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Tyrosinase (TYR) is a key enzyme that catalyzes the synthesis of melanin in plants, microorganisms and mammalian cells. Kojic acid (KA) is a well-known TYR inhibitor widely used as a popular cosmetic skin-lightening ingredient. However, KA is reported to have poor inhibitory activity against pigmentation within intact melanocytes or in clinical assays. In this study, a series of dihydropyrano[3,2-b] chromenedione (DHPC) (1a, 2a, 3a, 4a, 5a, 6a and 7a) and 1,8-dioxooctahydroxanthene (DOHX) (1b, 2b, 3b and 4b) derivatives were evaluated using a DPPH radical-scavenging assay. These results showed that 7a exhibited the most potent radical-scavenging activity. Compound 7a was characterised by X-ray crystallographic studies. Molecular docking was carried out to shed light on the mode of action and types of interaction between the compounds and the target. A metal-binding study suggested that these synthetic heterocyclic compounds may behave as competitive inhibitors for the L-DOPA binding site of the TYR. Finally, molecular modeling provided important insight into the mechanism of binding interactions with the TYR copper active site.

We report on the chemical analysis of a mud dauber wasp nest-associated fungus, Penicillium sp. CMB-MD22, leading to the discovery and structure elucidation of three known (1-3) and two new (4 and 5) anthrones, and a family of new and known bianthrones, neobulgarones 6-23. Detection and structure elucidation of 1-23 was supported by detailed spectroscopic analysis, as well as chemical (thermal) transformations, and global natural products social (GNPS) molecular networking. An empirical approach using HPLC retention times was effective at differentiating anti from syn bianthrone isomers, while a facile thermal equilibration was shown to favor anti over syn isomers. The neobulgarones 6-23 are natural products, and a crude extract rich in 6-23 exhibits selective antifungal activity against a co-isolated mud dauber wasp nest-associated fungus, suggestive of a possible ecological role as an antifungal chemical defense.

Using a molecular networking guided strategy, chemical analysis of the Australian mullet fish gastrointestinal tract-derived fungus Amauroascus sp. CMB-F713 yielded a family of polyketide pyrones, amaurones A-I (1-9), featuring an unprecedented carbon skeleton. Structures were assigned to 1-9 by detailed spectroscopic analysis (including X-ray analysis of 1), biosynthetic considerations, and chemical interconversions. For example, the orthoacetate 5 was unstable when stored dry at room temperature, transforming to the monoacetates 2 and 3, while mild heating (40 °C) prompted quantitative conversion of 3 to 2, via an intramolecular trans-acetylation. Likewise, during handling, the monoacetate 1 was prone to intramolecular trans-acetylation, leading to an equilibrium mixture with the isomeric monoacetate amaurone J (10), confirmed when partial hydrolysis of the diacetate 2 yielded the monoacetates 1 and 10 and the triol amaurone K (11).

The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

Mediator‐based extracellular electron transfer (EET) pathways can balance the redox metabolism of microbes. However, such electro‐biosynthesis processes are constrained by the unknown underlying EET mechanisms. In this paper, Pseudomonas putida was studied to systematically investigate its EET pathway to transition metal complexes (i. e., [Fe(CN)6]3−/4− and [Co(bpy)3]3+/2+; bpy=2,2′‐bipyridyl) under anaerobic conditions. Comparative proteomics showed the aerobic respiratory components were upregulated in a bioelectrochemical system without oxygen, suggesting their potential contribution to EET. Further tests found inhibiting cytochrome c oxidase activity by NaN3 and NADH dehydrogenase by rotenone did not significantly change the current output. However, the EET pathway was completely blocked, while cytochrome c reductase activity was inhibited by antimycin A. Although it cannot be excluded that cytochrome c and the periplasmic subunit of cytochrome c oxidase donate electrons to the transition metal complexes, these results strongly demonstrate that cytochrome c reductase is a key complex for the EET pathway., Liquid wires for biohybrid systems: The electron transfer pathway between redox metal‐complexes and biological respiratory proteins is systematically investigated and revealed. Such a pathway can be readily applied to many non‐electrogenic microorganisms and thus expand the application of microbial electrochemical technology in many fields such as industrial biotechnology and biosensors.

