Homo- and Heteronuclear Group 12 Metallothionein Type B Cluster Analogs: Synthesis, Structure, 1H NMR and ESI-MS.

Metallothioneins (MTs) are a ubiquitous class of small cysteine-rich metal-binding proteins involved in metal homeostasis and detoxification with highly versatile metal binding properties. Despite the long-standing association of MT with M3S3 and M4S5 metal clusters, synthetic complexes with these core architectures are exceptionally rare. Here, we demonstrate an approach to synthesizing and characterizing aggregates of group 12 metal ions with monocyclic M3S3 cores in acetonitrile solution without the protection of a protein. Multidentate monothiol ligand N,N-bis(2-pyridylmethyl)-2-aminoethanethiol (L1H) provided [Cd3(L1)3](ClO4)3 (1), the first structurally characterized nonproteinaceous aggregate with a metallothionein-like monocyclic Cd3S3 core. In addition, [Zn3(L1)3](ClO4)3·4CH3CN (2·4CH3CN) was characterized by X-ray crystallography. The complex cations of 1 and 2 had comparable structures despite being nonisomorphic. Variable temperature and concentration 1H NMR were used to investigate aggregation equilibria of 1, 2, and a precipitate with composition "Hg(L1)(ClO4)" (3). Cryogenic 1H NMR studies of 3 revealed a J(199Hg1H) coupling constant pattern consistent with an aggregate possessing a cyclic core. ESI-MS was used for gas-phase characterization of 1-3, as well as mixed-metal [M2M'(L1)3(ClO4)2]+ ions prepared in situ by pairwise acetonitrile solution combinations of the group 12 complexes of L1. Access to synthetic variants of metallothionein-like group 12 aggregates provides an additional approach to understanding their behavior.

Radical Complexes of Nickel(II)/Copper(II) and Redox Non-innocent MB-DIPY Ligands: Unusual Stability and Strong Near-Infrared Absorption at λmax ∼1300†nm.

The preparation of radicals with intense and redox-switchable absorption beyond 1000 nm is a long-standing challenge in the chemistry of functional dyes. Here we report the preparation of a series of unprecedented stable neutral nickel(II) and copper(II) complexes of "Manitoba dipyrromethenes" (MB-DIPYs) in which the organic chromophore is present in the radical-anion state. The new stable radicals have an intense absorption at λmax ∼1300 nm and can be either oxidized to regular [MII (MB-DIPY)]+ (M=Cu or Ni) or reduced to [MII (MB-DIPY)]- compounds. The radical nature of the stable [MII (MB-DIPY)] complexes was confirmed by EPR spectroscopy with additional insight into their electronic structure obtained by UV-Vis spectroscopy, electro- and spectroelectrochemistry, magnetic measurements, and X-ray crystallography. The electronic structures and spectroscopic properties of the radical-based chromophores were also probed by density functional theory (DFT) and time-dependent DFT (TDDFT) calculations. These nickel(II) and copper(II) complexes represent the first stable radical compounds with a MB-DIPY ligand.

HYSCORE Insights into the Distribution of the Unpaired Spin Density in an Engineered CuA Site in Azurin and Its His120Gly Variant.

