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Finding new antibiotics that can act synergistically with each other offers many benefits such as lower dosages used for each drug, improved pathogen clearance, and ability to act against multi-drug resistant strains. In this study, six peptides isolated from the tunicate Styela clava were evaluated for their synergistic interaction using the checkerboard assay and the time kill kinetics assay. Using two different tests, we report synergy between clavanin D and clavaspirin in both tests and synergy between clavanin A and B only in the checkerboard test when used against the multidrug resistant E. cloacae 0136. This work demonstrates the possible cooperativity between homologous AMPs from a single organism and the advantage of using two susceptibility tests instead of one when testing synergistic combinations., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

New 1,2-azolylamidino complexes fac -[RuCl(DMSO) 3 (NHC(R)az*-κ 2 N,N)]OTf [R = Me (2), Ph (3); az* = pz (pyrazolyl, a), indz (indazolyl, b)] are synthesized via chloride abstraction from their corresponding precursors cis , fac -[RuCl 2 (DMSO) 3 (az*H)] (1) after subsequent base-catalyzed coupling of the appropriate nitrile with the 1,2-azole previously coordinated. All the compounds are characterized by 1 H NMR, 13 C NMR and IR spectroscopy. Those derived from MeCN are also characterized by X-ray diffraction. Electrochemical studies showed several reduction waves in the range of -1.5 to -3 V. The electrochemical behavior in CO 2 media is consistent with CO 2 electrocatalytic reduction. The catalytic activity expressed as [ i cat (CO 2 )/ i p (Ar)] ranged from 1.7 to 3.7 for the 1,2-azolylamidino complexes at voltages of ca . -2.7 to -3 V vs. ferrocene/ferrocenium. Controlled potential electrolysis showed rapid decomposition of the Ru catalysts. Photocatalytic CO 2 reduction experiments using compounds 1b, 2b and 3b carried out in a CO 2 -saturated MeCN/TEOA (4 : 1 v/v) solution containing a mixture of the catalyst and [Ru(bipy) 3 ] 2+ as the photosensitizer under continuous irradiation (light intensity of 150 mW cm -2 at 25 °C, λ > 300 nm) show that compounds 1b, 2b and 3b allowed CO 2 reduction catalysis, producing CO and trace amounts of formate. The combined turnover number for the production of formate and CO is ca. 100 after 8 h and follows the order 1b < 2b ≈ 3b.

Antimicrobial peptides (AMPs) are essential components of innate immunity across all species. AMPs have become the focus of attention in recent years, as scientists are addressing antibiotic resistance, a public health crisis that has reached epidemic proportions. This family of peptides represents a promising alternative to current antibiotics due to their broad-spectrum antimicrobial activity and tendency to avoid resistance development. A subfamily of AMPs interacts with metal ions to potentiate antimicrobial effectiveness, and, as such, they have been termed metalloAMPs. In this work, we review the scientific literature on metalloAMPs that enhance their antimicrobial efficacy when combined with the essential metal ion zinc(II). Beyond the role played by Zn(II) as a cofactor in different systems, it is well-known that this metal ion plays an important role in innate immunity. Here, we classify the different types of synergistic interactions between AMPs and Zn(II) into three distinct classes. By better understanding how each class of metalloAMPs uses Zn(II) to potentiate its activity, researchers can begin to exploit these interactions in the development of new antimicrobial agents and accelerate their use as therapeutics.

We report the first total synthesis of 5-phenyl preacinetobactin and its characterization. The route was developed for the synthesis of preacinetobactin, the siderophore critical to the Gram-negative pathogen A. baumannii . It leverages a C5-substituted benzaldehyde as a key starting material and should enable the synthesis of similar analogs. 5-Phenyl preacinetobactin binds iron in a manner analogous to the natural siderophore, but it did not rescue growth in a strain of A. baumannii unable to produce preacinetobactin.

Antimicrobial peptides will be an essential component in combating the escalating issue of antibiotic resistance. Identifying synergistic combinations of two or more substances will increase the value of these peptides further. Several potential pitfalls in conducting synergy testing with peptides are discussed in detail. As case studies, we describe observations of AMP synergy with peptides, antibiotics, and metal ions as well as some of the mechanistic details that have been uncovered. The Bliss and Loewe models for synergy are presented prior to recommending protocols for conducting checkerboard, minimal inhibitory concentration, and time-kill assays. Establishing mechanisms of action and exploring the potential for resistance will be crucial to translate these studies into the clinic., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The need for optimized as well as standardized test systems of novel antimicrobial peptides (AMPs) was discussed by experts in the field at the International Meeting on Antimicrobial Peptides (IMAP) 2017 and the 2019 Gordon Research Conference (GRC) on Antimicrobial Peptides, and a survey related to this topic was circulated to participants to collate opinions. The survey included questions ranging from the relevance of susceptibility testing for understanding the mode of action of AMPs, to the importance of optimization and a degree of standardization of test methods and their clinical relevance. Based on the survey results, suggestions for future improvements in the research field are made.

In the search for novel broad-spectrum therapeutics to fight chronic infections, inflammation, and cancer, host defense peptides (HDPs) have garnered increasing interest. Characterizing their biologically-active conformations and minimum motifs for function represents a requisite step to developing them into efficacious and safe therapeutics. Here, we demonstrate that metallating HDPs with Cu 2+ is an effective chemical strategy to improve their cytotoxicity on cancer cells. Mechanistically, we find that prepared as Cu 2+ -complexes, the peptides not only physically but also chemically damage lipid membranes. Our testing ground features piscidins 1 and 3 (P1/3), two amphipathic, histidine-rich, membrane-interacting, and cell-penetrating HDPs that are α-helical bound to membranes. To investigate their membrane location, permeabilization effects, and lipid-oxidation capability, we employ neutron reflectometry, impedance spectroscopy, neutron diffraction, and UV spectroscopy. While P1-apo is more potent than P3-apo, metallation boosts their cytotoxicities by up to two- and seven-fold, respectively. Remarkably, P3-Cu 2+ is particularly effective at inserting in bilayers, causing water crevices in the hydrocarbon region and placing Cu 2+ near the double bonds of the acyl chains, as needed to oxidize them. This study points at a new paradigm where complexing HDPs with Cu 2+ to expand their mechanistic reach could be explored to design more potent peptide-based anticancer therapeutics.