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F–M (1–8) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure–activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F (1) was particularly noteworthy, with a 2.5 μM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).

Dysphania is an abundant genus of plants, many of which are endemic to the Australian continent, occurring primarily in arid and temperate zones. Despite their prevalence, very few investigations into the phytochemistry of native Dysphania have been undertaken. Described herein, is the isolation and elucidation of two enantiomeric diastereomers of humulene diepoxide C from D. kalpari and D. rhadinostachya, of which unassigned diastereomers of humulene diepoxide C have been previously reported as components in beer brewed from aged hops. In addition, two (+)-humulene diepoxiols (humulene diepoxiol C-I and C-II) were isolated from D. rhadinostachya. Analysis of Chinook hops oil confirmed the presence of both humulene diepoxide C-I and C-II as trace components, and in turn enabled GC-MS peak assignment to the relative stereochemistry. Anticancer assays did not reveal any significant activity for the (+)-humulene diepoxides. Antifungal assays showed good activity against a drug-resistant strain of C. auris, with MIC50 values of 8.53 and 4.91 μm obtained for (+)-humulene diepoxide C-I and C-II, respectively.

Chemical investigations into solid phase cultivations of an Australian sheep station pasture plant-derived Streptomyces sp. CMB-PB042, yielded the rare enamine naphthopyranoquinones BE-54238A (1) and BE-54238B (2), together with four new analogues, glenthenamines B–D (4–6) and F (8), and two handling artifacts, glenthenamines A (3) and E (7). Single crystal X-ray analyses of 1–2 resolved configurational ambiguities in the scientific literature, while detailed spectroscopic analysis and biosynthetic considerations assigned structures inclusive of absolute configuration to 3–8. We propose a plausible sequence of biosynthetic transformations linking structural and configurational features of 1–8, and apply a novel Schiff base "fishing" approach to detect a key deoxyaminosugar precursor. These enamine naphthopyranoquinone disclose a new P-gp inhibitory pharmacophore capable of reversing doxorubicin resistance in P-gp overexpressing colon carcinoma cells.

Chemical investigation on a Streptomyces sp. strain MS180069 isolated from a sediment sample collected from the South China Sea, yielded the new benzo[f]isoindole-dione alkaloid, bhimamycin J (1). The structure was determined by extensive spectroscopic analysis, including HRMS, 1D, 2D NMR, and X-ray diffraction techniques. A molecular docking study revealed 1 as a new molecular motif that binds with human angiotensin converting enzyme2 (ACE2), recently described as the cell surface receptor responsible for uptake of 2019-CoV-2. Using enzyme assays we confirm that 1 inhibits human ACE2 79.7 % at 25 µg/mL.

5-(2-Oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) is a natural product formed during bacterial synthesis of vitamin B2. It potently activates mucosal associated invariant T (MAIT) cells and has immunomodulatory, inflammatory, and anticancer properties. This highly polar and unstable compound forms a remarkably stable Schiff base with a lysine residue in major histocompatibility complex class I-related protein (MR1) expressed in antigen-presenting cells. Inspired by the importance of the ribityl moiety of 5-OP-RU for binding to both MR1 and the T cell receptor (TCR) on MAIT cells, each OH was removed in silico. DFT calculations and MD simulations revealed a very stable hydrogen bond between the C3'-OH and uracil N1H, which profoundly restricts flexibility and positioning of each ribityl-OH, potentially impacting their interactions with MR1 and TCR. By using deoxygenation strategies and kinetically controlled imine formation, four monodeoxyribityl and four monohydroxyalkyl analogues of 5-OP-RU were synthesised as new tools for probing T cell activation mechanisms.

The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, β-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (MoVI/V potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorTWT, precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in KMsulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations.