A comparative study of the 1H and 14N hyperfine interactions between the CuA site in an engineered CuA center in azurin (WT-CuAAz) and its His120Gly variant (H120G-CuAAz) using the two-dimensional ESEEM technique, HYSCORE, is reported. HYSCORE spectroscopy has clarified conflicting results in previous electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies and found clear differences between the two CuA azurins. Specifically, a hyperfine coupling AN⊥ of 15.3 MHz was determined for the first time from the frequencies of double-quantum transitions of 14N histidine nitrogens coordinated to CuA in WT-CuAAz. In contrast, such coupling was not observed in the spectra of H120G-CuAAz, indicating at least a several megahertz increase in AN⊥ for the coordinated nitrogen in this variant. In addition, 14N HYSCORE spectra of WT-CuAAz show interaction with only one type of weakly coupled nitrogen assigned to the remote Nε atom of coordinated imidazole residues based on the quadrupole coupling constant ( e2 Qq/4 h) of ∼0.4 MHz. The spectrum of H120G-CuAAz resolves additional features typical for backbone peptide nitrogens with larger e2 Qq/4 h values of ∼0.7 MHz. Hyperfine couplings with these nitrogens vary between ∼0.4 and 0.7 MHz. In addition, the two resolved cross-peaks from Cß protons in H120G-CuAAz display only ∼1 MHz shifts relative to the corresponding peaks in WT-CuAAz. These new findings have provided the first experimental evidence of the previous density functional theory analysis that predicted changes in the delocalized electron spin population of ∼0.02-0.03 (i.e., ∼10%) on copper and sulfur atoms of the CuA center in H120 variants relative to WT-CuAAz and resolved contradicting results between EPR and ENDOR studies of the valence distribution in CuAAz and its variants.

Nitrite Reductase Activity in Engineered Azurin Variants.

Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.

Mercury metallation of the copper protein azurin and structural insight into possible heavy metal reactivity.

Mercury(II) metallation of Pseudomonas aeruginosa azurin has been characterized structurally and biochemically. The X-ray crystal structure at 1.5Å of mercury(II) metallated azurin confirms the coordination of mercury at the copper binding active site and a second surface site. These findings are further validated by NMR, Matrix-assisted laser desorption/ionization spectrometry (MALDI), and UV-visible spectroscopic methods indicating copper displacement from the wild-type protein. Bioinformatic analysis has identified homologous human protein domains computationally, and compared them to the structure of azurin, providing a model for human mercury interactions. Study of the mercury-azurin adduct, in combination with other known examples of protein-heavy metal interactions, could provide further insight into the chemical mechanisms of toxicological interactions, leading toward a global understanding of the biological speciation of toxic heavy metals.

Structure and isomerization comparison of Zn(II), Cd(II) and Hg(II) perchlorate complexes of 2,6-bis([(2-pyridyl-methyl)amino]methyl)pyridine.

The divalent zinc triad perchlorate coordination chemistry of 2,6-bis([(2-pyridyl-methyl)amino]methyl)pyridine (L) was investigated by X-ray crystallography and solution state (1)H NMR. New complexes [HgL(ClO4)2] (1) and [CdL(ClO4)2] (2) were isolated as bicapped distorted square pyramidal racemates, contrasting with the approximate trigonal bipyramidal structure of [ZnL](ClO4)2 (3). Although rapid inter- and intramolecular exchange is common for simple complexes of zinc triad metal ions, conditions for slow intramolecular isomerization on both the δ and J(HH) time scales were established for 1, 2 and 3. Trends in geminal (1)H coupling suggested that an asymmetric structure was favored for all three metal ions at or below 40 °C. Contributions of a symmetric structure to solution equilibria were both temperature- and metal ion-dependent. Spectral trends were consistent with interconversion of a pair of enantiomeric square pyramidal ligand conformers through a symmetric trigonal bipyramidal ligand conformer by M-N bond cleavage and nitrogen inversion. Racemization was slower than the coupling constant time scale up to 40 °C for all complexes. Differential stabilization of specific small ligand conformations in solution has potential toxicological significance.

Isomorphic deactivation of a Pseudomonas aeruginosa oxidoreductase: The crystal structure of Ag(I) metallated azurin at 1.7 Å.

Multiple biophysical methods demonstrate that silver effectively metallates Pseudomonas aeruginosa apo-azurin in solution. X-ray crystallography of the silver-modified protein reveals that silver binds to azurin at the traditional copper mediated active site with nearly identical geometry. Cyclic voltammetry indicates that the silver adduct is redox inert. Our results suggest that a potential mechanism for the microbial toxicity of silver is the deactivation of copper oxidoreductases by the effective binding and structural mimicry by silver without the corresponding function.

Stereoselective synthesis of pyroglutamate natural product analogs from α- aminoacids and their anti-cancer evaluation.