The development of antimicrobial peptides (AMPs) as potential therapeutics requires resolving the foundational principles behind their structure-activity relationships. The role of histidine residues within AMPs remains a mystery despite the fact that several potent peptides containing this amino acid are being considered for further clinical development. Gaduscidin-1 (Gad-1) is a potent AMP from Atlantic cod fish that has a total of five His residues. Herein, the role of His residues and metal-potentiated activity of Gad-1 was studied. The five His residues contribute to the broad-spectrum activity of Gad-1. We demonstrated that Gad-1 can coordinate two Cu 2+ ions, one at the N-terminus and one at the C-terminus, where the C-terminal binding site is a novel Cu 2+ binding motif. High affinity Cu 2+ binding at both sites was observed using mass spectrometry and isothermal titration calorimetry. Electron paramagnetic resonance was used to determine the coordination environment of the Cu 2+ ions. Cu 2+ binding was shown to be responsible for an increase in antimicrobial activity and a new mode of action. Along with the traditional AMP mode of action of pore formation, Gad-1 in the presence of Cu 2+ (per)oxidizes lipids. Importantly, His3, His11, His17, and His21 were found to be important to lipid (per)oxidation. This insight will help further understand the inclusion and role of His residues in AMPs, the role of the novel C-terminal binding site, and can contribute to the field of designing potent AMPs that bind metal ions to potentiate activity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inspired by the architecture of the macrocycle of heme d1, a series of synthetic mono-, di- and tri-β-oxo-substituted porphyrinoid cobalt(ii) complexes were evaluated as electrocatalytic CO2 reducers, identifying complexes of unusually high efficiencies in generating multi-electron reduction products, including CH4.

Bacterial infections in cystic fibrosis (CF) patients are an emerging health issue and lead to a premature death. CF is a hereditary disease that creates a thick mucus in the lungs that is prone to bacterial biofilm formation, specifically Pseudomonas aeruginosa biofilms. These biofilms are very difficult to treat because many of them have antibiotic resistance that is worsened by the presence of extracellular DNA (eDNA). eDNA helps to stabilize biofilms and can bind antimicrobial compounds to lessen their effects. The metallo-antimicrobial peptide Gaduscidin-1 (Gad-1) eradicates established P. aeruginosa biofilms through a combination of modes of action that includes nuclease activity that can cleave eDNA in biofilms. In addition, Gad-1 exhibits synergistic activity when used with the antibiotics kanamycin and ciprofloxacin, thus making Gad-1 a new lead compound for the potential treatment of bacterial biofilms in CF patients., (© 2021 Wiley-VCH GmbH.)

The atomic-level tunability of molecular structures is a compelling reason to develop homogeneous catalysts for challenging reactions such as the electrochemical reduction of carbon dioxide to valuable C 1 -C n products. Of particular interest is methane, the largest component of natural gas. Herein, we report a series of three isomeric rhenium tricarbonyl complexes coordinated by the asymmetric diimine ligands 2-(isoquinolin-1-yl)-4,5-dihydrooxazole ( quin-1-oxa ), 2-(quinolin-2-yl)-4,5-dihydrooxazole ( quin-2-oxa ), and 2-(isoquinolin-3-yl)-4,5-dihydrooxazole ( quin-3-oxa ) that catalyze the reduction of CO 2 to carbon monoxide and methane, albeit the latter with a low efficiency. To our knowledge, these complexes are the first examples of rhenium(I) catalysts capable of converting carbon dioxide into methane. Re(quin-1-oxa)(CO) 3 Cl ( 1 ), Re(quin-2-oxa)(CO) 3 Cl ( 2 ), and Re(quin-3-oxa)(CO) 3 Cl ( 3 ) were characterized and studied using a variety of electrochemical and spectroscopic techniques. In bulk electrolysis experiments, the three complexes reduce CO 2 to CO and CH 4 . When the controlled-potential electrolysis experiments are performed at -2.5 V (vs Fc +/0 ) and in the presence of the Brønsted acid 2,2,2-trifluoroethanol, methane is produced with turnover numbers that range from 1.3 to 1.8. Isotope labeling experiments using 13 CO 2 atmosphere produce 13 CH 4 ( m / z = 17) confirming that methane originates from CO 2 reduction. Theoretical calculations are performed to investigate the mechanistic aspects of the 8e - /8H + reduction of CO 2 to CH 4 . A ligand-assisted pathway is proposed to be an efficient pathway in the formation of CH 4 . Delocalization of the electron density on the (iso)quinoline moiety upon reduction stabilizes the key carbonyl intermediate leading to additional reactivity of this ligand. These results should aid the development of more robust catalytic systems that produce CH 4 from CO 2 .

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

New cis -(1,2-azole)-aquo bis(2,2'-bipyridyl)ruthenium(II) (1,2-azole (az*H) = pzH (pyrazole), dmpzH (3,5-dimethylpyrazole), and indzH (indazole)) complexes are synthesized via chlorido abstraction from cis -[Ru(bipy) 2 Cl(az*H)]OTf. The latter are obtained from cis -[Ru(bipy) 2 Cl 2 ] after the subsequent coordination of the 1,2-azole. All the compounds are characterized by 1 H, 13 C, 15 N NMR spectroscopy as well as IR spectroscopy. Two chlorido complexes (pzH and indzH) and two aquo complexes (indzH and dmpzH) are also characterized by X-ray diffraction. Photophysical and electrochemical studies were carried out on all the complexes. The photophysical data support the phosphorescence of the complexes. The electrochemical behavior of all the complexes in an Ar atmosphere indicate that the oxidation processes assigned to Ru(II) → Ru(III) occurs at higher potentials in the aquo complexes. The reduction processes under Ar lead to several waves, indicating that the complexes undergo successive electron-transfer reductions that are centered in the bipy ligands. The first electron reduction is reversible. The electrochemical behavior in CO 2 media is consistent with CO 2 electrocatalyzed reduction, where the values of the catalytic activity [ i cat (CO 2 )/ i p (Ar)] ranged from 2.9 to 10.8. Controlled potential electrolysis of the chlorido and aquo complexes affords CO and formic acid, with the latter as the major product after 2 h. Photocatalytic experiments in MeCN with [Ru(bipy) 3 ]Cl 2 as the photosensitizer and TEOA as the electron donor, which were irradiated with >300 nm light for 24 h, led to CO and HCOOH as the main reduction products, achieving a combined turnover number (TON CO+HCOO - ) as high as 107 for 2c after 24 h of irradiation.