As recently as 2006 the mitochondrial amidoxime reducing component (mARC) was identified as the fourth and last Mo enzyme present in humans. Its physiological role remains unknown. mARC is capable of reducing a variety of N-hydroxylated compounds such as amidoximes to their corresponding amidine and there is considerable interest in this enzyme from a pharmaceutical perspective. mARC is a target for N-hydroxylated pro-drugs that may be reductively activated intracellularly to release potent drugs such as cationic amidinium ions, which exhibit a broad spectrum of activity as antithrombotics and against various bacteria and parasites. In the quest for a rapid screen of new mARC substrates and inhibitors we present an electrochemical method which utilizes the natural electron partner of mARC, cytochrome b5, coupled to an electrochemical electrode. Mediated electron transfer from the electrode via cytochrome b5 to mARC results in a catalytic current in the presence of substrate. Keywords: mARC, Molybdenum, Enzyme, Voltammetry, Cytochrome

Air oxidation of methanolic solutions of biologically active tridentate pyridyl thiosemicarbazone (TSC) complexes of the general formula [FeII(TSC)2] has been studied at varying dioxygen concentrations, temperatures, and pressures. The data collected indicate that the activation entropy of the reaction increases linearly with the redox potential of the complexes in a more definite way than the activation enthalpy. However, a very distinct behavior is observed for the values of the activation volumes, which do not follow the expected entropy-volume parallel trend for all of the systems studied. The involvement of important interactions between the terminal NH groups of the coordinated TSC ligand and molecular dioxygen has been found to be significant by measurements carried out at varying hydrostatic pressures. Kinetic experiments run on analogous N-deuterated complexes confirm the importance of these noncovalent interactions, which are weaker for the less acidic ND groups. These interactions show the existence of an ordering/expansion process upon going from the reactants to the transition state, whenever an interaction between the polar terminal amino groups and dioxygen can be established.

International audience; The first stereocontrolled Cu-catalyzed sequential 1,6/1,4-asymmetric conjugate addition (ACA) of C-metalated hard nucleophiles to cyclic dienones is reported. The use of DiPPAM (diphenylphosphinoazomethinylate) followed by a phosphoramidite as the stereoinducing ligands facilitated both high ee values for the 1,6-ACA and high de values for the 1,4-ACA reaction components, which thus gave enantioenriched 1,3-dialkylated moieties. The absolute configurations were determined by using vibrational circular dichroism (VCD) and optical rotatory dispersion (ORD) spectroscopy, in combination with DFT calculations and X-ray analysis. Interestingly, DFT calculations for the mechanism of enantioselective 1,6-addition by using an unprecedented Cu-Zn bimetallic catalytic system confirmed this attribution. Lastly, exploring intramolecular cyclization avenues for enantioenriched 1,3-dialkylated products provided access to the challenging drimane skeleton.

The widely occurring tetranortriterpene natural product gedunin has a well-established range of important biological properties. Although this molecule has received substantial semi-synthetic attention from medicinal chemists, it has not received devoted de novo synthetic attention since it was first isolated in 1960. A key feature of the approach presented herein was the decision to utilize a Robinson annulation reaction, which facilitated the installation of the challenging 7-acetoxy functional group and the completion of the fully functionalized ABC ring system.

Phorbol esters are long regarded as tumor promotors, due to protein kinase C (PKC) activation, but more recently higher oxidized natural derivatives have been shown to display antitumor activity. Given the synthetic difficulty, systematic non-natural systems are not readily available to further interrogate PKC binding. Herein reported is the concise construction of a considerably advanced intermediate toward D-ring inverted phorbol esters, enabled by a rhodium-catalyzed [4 + 3] cycloaddition involving a highly functionalized tetrahydrobenzofuran.

CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH−. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

We report the first electrochemical study of a lanthanoid-dependent methanol dehydrogenase (Eu-MDH) from the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV with its own physiological cytochrome cGJ electron acceptor. Eu-MDH harbours a redox active 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) cofactor which is non-covalently bound but coordinates trivalent lanthanoid elements including Eu3+ . Eu-MDH and the cytochrome were co-adsorbed with the biopolymer chitosan and cast onto a mercaptoundecanol (MU) monolayer modified Au working electrode. Cyclic voltammetry of cytochrome cGJ reveals a well-defined quasi-reversible FeIII/II redox couple at +255 mV vs. NHE at pH 7.5 and this response is pH independent. The reversible one-electron response of the cytochrome cGJ transforms into a sigmoidal catalytic wave in the presence of Eu-MDH and its substrates (methanol or formaldehyde). The catalytic current was pH-dependent and pH 7.3 was found to be optimal. Kinetic parameters (pH dependence, activation energy) obtained by electrochemistry show the same trends as those obtained from an artificial phenazine ethosulfate/dichlorophenol indophenol assay.

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV2+) and anthraquinone sulfonate (AQS−) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.

MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized.

Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the Fe III/II redox couple of the heme domain. In this work, midpoint potentials ( E m ) assigned to the Fe III/II redox couple of the heme domain of FixL from Rhizobium etli ( Re FixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded Re FixL protein (+ 19 mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. E m = − 51, − 57 and − 156 mV for O 2 , imidazole and CN − , respectively. Upon binding to CO, which does not affect the kinase active, E m was observed at + 21 mV. The potential values observed for Fe III/II of the heme domain of Re FixL upon binding to CO and O 2 do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the Fe II state. Our results suggest that the conformational changes that switch off kinase activity upon O 2 binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the Fe II state and counterbalances the Fe II -stabilizing influence of the O 2 ligand.

Tin(IV) complexes of formulae, [Sn(acpysme)(C6H5)2Cl], [Sn(acpysbz)(C6H5)2Cl], [Sn(acpysbz)(C6H5)Cl2] and [Sn(acpysbz)I3] (acpysme and acpysbz = anionic forms of the 2-acetylpyrazine Schiff bases of S-methyldithiocarbazate and S-benzyldithiocarbazate, respectively) have been synthesized and structurally characterized by a variety of physico-chemical techniques and X-ray diffraction measurements. In all these complexes the tin atom adopts a distorted octahedral geometry with the Schiff bases coordinating with the tin(IV) ion as uninegatively charged tridentate NNS chelating agents via one of the pyrazine nitrogen atoms, the azomethine nitrogen atom and the thiolate sulfur atom. In the [Sn(NNS)Ph2Cl] complexes, the three NNS donor atoms of the Schiff base and a chloride ion occupy equatorial positions whereas the two phenyl groups take up axial positions of a distorted octahedron. The complexes, [Sn(acapsbz)(Ph)Cl2] and [Sn(acapsme)I3] have also distorted octahedral configurations but in this case the halido ligands are in axial positions. The Schiff bases, Hacapsme and Hacapsbz and the complex, [Sn(acpysme)I3] exhibit strong cytotoxic activity against the human breast cancer cell lines, MDA-MB-231.

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC–MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC–MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC–MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of th...

We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

Cubane was recently validated as a phenyl ring (bio)isostere, but highly strained caged carbocyclic systems lack π character, which is often critical for mediating key biological interactions. This electronic property restriction associated with cubane has been addressed herein with cyclooctatetraene (COT), using known pharmaceutical and agrochemical compounds as templates. COT either out performed or matched cubane in multiple cases suggesting that versatile complementarity exists between the two systems for enhanced bioactive molecule discovery.

Viridicatumtoxins, which belong to a rare class of fungal tetracycline-like mycotoxins, were subjected to comprehensive spectroscopic and chemical analysis, leading to reassignment/assignment of absolute configurations and characterization of a remarkably acid-stable antibiotic scaffold. Structure activity relationship studies revealed exceptional growth inhibitory activity against vancomycin-resistant Enterococci (IC50 40 nM), >270-fold more potent than the commercial antibiotic oxytetracycline.

An efficient method to directly access the bicyclo[5.3.0]decane core was achieved by rhodium-catalyzed reaction of a novel donor-acceptor cyclopentenyl diazocarboxylate with a variety of furans. As this motif is commonly found within bioactive antitumor natural products, selected systems were further manipulated and evaluated against cancer cell lines sensitive to protein kinase C (PKC) activation. A cyclic donor-acceptor diazo compound was synthesized for the first time, facilitating direct access to the bicyclo[5.3.0]decane motif, which is commonly seen in a number of prominent natural products active against cancer. Some examples were evaluated against various PKC cancer cell lines.