Alkylation of α-amino acid derived iminoesters with Baylis-Hillman (BH) reaction template based allyl bromides/allyl acetates followed by acidic hydrolysis furnished α-methylene-ß-substituted-pyroglutamates and α-alkylidene pyroglutamates respectively. Application of these methodologies has been demonstrated in the synthesis of fused [3.2.0]-γ-lactam-ß-lactones. Further, substrate controlled stereoselective alkylation of L-threonine derived oxazoles with BH reaction based allyl bromides and acetates yielded optically pure α-methylene-ß-substituted pyroglutamates, and α-alkylidene pyroglutamates. These methodologies have been applied in the preparation of chiral [3.2.0] heterobicyclic pyroglutamates containing hydroxyethyl side chain. All the synthesized pyroglutamates have been evaluated for their anti-cancer and enzyme proteasome inhibition activity.

Carbonate-templated self-assembly of an alkylthiolate-bridged cadmium macrocycle.

In the presence of Cd(ClO4)2 and a base, a new mixed N,S-donor alkylthiolate ligand supported both carbonate formation from atmospheric CO2 and the self-assembly of a novel bicapped puckered (CdS)6 molecular wheel. The remarkable stability of the complex was demonstrated by slow intermolecular ligand exchange on the (2)J(HH) and J((111/113)Cd(1)H) time scales at elevated temperature. Both CO2 and the base were required to convert amorphous "CdLClO4" precipitated in the absence of air to the carbonate complex. The complex shares structural features with the ζ-carbonic anhydrase class associating cadmium(II) with the biogeochemical cycling of carbon and is the first structurally characterized carbonate complex of any metal involving an alkylthiolate ligand.

Incorporation of the red copper nitrosocyanin binding loop into blue copper azurin.

Loop-directed mutagenesis was applied to the blue copper protein azurin to replace its copper binding loop with that from the red copper protein nitrosocyanin. A ten amino acid long loop that provides three of the four copper ligands from nitrosocyanin was incorporated into azurin to make a variant called NC-azurin. The chimeric protein displayed a red color, and UV-vis absorption and EPR spectra that closely resembled those of the loop parent, nitrosocyanin. We added the fourth ligand from nitrosocyanin into NC-azurin, a carboxylate-containing amino acid, but the proteins had altered stability and spectroscopic properties that did not resemble those of either parent copper protein. The loop alone, however, was enough to impart red copper site characteristics to the NC-azurin protein. Finally, the reduction potential of the variant was found to be between the reduction potentials of the parent proteins and about 50 mV below that of wild-type azurin.

Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations.

Recent evidence has shown that the properties of metal binding sites can be tuned by more than the ligands in the primary coordination sphere. We investigated the incorporation of four phenylalanine residues into the secondary coordination sphere of the small soluble blue copper protein azurin. The locations for placement of these residues in azurin were based on the structure of the highly hydrophobic blue copper protein rusticyanin, which is known to have a significantly higher reduction potential than azurin. Using site-directed mutagenesis, these residues in close proximity to the copper binding site were mutated to large hydrophobic phenylalanine residues individually and in combination. We also added the Met121Leu mutation on top of the Phe mutations to construct a total of 13 variants. We found little change in the UV-visible absorption and EPR data for these proteins, however modest increases in reduction potential were observed with increases by as much as 30mV per Phe residue. Furthermore, we observed the increases in potential to be additive.

A synthetic model of Hg(II) sequestration.

Tridentate ligand N-(2-pyridylmethyl)-N-(2-(ethylthiolato)amine (L) forms the novel complex [Hg(5)(L)(6)](ClO(4))(4).toluene () with a bicyclo[3.3.3] Hg(5)S(6) core and 4-, 5- and 6-coordinate metal centers; characterization of a solution of by ESI-MS revealed elaborate speciation involving [Hg(n)L(n+1)(ClO(4))(n-2)](+), [Hg(n)L(n)(ClO(4))(n-1)](+) and [Hg(n)L(n-1)(ClO(4))(n)](+) ion families.