Antimicrobial photodynamic therapy (APDT) has gained increased attention because of its broad spectrum activity and lower likelihood to elicit bacterial resistance. Although many photosensitizers excel at eradicating Gram-positive bacterial infections, they are generally less potent when utilized against Gram-negative bacteria. We hypothesized that conjugating the DNA-targeting, antimicrobial peptide buforin II to a metal-based photosensitizer would result in a potent APDT agent. Herein, we present the synthesis and characterization of a buforin II-[Ru(bpy) 3 ] 2+ bioconjugate ( 1 ). The submicromolar activity of 1 against the multidrug-resistant strains Escherichia coli AR 0114 and Acinetobacter baumannii Naval-17 indicates strong synergy between the ruthenium complex and buforin II. Our mechanistic studies point to an increased rate of DNA damage by 1 compared to [Ru(bpy) 3 ] 2+ . These results suggest that conjugating metal complexes to antimicrobial peptides can lead to potent antimicrobial agents.

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials., (Copyright © 2020 Mercer, Torres, Duay, Lovie, Simpson, von Köckritz-Blickwede, de la Fuente-Nunez, O'Neil and Angeles-Boza.)

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (Δ recA::kan ) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Antimicrobial peptides (AMPs) represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Optimized synthetic versions incorporating additional design principles are needed. Here, we engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of two AMPs, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the in silico selected lead template, CM15, and its ATCUN analogs increased bacterial cell death. Mass spectrometry, circular dichroism, and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not increase the helical tendencies of the AMPs. CM15 ATCUN variants, when combined with Meropenem, streptomycin, or chloramphenicol, showed synergistic effects against E. coli (KpC+ 1812446) biofilms. Motif addition also reduced the hemolytic activity of the wild-type AMP and improved the survival rate of mice in a systemic infection model. The dependence of these bioactivities on the particular amino acids of the ATCUN motif highlights the possible use of size, charge, and hydrophobicity to fine-tune AMP biological function. Our data indicate that incorporating metal-binding motifs into peptide sequences leads to synthetic variants with modified biological properties. These principles may be applied to augment the activities of other peptide sequences.

Clavanin A (ClavA) is an antimicrobial peptide (AMP) whose antimicrobial activity is enhanced in the presence of Zn(II) ions. The antimicrobial activity of ClavA has been shown to increase 16-fold in the presence of Zn(II) ions. In this study, we investigate the potential sources of this enhancement, namely, the effect of Zn(II) binding on the helical conformation of ClavA and on the ClavA interaction with a model for gram-negative bacterial membranes. In addition, we investigate the effect of Zn(II) on the membrane mechanical properties. We employed all-atom equilibrium molecular dynamics simulations initiated from both fully helical and random coil structures of ClavA. We observe that Zn(II) can stabilize an existing helical conformation in the Zn(II)-binding region, but we do not observe induction of helical conformations in systems initiated in random coil configurations. Zn(II) binding to ClavA provides more favorable electrostatics for membrane association in the C-terminal region. This is evidenced by longer and stronger C-terminal-lipid interactions. Zn(II) is also capable of modulating the membrane properties in a manner which favors ClavA insertion and the potential for enhanced translocation into the cell. This work provides insights into the role of divalent metal cations in the antimicrobial activity of ClavA. This information can be used for the development of synthetic AMPs containing motifs that can bind metals (metalloAMPs) for therapeutic and medical purposes.

We report polymer-promoted cooperative catalysis of Cu for oxygen activation. A series of random copolymers containing dipicolylamine as binding motifs are designed to coordinate type-3 Cu sites. The Cu-copolymers show a 6-8-fold activity enhancement, compared to the molecular complex of Cu with an identical coordination site. Michaelis-Menten analysis demonstrates that the kinetic enhancement results from flexible polymer-promoted cooperative catalysis among multi-Cu sites despite the imposed thermodynamic barrier. These observations provide guidance for the bioinspired design of metallopolymers as soluble catalysts with high activity.

Copper (Cu) ions are critical in controlling bacterial infections, and successful pathogens like Mycobacterium tuberculosis (Mtb) possess multiple Cu resistance mechanisms. We report, as proof of concept, that a novel Cu hypersensitivity phenotype can be generated in mycobacteria, including Mtb, through a peptide, DAB-10, that is able to form reactive oxygen species (ROS) following Cu-binding. DAB-10 induces intramycobacterial oxidative stress in a Cu-dependent manner in vitro and during infection. DAB-10 penetrates murine macrophages and encounters intracellular mycobacteria. Significant intracellular Cu-dependent protection was observed when Mtb-infected macrophages were treated with DAB-10 alongside a cell-permeable Cu chelator. Treatment with the Cu chelator reversed the intramycobacterial oxidative shift induced by DAB-10. We conclude that DAB-10 utilizes the pool of phagosomal Cu ions in the host-Mtb interface to augment the mycobactericidal activity of macrophages while simultaneously exploiting the susceptibility of Mtb to ROS. DAB-10 serves as a model with which to develop next-generation, multifunctional antimicrobials.