A series of novel sesterterpenes (2-6) have been isolated from the roots of Aletris farinosa and structurally characterized by MS, NMR, and X-ray crystallography in conjunction with computational modeling. Their structures provide new insights into the mechanisms of sesterterpene biosynthesis. Specifically, we propose with support from density functional theory computations that the configuration at a single stereocenter determines the fate of a key tetracyclic carbocationic intermediate, derived from an oxidogeranylfarnesol precursor. Whereas one epimer of the carbocation undergoes H+ elimination to give 6, the other undergoes a spectacular cascade of seven 1,2-methyl and hydride migrations leading to the previously unreported carbon skeleton of 5. Theoretical calculations suggest that the cascade is triggered by substrate preorganization in the enzyme active site.

Chemical analysis of a marine-derived fungus, Chaunopycnis sp. (CMB-MF028), isolated from the inner tissue of a pulmonate false limpet Siphonaria sp., collected from rock surfaces in the intertidal zone of Moora Park, Shorncliffe, Queensland, yielded the tetramic acid F-14329 (1) and new analogues, chaunolidines A-C (2-4), together with the new pyridinone chaunolidone A (5), and pyridoxatin (6). Structures inclusive of absolute configurations were assigned to 1-6 on the basis of detailed spectroscopic analysis, X-ray crystallography, electronic circular dichroism (ECD), biosynthetic considerations and chemical interconversion. Chaunolidine C (4) exhibits modest Gram-positive antibacterial activity (IC50 5-10 μM), while chaunolidone A (5) is a selective and potent inhibitor (IC50 0.09 μM) of human non-small cell lung carcinoma cells (NCI-H460). Tetramic acids 1-4 form metal chelates with Fe(III), Al(III), Cu(II), Mg(II) and Zn(II).

This study describes an investigation into secondary metabolites that are produced by a marine red alga, Symphyocladia latiuscula, which was collected from coastal waters off Qingdao, China. A combination of normal, reversed phase, and gel chromatography was used to isolate six citric acid derived natural products, aconitates A-F (1-6), together with two known and ten new polybrominated phenols, symphyocladins C/D (7a/b), and symphyocladins H-Q (8a/b, 9a/b and 10-15), respectively. Structure elucidation was achieved by detailed spectroscopic (including X-ray crystallographic) analysis. We propose a plausible and convergent biosynthetic pathway involving a key quinone methide intermediate, linking aconitates and symphyocladins.

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Attempted synthesis of the imidazylate derivative of 1,2-O-isopropylidene- α-D-glucurono-6,3-lactone (2) via treatment with sulfuryl chloride in the presence of excess imidazole in DMF at either -40°C or -70°C resulted in the unexpected formation of 5-chloro-5-deoxy-1,2-O-isopropylidene-β-l- idurono-6,3-lactone (7). Chloride 7 presumably forms via the rapid S N2 displacement by a chloride ion of an initially formed chlorosulfate ester intermediate, which is evidently unusually reactive. The identity of the product was confirmed by a single-crystal X-ray structure determination.

The tachykinin neuropeptide family, which includes substance P and neurokinin B, is involved in a wide array of biological functions. Among these is the ability to protect against the neurotoxic processes in Alzheimer's Disease, but the mechanisms driving neuroprotection remain unclear. Dysregulation of metal ions, particularly copper, iron and zinc is a common feature of Alzheimer's Disease, and other amyloidogenic disorders. Copper is known to be released from neurons and recent work has shown that some tachykinins can bind Cu(II) ions, and that neurokinin B can inhibit copper uptake into astrocytes. We have now examined whether neurokinin B is capable of binding Cu(I), which is predicted to be available in the synapse. Using a combination of spectroscopic techniques including cyclic voltammetry and magnetic resonance we show that neurokinin B can bind Cu(I) either directly from added CuCl or by reduction of Cu(II)-bound neurokinin B. The results showed that the Cu(I) binding site differs greatly to that of Cu(II) and involves thioether coordination via Met2 and Met10 and an imidazole nitrogen ligand from His3. The Cu(I) coordination is also different to the site adopted by Ag(I). During changes in oxidation state, copper remains bound to neurokinin B despite large changes to the inner coordination sphere. We predict that neurokinin B may be involved in synaptic copper homeostasis.