9-O-Ethyl-berberrubinium iodide monohydrate.

IN THE TITLE COMPOUND (SYSTEMATIC NAME: 9-eth-oxy-10-meth-oxy-5,6-dihydro-1,3-dioxolo[4,5-g]isoquinolino-[3,2-a]isoquin-olin-7-ium iodide monohydrate), 2C(21)H(20)NO(4) (+)·2I(-)·H(2)O, two independent mol-ecules pack in the unit cell, where interactions between the molecules are stabilized by weak inter-molecular π-π stacking inter-actions [centroid-centroid distances in the range 3.571 (4) to 3.815 (4)Å]. Inter-molecular C-H⋯O inter-actions are also observed. The iodide anions are disordered with occupancy ratios of 0.94 (1):0.06 (1) and 0.91 (1):0.09 (1). The cationic molecule is planar in structure with a small torsion resulting from the dihydropyridine ring.

Structural and NMR characterization of Sm(III), Eu(III), and Yb(III) complexes of an amide based polydentate ligand exhibiting paramagnetic chemical exchange saturation transfer abilities.

Complexes of Sm(III), Eu(III), and Yb(III) with a new polydentate ether ligand with amide arms were synthesized. Solid state X-ray structures of the complexes reveal all three complexes crystallize in monoclinic unit cells. The mononuclear complexes have nine coordinate tricapped trigonal prismatic geometries with coordination of all four amide carbonyl oxygen and all three of the backbone ether oxygen atoms. The molecules possess a pseudo C(2) symmetry axis. The complexes were characterized by solution and solid state emission spectroscopy and IR spectroscopy. Solution state behavior of the complexes was further explored using NMR. The (1)H NMR spectra show 16 peaks suggesting the complexes are slow in exchanging on the NMR time scale and that the C(2) symmetry axis is maintained. The NMR spectra were assigned using (1)H, (13)C, COSY, and HMQC experiments. The Eu(III) complex was tested for the recently explored Magnetic Resonance Imaging phenomenon called paramagnetic chemical exchange saturation transfer (PARACEST). At physiological pH and temperature two CEST peaks were observed that caused a decrease in the bulk water molecule signal intensity of 10 and 16%.

Models of noncoupled dinuclear copper centers in azurin.

The successful modeling of metalloproteins is an important step in understanding their structure and function. Toward this goal, models of the noncoupled copper centers found in the enzymes peptidyl alpha-hydroxylating monooxygenase (PHM), dopamine beta-monooxygenase (DBM), and nitrite reductase (NiR) were designed into the small soluble protein azurin. The models are significant because they maintain the existing type 1 (T1) copper, electron transfer site of azurin while including the second designed type 2 (T2) copper center that mimics the T2 catalytic sites in the target enzymes. UV-vis absorption and EPR spectroscopy data of the model sites are consistent with T2 centers and establish copper binding at the sites, thus modeling those found in PHM/DBM and NiR. Importantly the models' approximate 11-13 A separation between the T1 and T2 copper sites is comparable with the separations in the native systems. This, along with the power to tune the T1 site redox potential in azurin, allows for the future evaluation of relevant activity assays in these models.

Bis-tridentate Chelates of an Asymmetric Ligand: X-ray Structures and Solution NMR Characterization of Divalent Zinc Triad Metal Ion Complexes of N-(2-pyridylmethyl)-N-(2-(methylthio)ethyl)amine.

Divalent zinc triad metal ion complexes of type M(L)(2)(ClO(4))(2) (L = N-(2-pyridylmethyl)-N-(2-(methylthio)ethyl)amine) with N(4)S(2) metal coordination spheres were isolated and characterized by X-ray crystallography and variable temperature proton NMR. Although bis-tridentate chelates have nine geometric isomers, the crystallographically characterized complexes of all three metal ions had trans facial octahedral coordination geometry with C(i) symmetry. Despite the low coordination number and geometric preferences of d(10) metal ions, which facilitate inter- and intramolecular exchange processes, dilute solutions of these bis-tridentate chelates exhibited slow geometric isomerization. Symmetry, sterics and shielding arguments supported specific isomeric assignments for the major and minor chemical shift environments observed at low temperature. At elevated temperature, rapid intramolecular exchange occurred for all three complexes but slow intermolecular exchange on the coupling constant time scale was evidenced through detection of J(HgH) interactions for Hg(L)(2) (2+). These unusual observations are discussed in the context of the zinc triad metal ion coordination chemistry of related bis-tridentate chelates.