Understanding the mechanism of water oxidation to dioxygen represents the bottleneck towards the design of efficient energy storage schemes based on water splitting. The investigation of kinetic isotope effects has long been established for mechanistic studies of various such reactions. However, so far natural isotope abundance determination of O 2 produced at solid electrode surfaces has not been applied. Here, we demonstrate that such measurements are possible. Moreover, they are experimentally simple and sufficiently accurate to observe significant effects. Our measured kinetic isotope effects depend strongly on the electrode material and on the applied electrode potential. They suggest that in the case of iron oxide as the electrode material, the oxygen evolution reaction occurs via a rate-determining O-O bond formation via nucleophilic water attack on a ferryl unit.

In this work, we examine the use of competitive 13C kinetic isotope effects (13C KIEs) on CO2 reduction reactions that produce CO and formic acid as a means to formulate reaction mechanisms. The findings reported here mark a further advancement in the combined 13C KIE measurements and theoretical calculations methodology for probing CO2 conversion reactions.

Host-defense peptides (HDPs) feature evolution-tested potency against life-threatening pathogens. While piscidin 1 (p1) and piscidin 3 (p3) are homologous and potent fish HDPs, only p1 is strongly membranolytic. Here, we hypothesize that another mechanism imparts p3 strong potency. We demonstrate that the N-termini of both peptides coordinate Cu 2+ and p3-Cu cleaves isolated DNA at a rate on par with free Cu 2+ but significantly faster than p1-Cu. On planktonic bacteria, p1 is more antimicrobial but only p3 features copper-dependent DNA cleavage. On biofilms and persister cells, p3-Cu is more active than p1-Cu, commensurate with stronger peptide-induced DNA damage. Molecular dynamics and NMR show that more DNA-peptide interactions exist with p3 than p1, and the peptides adopt conformations simultaneously poised for metal- and DNA-binding. These results generate several important conclusions. First, homologous HDPs cannot be assumed to have identical mechanisms since p1 and p3 eradicate bacteria through distinct relative contributions of membrane and DNA-disruptive effects. Second, the nuclease and membrane activities of p1 and p3 show that naturally occurring HDPs can inflict not only physicochemical but also covalent damage. Third, strong nuclease activity is essential for biofilm and persister cell eradication, as shown by p3, the homolog more specific toward bacteria and more expressed in vascularized tissues. Fourth, p3 combines several physicochemical properties (e.g., Amino Terminal Copper and Nickel binding motif; numerous arginines; moderate hydrophobicity) that confer low membranolytic effects, robust copper-scavenging capability, strong interactions with DNA, and fast nuclease activity. This new knowledge could help design novel therapeutics active against hard-to-treat persister cells and biofilms., (© 2017 Federation of European Biochemical Societies.)

A heterogeneous copper oxide supported on mesoporous manganese oxide (meso Cu/MnO x ) was explored for Ullmann-type cross-coupling reactions. An inverse micelle-templated evaporation-induced self-assembly method with in situ addition of copper was adopted to synthesize the mesoporous catalyst. Broad substrate scope and excellent functional group tolerability in C-O, C-N, and C-S bond formation reactions were observed using the optimized reaction conditions. The catalytic protocol was ligand free, and the catalyst was reusable without any significant loss of activity. The kinetic and Hammett analyses provided evidence for oxidative addition to a Cu(I) reaction center followed by nucleophilic addition and reductive elimination at the active copper oxide surface. Rate acceleration was observed for aryl halides with electron-withdrawing groups. The Hammett analysis determined ρ = +1.0, indicative of an oxidative addition, whereas the electronic effect in the phenol ring (ρ = -2.9) was indicative of coordination to a metal ion. Theoretically, the oxidative addition of the aryl halides is assisted by the ligand environment of the copper center. Relevant mechanistic implications are discussed on the basis of the experimental and computational results.

The biophysical properties of cellular membranes intimately influence the delivery of cargoes into cells by cell-penetrating peptides (CPPs) and the bactericidal activity of antimicrobial peptides (AMPs). Here, we discuss how lipid oxidation creates important chemical and biophysical changes in membranes, and hypothesize about the observed synergy between oxidized membranes and membrane-active peptides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Metal-containing single chain polymeric nanoparticles (SCPNs) can be used as synthetic mimics of metalloenzymes. Currently, the role of the folded polymer backbones on the activity and selectivity of metal sites is not clear. Herein, we report our findings on how polymeric frameworks modulate the coordination of Cu sites and the catalytic activity/selectivity of Cu-containing SCPNs mimicking monophenol hydroxylation reactions. Imidazole-functionalized copolymers of poly(methyl methacrylate- co -3-imidazolyl-2-hydroxy propyl methacrylate) were used for intramolecular Cu-imidazole binding that triggered the self-folding of polymers. Polymer chains imposed steric hindrance which yielded unsaturated Cu sites with an average coordination number of 3.3. Cu-containing SCPNs showed a high selectivity for the hydroxylation reaction of phenol to catechol, >80%, with a turnover frequency of >870 h -1 at 60 °C. The selectivity was largely influenced by the flexibility of the folded polymer backbone where a more flexible polymer backbone allows the cooperative catalysis of two Cu sites. The second coordination sphere provided by the folded polymer that has been less studied is therefore critical in the design of active mimics of metalloenzymes.

Cell delivery or cell killing processes often involve the crossing or disruption of cellular membranes. We review how, by modifying the composition and properties of membranes, membrane oxidation can be exploited to enhance the delivery of macromolecular cargoes into live human cells. We also describe how membrane oxidation can be utilized to achieve efficient killing of bacteria by antimicrobial peptides. Finally, we present recent evidence highlighting how membrane oxidation is intimately engaged in natural biological processes such as antigen delivery in dendritic cells and in the killing of bacteria by antimicrobial peptides. Overall, the insights that have been recently gained in this area should facilitate the development of more effective delivery technologies and antimicrobial therapeutic approaches.

