ni-SOD(red)), Ni(II) is ligated to a primary amine-N from His1, anionic carboxamido-N/thiolato-S from Cys2, and a second thiolato-S from Cys6 to complete a NiN(2)S(2) square-planar coordination motif. Utilizing the dipeptide N(2)S(2-) ligand, H(2)N-Gly-l-Cys-OMe (GC-OMeH(2)), an accurate model of the structural and electronic contributions provided by His1 and Cys2 in Ni-SOD(red), we constructed the dinuclear sulfur-bridged metallosynthon, [Ni(2)(GC-OMe)(2)] (1). From 1 we prepared the following monomeric Ni(II)-N(2)S(2) complexes: K[Ni(GC-OMe)(SC(6)H(4)-p-Cl)] (2), K[Ni(GC-OMe)(S(t)Bu)] (3), K[Ni(GC-OMe)(SC(6)H(4)-p-OMe)] (4), and K[Ni(GC-OMe)(SNAc)] (5). The design strategy in utilizing GC-OMe(2-) is analogous to one which we reported before (see Inorg. Chem. 2009, 48, 5620 and Inorg. Chem. 2010, 49, 7080) where Ni-SOD(red) active site mimics can be assembled at will with electronically variant RS(-) ligands. Discussed herein is our initial account pertaining to the aqueous behavior of isolable, small-molecule Ni-SOD model complexes (non-maquette based). Spectroscopic (FTIR, UV-vis, ESI-MS, XAS) and electrochemical (CV) measurements suggest that 2-5 successfully simulate many of the electronic features of Ni-SOD(red). Furthermore, the aqueous studies reveal a dynamic behavior with regard to RS(-) lability and bridging interactions, suggesting a stabilizing role brought about by the protein architecture.

Exploring the effects of H-bonding in synthetic analogues of nickel superoxide dismutase (Ni-SOD): experimental and theoretical implications for protection of the Ni-SCys bond.

Nickel superoxide dismutase (Ni-SOD) is a recently discovered SOD obtained from soil microbes and cyanobacteria that shares no structural or spectroscopic similarities with other isoforms of SOD. The enzyme is found in both the Ni(II) (Ni-SOD(red)) and Ni(III) (Ni-SOD(ox)) oxidation states in "as isolated" preparations of the enzyme from two separate and independently crystallized Streptomyces strains. Ni-SOD contains an unusual and unprecedented biological coordination sphere comprised of Cys-S and peptido-N donors. To understand the role of these donors, we have previously synthesized the monomeric Ni(II)N(2)S(2) complexes, (Et(4)N)[Ni(nmp)(SC(6)H(4)-p-Cl)] (2) and (Et(4)N)[Ni(nmp)(S(t)Bu)] (3) as Ni-SOD(red) models arising from the S,S-bridged precursor molecule, [Ni(2)(nmp)(2)] (1) (where nmp(2-) = doubly deprotonated form of N-2-(mercaptoethyl)picolinamide). In addition to 2 and 3, we report here three new complexes, (Et(4)N)[Ni(nmp)(S-o-babt)] (4), (Et(4)N)[Ni(nmp)(S-meb)] (5), and K[Ni(nmp)(S-NAc)] (6) (where (-)S-o-babt = thiolate of o-benzoylaminobenzene thiol; (-)S-meb = thiolate of N-(2-mercaptoethyl)benzamide; and (-)S-NAc = thiolate of N-acetyl-L-cysteine methyl ester), that provide a unique comparison as to the structural and reactivity effects imparted by H-bonding in square planar asymmetrically coordinated Ni(II)N(2)S(2) complexes. X-ray structural analysis in combination with cyclic voltammetry (CV), spectroscopic measurements, density functional theory (DFT) calculations, and reactivity studies with O(2) and various ROS were employed to gain insight into the role that H-bonding plays in NiN(2)S(2) complexes related to Ni-SOD. The experimental results coupled with theoretical analysis demonstrate that H-bonding to coordinated thiolates stabilizes S-based molecular orbitals relative to those arising from Ni(II), allowing for enhanced Ni contribution to the highest occupied molecular orbital (HOMO), which is predominantly of S-Ni pi* character. These studies provide a unique perspective on the role played by electronically different thiolates regarding the intimately coupled interplay and delicate balance of Ni- versus S-based reactivity in Ni-SOD model complexes. The reported results have offered new insight into the chemistry that H-bonding/thiolate protonation imparts upon the Ni-SOD active site during catalysis, in particular, as a protective mechanism against oxidative modification/degradation.

Versatile methodology toward NiN(2)S(2) complexes as nickel superoxide dismutase models: structure and proton affinity.

Structural features of the reduced form of the nickel superoxide dismutase (Ni-SOD) active site have been modeled with asymmetric NiN(2)S(2) complexes (Et(4)N)[Ni(nmp)(SR)] (R = C(6)H(4)-p-Cl (2) and (S(t)Bu) (3)) obtained via S,S-bridge splitting of the dimeric metallosynthon, [Ni(2)(nmp)(2)] (1). Complexes 2 and 3 are irreversibly oxidized at potentials within the window needed for SOD activity, 236 and 75 mV versus Ag/AgCl, respectively. The exogenous thiolato-S in 2 and 3 serves as a proton acceptor, suggesting potential involvement of Cys6 in Ni-SOD for H(+) storage between SOD half reactions.