Reduction potential tuning of the blue copper center in Pseudomonas aeruginosa azurin by the axial methionine as probed by unnatural amino acids.

The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in < 6 nm shifts in the S(Cys)-Cu charge transfer (CT) band in the electronic absorption spectra and < 8 gauss changes in the copper hyperfine coupling constants (AII) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of AII differs, suggesting additional factors influencing AII. In contrast to the small changes in the UV-vis and EPR spectra, a large variation of > 227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.

Investigation of group 12 metal complexes with a tridentate SNS ligand by X-ray crystallography and 1H NMR spectroscopy.

Two series of zinc triad complexes containing the ligand 2,6-bis(methylthiomethyl)pyridine (L1) were synthesized and characterized by X-ray crystallography and solution-state 1H NMR spectroscopy. The distorted meridional octahedral M(L1)2(ClO4)2 series includes the first structurally characterized Zn(II) and Cd(II) complexes with N2(SR2)4 coordination spheres. Coordination of HgCl2 and ZnCl2 with 1 equiv of ligand afforded mononuclear, five-coordinate species Hg(L1)Cl2 and Zn(L1)Cl2, respectively, with distorted square-pyramidal and trigonal-bipyramidal geometries. With CdCl2, the dimeric [Cd(L1)Cl(mu-Cl)]2 complex was obtained. The distorted octahedral coordination geometry of each Cd(II) center in this complex is formed by one tridentate ligand, two bridging chloride ions, and one terminal chloride ion. NMR spectra indicate that the intermolecular ligand-exchange rate of [M(L1)2](2+) decreased in the order Cd(II) > Zn(II) > Hg(II). Slow intermolecular ligand-exchange conditions on the chemical-shift time scale were found for 1:2 metal-to-ligand complexes of L(1) with Hg(II) and Zn(II) but not Cd(II). Slow intermolecular ligand-exchange conditions in acetonitrile-d(3) solutions permitting detection of (3-5)J(199Hg1H) were found for 1:1 and 1:2 Hg(ClO4)2/L1 complexes, but not for the related Cd(ClO4)2) complexes. The magnitudes of J(199Hg1H) for equivalent protons were smaller in [Hg(L1)2](2+) than in [Hg(L1)(NCCH3)x](2+). The relative intermolecular ligand-exchange rates of the zinc triad complexes investigated here suggest that the toxicity of Hg(II) is accentuated by the relative difficulty of displacing it from the coordination sites encountered.

Axial methionine has much less influence on reduction potentials in a CuA center than in a blue copper center.

The role of the highly conserved axial methionine of the purple CuA center in an engineered CuA azurin on modulating the reduction potentials of the copper center was investigated by a systematic replacement of the methionine with glutamate, aspartate, and leucine. In contrast to the same substitutions in the structurally related blue copper azurin, much smaller changes in reduction potential were observed in the CuA azurin upon replacing the methionine ligand with negatively charged Glu (-8 mV) and Asp (-5 mV) and more hydrophobic Leu (+16 mV). These findings are important in understanding the different roles of the two cupredoxins. The diamond core Cu2S2(Cys) structure of the CuA is much more resistant to variations of axial ligand interactions than the distorted tetrahedral structure of the blue copper protein. This difference may translate into a much wider range of reduction potentials (>1000 mV) for blue copper proteins that transfer electrons to a variety of partners in many different biological systems and a much narrower range of reduction potentials (<40 mV) for CuA proteins where a small difference in reduction potentials between the CuA and its redox partners is required.