A series of rhenium tricarbonyl complexes coordinated by asymmetric diimine ligands containing a pyridine moiety bound to an oxazoline ring were synthesized, structurally and electrochemically characterized, and screened for CO 2 reduction ability. The reported complexes are of the type Re(N-N)(CO) 3 Cl, with N-N = 2-(pyridin-2-yl)-4,5-dihydrooxazole (1), 5-methyl-2-(pyridin-2-yl)-4,5-dihydrooxazole (2), and 5-phenyl-2-(pyridin-2-yl)-4,5-dihydrooxazole (3). The electrocatalytic reduction of CO 2 by these complexes was observed in a variety of solvents and proceeds more quickly in acetonitrile than in dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). The analysis of the catalytic cycle for electrochemical CO 2 reduction by 1 in acetonitrile using density functional theory (DFT) supports the C-O bond cleavage step being the rate-determining step (RDS) (ΔG ⧧ = 27.2 kcal mol -1 ). The dependency of the turnover frequencies (TOFs) on the donor number (DN) of the solvent also supports that C-O bond cleavage is the rate-determining step. Moreover, the calculations using explicit solvent molecules indicate that the solvent dependence likely arises from a protonation-first mechanism. Unlike other complexes derived from fac-Re(bpy)(CO) 3 Cl (I; bpy = 2,2'-bipyridine), in which one of the pyridyl moieties in the bpy ligand is replaced by another imine, no catalytic enhancement occurs during the first reduction potential. Remarkably, catalysts 1 and 2 display relative turnover frequencies, (i cat /i p ) 2 , up to 7 times larger than that of I.

Tunicates have been used as primitive models for understanding cell-mediated and humoral immunity. Clavanin A (ClavA) is one member of a family of antimicrobial peptides produced by the solitary tunicate Styela clava. In this work, we demonstrate that ClavA utilizes Zn 2+ ions to potentiate its antimicrobial activity not only by reducing the concentration at which the peptide inhibits the growth of bacteria but also by increasing the rate of killing. Membrane depolarization, β-galactosidase leakage, and potassium leakage assays indicate that ClavA is membrane active, forms small pores, but induces cell death by targeting an intracellular component. ClavA and ClavA-Zn 2+ added to Escherichia coli and imaged by confocal microscopy translocate across the cell membrane. E. coli mutants lacking the functional Zn 2+ import system are less susceptible to ClavA, suggesting that the synergistic activity between ClavA and Zn 2+ has a cytoplasmic target, which is further supported by its nucleolytic activity. Overall, these studies identify a remarkable new mechanism by which zinc contributes to the immune response in the tunicate S. clava.

Herein, we report a heterogeneous, aerobic, additive-free and environmentally benign catalytic protocol for oxidative aromatization of saturated nitrogen-heterocycles using a mesoporous manganese oxide material. The aromatized products can be separated by easy filtration and the catalyst is reusable for at least four cycles. Mechanistic investigation provides evidence for radical intermediates, a multi-electron redox cycle between Mn centers, and an oxygen exchange mechanism.

Ticks transmit multiple pathogens to different hosts without compromising their health. Their ability to evade microbial infections is largely a result of their effective innate immune response including various antimicrobial peptides. Therefore, a deep understanding of how ticks (and other arthropod vectors) control microbial loads could lead to the design of broad-spectrum antimicrobial agents. In this paper we study the role of the amino-terminal copper and nickel (ATCUN)-binding sequence in the peptide ixosin, isolated from the salivary glands of the hard tick Ixodes sinensis. Our results indicate that the ATCUN motif is not essential to the potency of ixosin, but is indispensable to its oxidative mechanism of action. Specifically, the ATCUN motif promotes dioxygen- and copper-dependent lipid (per)oxidation of bacterial membranes in a temporal fashion coinciding with the onset of bacterial death. Microscopy and studies on model membranes indicate that the oxidized phospholipids are utilized as potential targets of ixosin B (another tick salivary gland peptide) involving its delocalization to the bacterial membrane, thus resulting in a synergistic effect. Our proposed mechanism of action highlights the centrality of the ATCUN motif to ixosin's mechanism of action and demonstrates a novel way in which (tick) antimicrobial peptides (AMPs) utilize metal ions in its activity. This study suggests that ticks employ a variety of effectors to generate an amplified immune response, possibly justifying its vector competence.

Piscidins were the first antimicrobial peptides discovered in the mast cells of vertebrates. While two family members, piscidin 1 (p1) and piscidin 3 (p3), have highly similar sequences and α-helical structures when bound to model membranes, p1 generally exhibits stronger antimicrobial and hemolytic activity than p3 for reasons that remain elusive. In this study, we combine activity assays and biophysical methods to investigate the mechanisms underlying the cellular function and differing biological potencies of these peptides, and report findings spanning three major facets. First, added to Gram-positive (Bacillus megaterium) and Gram-negative (Escherichia coli) bacteria at sublethal concentrations and imaged by confocal microscopy, both p1 and p3 translocate across cell membranes and colocalize with nucleoids. In E. coli, translocation is accompanied by nonlethal permeabilization that features more pronounced leakage for p1. Second, p1 is also more disruptive than p3 to bacterial model membranes, as quantified by a dye-leakage assay and (2)H solid-state NMR-monitored lipid acyl chain order parameters. Oriented CD studies in the same bilayers show that, beyond a critical peptide concentration, both peptides transition from a surface-bound state to a tilted orientation. Third, gel retardation experiments and CD-monitored titrations on isolated DNA demonstrate that both peptides bind DNA but p3 has stronger condensing effects. Notably, solid-state NMR reveals that the peptides are α-helical when bound to DNA. Overall, these studies identify two polyreactive piscidin isoforms that bind phosphate-containing targets in a poised amphipathic α-helical conformation, disrupt bacterial membranes, and access the intracellular constituents of target cells. Remarkably, the two isoforms have complementary effects; p1 is more membrane active, while p3 has stronger DNA-condensing effects. Subtle differences in their physicochemical properties are highlighted to help explain their contrasting activities.

The emergence of antibiotic resistant strains of bacteria has resulted in the need to develop more potent antimicrobials that target microorganisms in a novel manner. Antimicrobial Peptides (AMPs) show great potential for drug development because of their broad activity and unique mechanism of action. Several AMPs contain an Amino Terminal Copper and Nickel (ATCUN) binding motif; however, its function has not yet been determined. We have previously demonstrated that the activity of a truncated version of Buforin II (sh-Buforin, RAGLQFPVGRVHRLLRK-NH2) increases by the addition of an ATCUN motif. We now focus our current studies on understanding the effect of: 1) a positively charged ATCUN sequence, and 2) l-to-d amino acid substitution on the hybrid peptides. We identified that the addition of a positively charged ATCUN motif increases the affinity of the ATCUN-AMP for DNA but does not always result in an enhanced antimicrobial activity over a neutral ATCUN motif. The all-d peptides exhibited up to a 32-fold increase in antimicrobial activity compared to the all-l peptides. The larger activity of the all-d peptides is the result of a larger DNA cleavage activity and higher stability towards proteolysis. Cytotoxicity assays determined that, at their MIC, these peptides caused less than 8% hemolysis and, at 128 μM, no toxicity to HeLa and HEK293 cell lines. These results indicate that the ATCUN-AMP hybrids are an attractive alternative for treating infectious diseases and provide key insights into the role of the ATCUN motif in naturally-occurring AMPs., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Competitive (13)C and (18)O kinetic isotope effects (KIEs) on CO2 reduction catalyzed by Re(bpy)(CO)3Cl () under photocatalytic conditions and using Na(Hg) amalgam as a sacrificial reducing agent are reported. The analysis of the measured KIEs rules out the outer-sphere electron transfer mechanism and indicates that CO2 binding to the reduced rhenium complex is the rate determining step in the reduction of CO2 by under these conditions.

Antimicrobial peptides (AMPs) are broad spectrum antimicrobial agents that act through diverse mechanisms, this characteristic makes them suitable starting points for development of novel classes of antibiotics. We have previously reported the increase in activity of AMPs upon addition of the Amino Terminal Copper and Nickel (ATCUN) Binding Unit. Herein we synthesized the membrane active peptide, Anoplin and two ATCUN-Anoplin derivatives and show that the increase in activity is indeed due to the ROS formation by the Cu(II)-ATCUN complex. We found that the ATCUN-Anoplin peptides were up to four times more potent compared to Anoplin alone against standard test bacteria. We studied membrane disruption, and cellular localization and found that addition of the ATCUN motif did not lead to a difference in these properties. When helical content was calculated, we observed that ATCUN-Anoplin had a lower helical composition. We found that ATCUN-Anoplin are able to oxidatively damage lipids in the bacterial membrane and that their activity trails the rate at which ROS is formed by the Cu(II)-ATCUN complexes alone. This study shows that addition of a metal binding tripeptide motif is a simple strategy to increase potency of AMPs by conferring a secondary action., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon metal binding. The combination of these two peptidic constructs could lead to a novel class of dual-acting antimicrobial agents. To test this hypothesis, a set of ATCUN binding motifs were screened for their ability to induce ROS formation, and the most potent were then used to modify AMPs with different modes of action. ATCUN binding motif-containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apoptotic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin (RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be more active than the parent AMPs against a panel of clinically relevant bacteria. The lower minimum inhibitory concentration (MIC) values for the ATCUN-anoplin peptides are attributed to the higher pore-forming activity along with their ability to cause ROS-induced membrane damage. The addition of the ATCUN motifs to PAP also increases its ability to disrupt membranes. DNA damage is the major contributor to the activity of the ATCUN-sh-buforin peptides. Our findings indicate that the addition of ATCUN motifs to AMPs is a simple strategy that leads to AMPs with higher antibacterial activity and possibly to more potent, usable antibacterial agents., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The mechanism of O(2) reduction by copper amine oxidase from Arthrobacter globiformus (AGAO) is analyzed in relation to the cobalt-substituted protein. The enzyme utilizes a tyrosine-derived topaquinone cofactor to oxidize primary amines and reduce O(2) to H(2)O(2). Steady-state kinetics indicate that amine-reduced CuAGAO is reoxidized by O(2) >10(3) times faster than the CoAGAO analogue. Complementary spectroscopic studies reveal that the difference in the second order rate constant, k(cat)/K(M)(O(2)), arises from the more negative redox potential of Co(III/II) in relation to Cu(II/I). Indistinguishable competitive oxygen-18 kinetic isotope effects are observed for the two enzymes and modeled computationally using a calibrated density functional theory method. The results are consistent with a mechanism where an end-on (η(1))-metal bound superoxide is reduced to an η(1)-hydroperoxide in the rate-limiting step.

Molecular oxygen is produced from water via the following reaction of potassium ferrate (K(2)FeO(4)) in acidic solution: 4[H(3)Fe(VI)O(4)](+) + 8H(3)O(+) → 4Fe(3+) + 3O(2) + 18H(2)O. This study focuses upon the mechanism by which the O-O bond is formed. Stopped-flow kinetics at variable acidities in H(2)O and D(2)O are used to complement the analysis of competitive oxygen-18 kinetic isotope effects ((18)O KIEs) upon consumption of natural abundance water. The derived (18)O KIEs provide insights concerning the identity of the transition state. Water attack (WA) and oxo-coupling (OC) transition states were evaluated for various reactions of monomeric and dimeric ferrates using a calibrated density functional theory protocol. Vibrational frequencies from optimized isotopic structures are used here to predict (18)O KIEs for comparison to experimental values determined using an established competitive isotope-fractionation method. The high level of agreement between experimental and theoretic isotope effects points to an intramolecular OC mechanism within a di-iron(VI) intermediate, consistent with the analysis of the reaction kinetics. Alternative mechanisms are excluded based on insurmountably high free energy barriers and disagreement with calculated (18)O KIEs.

Oxygen isotope fractionation is applied for the first time to probe the catalytic oxidation of water using a widely studied ruthenium complex, [Ru(II)(tpy)(bpy)(H(2)O)](ClO(4))(2) (bpy = 2,2'-bipyridine; tpy = 2,2';6",2"-terpyridine). Competitive oxygen-18 kinetic isotope effects ((18)O KIEs) derived from the ratio of (16,16)O(2) to (16,18)O(2) formed from natural-abundance water vary from 1.0132 ± 0.0005 to 1.0312 ± 0.0004. Experiments were conducted with cerium(IV) salts at low pH and a photogenerated ruthenium(III) tris(bipyridine) complex at neutral pH as the oxidants. The results are interpreted within the context of catalytic mechanisms using an adiabatic formalism to ensure the highest barriers for electron-transfer and proton-coupled electron-transfer steps. In view of these contributions, O-O bond formation is predicted to be irreversible and turnover-limiting. The reaction with the largest (18)O KIE exhibits the greatest degree of O-O coupling in the transition state. Smaller (18)O KIEs are observed due to multiple rate-limiting steps or transition-state structures which do not involve significant O-O motion. These findings provide benchmarks for systematizing mechanisms of O-O bond formation, the critical step in water oxidation by natural and synthetic catalysts. In addition, the measurements introduce a new tool for calibrating computational studies using relevant experimental data., (© 2012 American Chemical Society)

Rice α-(di)oxygenase mediates the regio- and stereospecific oxidation of fatty acids using a persistent catalytic tyrosyl radical. Experiments conducted in the physiological O(2) concentration range, where initial hydrogen atom abstraction from the fatty acid occurs in a kinetically reversible manner, are described. Our findings indicate that O(2)-trapping of an α-carbon radical is likely to reversibly precede reduction of a 2-(R)-peroxyl radical intermediate in the first irreversible step. A mechanism of concerted proton-coupled electron transfer is proposed on the basis of natural abundance oxygen-18 kinetic isotope effects, deuterium kinetic isotope effects, and calculations at the density functional level of theory, which predict a polarized transition state in which electron transfer is advanced to a greater extent than proton transfer. The approach outlined should be useful for identifying mechanisms of concerted proton-coupled electron transfer in a variety of oxygen-utilizing enzymes.

The steady-state catalytic mechanism of a fatty acid α-(di)oxygenase is examined, revealing that a persistent tyrosyl radical (Tyr379(•)) effects O(2) insertion into C(α)-H bonds of fatty acids. The initiating C(α)-H homolysis step is characterized by apparent rate constants and deuterium kinetic isotope effects (KIEs) that increase hyperbolically upon raising the concentration of O(2). These results are consistent with H(•) tunneling, transitioning from a reversible to an irreversible regime. The limiting deuterium KIEs increase from ∼30 to 120 as the fatty acid chain is shortened from that of the native substrate. In addition, activation barriers increase in a manner that reflects decreased fatty acid binding affinities. Anaerobic isotope exchange experiments provide compelling evidence that Tyr379(•) initiates catalysis by H(•) abstraction. C(α)-H homolysis is kinetically driven by O(2) trapping of the α-carbon radical and reduction of a putative peroxyl radical intermediate to a 2(R)-hydroperoxide product. These findings add to a body of work which establishes large-scale hydrogen tunneling in proteins. This particular example is novel because it involves a protein-derived amino acid radical.

We describe the synthesis and cellular delivery properties of multivalent and branched delivery systems consisting of cell-penetrating peptides assembled onto a peptide scaffold using native chemical ligation. A trimeric delivery system presenting three copies of the prototypical cell-penetrating peptide TAT shows an endosomolytic activity much higher than its monomeric and dimeric counterparts. This novel reagent promotes the endosomal release of macromolecules internalized into cells by endocytosis, and as a result, it can be used to achieve cytosolic delivery of bioactive but cell-impermeable macromolecules in either cis (covalent conjugation) or trans (simple coincubation).

A series of dirhodium(II,II) complexes of the type cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppn)(L)](2+), where dppn = benzo[i]dipyrido[3,2-a:2',3'-h]quinoxaline and L = 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-f:2'3'-h]quinoxaline (dpq, 3), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4), and dppn (5), were synthesized and their photophysical properties investigated to probe their potential usefulness as photodynamic therapy agents. The ability of the complexes to bind and photocleave DNA was also probed, along with their toxicity toward human skin cells in the dark and when irradiated with visible light. Nanosecond time-resolved absorption measurements established that the lowest energy excited state in 1-5 is dppn-localized (3)pipi* with lifetimes of 2.4-4.1 micros in DMSO, with spectral features similar to those of the free dppn ligand (tau = 13.0 micros in CHCl(3)). Complexes 1-4 photocleave DNA efficiently via a mechanism that is mostly mediated by reactive oxygen species, including (1)O(2) and O(2)(-). The DNA photocleavage by 5 is significantly lower than those measured for 1-4 in air, and the absence of O(2) in solution or the addition of azide, SOD (superoxide dismutase), or D(2)O does not affect the efficiency of the photocleavage reaction. The toxicity of 1-5 toward Hs-27 human skin fibroblasts is significantly greater upon irradiation with visible light than in the dark. In contrast to the DNA photocleavage results, 5 exhibits the largest increase in toxicity upon irradiation within the series. These results are explained in terms of the known ability of the complexes to transverse cellular membranes, the toxicity of the complexes in the dark, and their photophysical properties.

The series of complexes cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppn)(L)](2+), where dppn = benzo[i]dipyrido[3,2-a:2',3'-c] phenazine, and L = bpy (2,2'-bipyridine) (1), phen (1,10-phenanthroline) (2), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline) (3), dppz (dipyrido[3,2-a:2',3'-c]phenazine) (4), and dppn (5) were synthesized and their effect on the human cancer cells HeLa and COLO-316 was monitored. Complexes 1 and 2 interact with DNA through intercalation, whereas compounds 3-5 bind only electrostatically. It was found that the dirhodium complex 4 is the most effective compound at inhibiting cell viability of the human cancer cells HeLa and COLO-316. A general conclusion is that the hydrophobicity of the compounds correlates with their in cellulo activity in both cell lines. The ability of the compounds to reach nuclear DNA and form adducts was explored using the comet assay. The results indicate that compounds 1-5 either do not form adducts with DNA that are detrimental to the cell or that they are successfully repaired by the cellular machinery. The results of an annexin V assay indicate that compounds 1-4 trigger apoptosis, whereas compound 5 clearly does not. These findings are significant because they support the contention that dirhodium complexes can be tuned to direct their effect to cellular targets other than nuclear DNA.

The promising antitumor activity of dirhodium complexes has been known for over 30 years. There remains, however, a general lack of understanding of their activity in cellulo. In this study, we report the DNA interactions and activity in living cells of six monosubstituted dirhodium(II,II) complexes of general formula [Rh(2)(mu-O(2)CCH(3))(2)(eta(1)-O(2)CCH(3))(L)(CH(3)OH)](+), where L = bpy (2,2'-bipyridine) (1), phen (1,10-phenanthroline) (2), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline) (3), dppz (dipyrido[3,2-a:2',3'-c]phenazine) (4), dppn (benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (5), and dap (4,7-dihydrodibenzo[de,gh][1,10]phenanthroline) (6). DNA interactions were investigated by UV/visible spectroscopy, relative viscosity measurements, and electrophoretic mobility shift assay. These measurements indicate that compound 5 exhibits the strongest interaction with DNA. Compound 5 also causes the most damage to DNA after cellular internalization, as evaluated by the alkaline comet assay. Compound 5, however, is not the most effective at inhibiting cell viability of the human cancer cells HeLa and COLO-316. The greater hydrophobicity of 5 as compared to that of 4, which is the most effective compound in the series, hinders its ability to reach its cellular target(s). Data from modulation studies of glutathione using N-acetylcysteine and L-buthionine-sulfoximine indicate that changes in glutathione levels do not affect the activity of these particular dirhodium complexes. These results suggest that glutathione is not the only agent involved in the deactivation of these dirhodium complexes.

The series of dirhodium(II) complexes cis-[Rh(2)(O(2)CCH(3))(2)(R(1)R(2)dppz)(2)](2+) 1-6 (R(1) = R(2) = H, MeO, Me, Cl, NO(2) for 1-4, 6, respectively, and R(1)= H, R(2) = CN for 5), coordinated to R(1)R(2)dppz ligands with electron-donating or -withdrawing substituents at positions 7,8 of dppz (dppz = dipyrido[3,2-a:2',3'-c]phenazine), were synthesized and their effect on the transcription process in vitro was monitored. Complexes 1-6 are easily reduced, readily oxidize cysteine, and engage in redox-based reactions with T7-RNA Polymerase (T7-RNAP), which contains accessible thiol groups. Transcription is inhibited in vitro by 1-6 via formation of intra- and inter-T7-RNAP disulfide bonds that affect the enzyme critical sulfhydryl cysteine groups. The progressively increasing electron-withdrawing character of the dppz substituents (MeO < Me < H < Cl < CN < NO(2)) gives rise to the order 2 < 3 < 1 < 4 < 5 < 6 for the measured IC(50) values of 1-6. The ease of reduction for 1-6 is consistent with the energies of the dppz-centered lowest unoccupied molecular orbitals (LUMOs), which decrease with the electron-withdrawing character of the dppz substituents. The ligand-centered reductions for 1-6 are supported by electron paramagnetic resonance (EPR) studies which support the conclusion that reduction of 1-6 leads to the formation of dppz centered radicals [Rh(2)(O(2)CCH(3))(2)(R(1)R(2)dppz)(2)](*+) with isotropic g values approximately 2.003 which are essentially identical to the reported value for the free radical dppz anions. The EPR results are corroborated by density functional theory (DFT) calculations, which indicate that the complexes contain dppz-based LUMOs primarily phenazine (phz) in character; the unpaired electron is completely delocalized in the phenazine orbitals in 4-6. The low IC(50) values for 1-6 lend further support to the fact that they exhibit redox-based activity with the enzyme and lead to the conclusion that the complexes constitute a sensitive redox-regulated series of T7-RNAP inhibitors with the potential to control or inhibit other important biochemical processes.

The ultrafast kinetics of ligand exchange of cis-[Ru(bpy)(2)(CH(3)CN)(2)](2+) were measured in H(2)O and CH(3)CN. The formation of the (3)MLCT excited-state and a five-coordinate intermediate are observed in both solvents within 2 ps after excitation (310 nm, fwhm approximately 300 fs). The (3)MLCT excited-state undergoes vibrational cooling (5-6 ps), then decays to regenerate the ground-state with a lifetime of approximately 50 ps. In CH(3)CN, ligand recombination takes place in 28 ps, while the formation of cis-[Ru(bpy)(2)(CH(3)CN)(H(2)O)](2+) in H(2)O takes place with tau = 77 ps.

The reactivities toward biomolecules of a series of three dirhodium(II,II) complexes that possess an increasing number of accessible axial coordination sites are compared. In cis-[Rh2(OAc)2(np)2]2+ (1; np=1,8-naphthyridine) both axial sites are available for coordination, whereas for cis-[Rh2(OAc)2(np)(pynp)]2+ (2; pynp=2-(2-pyridyl)1,8-naphthyridine) and cis-[Rh2(OAc)2(pynp)2]2+ (3) the bridging pynp ligand blocks one and two of the axial coordination sites in the complexes, respectively. The electronic absorption spectra of the complexes are consistent with strong metal-to-ligand charge transfer transitions at low energy and ligand-centered peaks localized on the np and/or pynp ligands in the UV and near-UV regions. Time-dependent density functional theory calculations were used to aid in the assignments. The three complexes exhibit metal-centered oxidations and reductions, localized on the aromatic ligands. The ability of the complexes to stabilize duplex DNA and to inhibit transcription in vitro is greatly affected by the availability of an open axial coordination site. The present work shows that open axial coordination sites on the dirhodium complexes are necessary for biological activity.