The Schiff bases formed by condensation of S-methyl- and S-benzyldithiocarbazate with 2-pyridinecarboxaldehyde react with zinc(II) and cadmium(II) acetates to form stable metal complexes of general formulas, [Zn(NNS)(CH3COO)]2 and [M(NNS)2] (M = Zn2+, Cd2+; NNS− = anionic forms of the 2-pyridinecarboxaldenhdye Schiff bases of S-methyl- and S-benzyldithiocarbazate, respectively) which have been characterized by a variety of physico-chemical techniques and X-ray crystallographic structure analysis. The complexes, [Zn(NNS)(CH3COO)]2 are acetate anion-bridged centrosymmetric dimers in which each of the Schiff bases is coordinated to the zinc(II) ions as a uninegatively charged NNS tridentate chelating agent coordinating via the pyridine nitrogen atom, the azomethine nitrogen atom and the thiolate sulfur atom. The acetate anion acts as a bridging bidentate ligand coordinating with the two zinc atoms in a syn–syn manner. Each zinc(II) ion in the dimer adopts an approximately square-pyramidal geometry. The bis-ligand complexes, [M(NNS)2] (M = Zn2+, Cd2+) have distorted octahedral structures in which the two anionic Schiff bases are coordinated to the metal ions meridionally as NNS tridentate chelating agents via the pyridine nitrogen atom, the azomethine nitrogen atom and the thiolate sulfur atom. Both the mono- and bis-ligand zinc(II) complexes exhibit strong cytotoxic activity against the HELA (human cervical cancer) cell lines. Some of these compounds exhibit stronger cytotoxicity against HELA than the commercially important anticancer drug, Tamoxifen.

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a Ki value of 4μM, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site.

The isolation and characterisation of a new macrocyclic hexaamine trans-6,13-bis(ferrocenylmethylamino)-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane (L2) bearing two ferrocenyl groups appended to its exocyclic amines is reported. The crystal structures of L2 and its dihydrochloride salt L2·2HCl·2H2O have been determined. In the latter case cation–anion hydrogen bonding is observed in the solid state. Substrate binding by the electroactive L2 in MeCN–CH2Cl2 solution has been examined by cyclic voltammetry and reveals the receptor electrochemically to recognise benzoate and chloride anions. The macrocyclic N-donors may also bind transition metal cations such as CuII and ZnII.

Thiosemicarbazone chelators, including the 2'-benzoylpyridine thiosemicarbazones (BpT) class, show marked potential as anticancer agents. Importantly, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) has been investigated in20 phase I and II clinical trials. However, side effects associated with 3-AP administration include methemoglobinemia. Considering this problem, novel BpT analogues were designed bearing hydrophobic, electron-donating substituents at the para position of the phenyl group (RBpT). Their Fe(III/II) redox potentials were all within the range accessible to cellular oxidants and reductants, suggesting they can redox cycle. These RBpT ligands exhibited potent and selective antiproliferative activity, which was comparable or exceeded their BpT counterparts. Major findings include that methemoglobin formation mediated by the lipophilic t-BuBpT series was significantly (p0.05-0.001) decreased in comparison to 3-AP in intact red blood cells and were generally comparable to the control. These data indicate the t-BuBpT ligands may minimize methemoglobinemia, which is a marked advantage over 3-AP and other potent thiosemicarbazones.

New mixed-ligand complexes of the general formula, [M(L)(sac)] (M = Ni2+, Cu2+; L− = anionic forms of the salicylaldehyde Schiff base of N-methyl-S-methyldithiocarbazate (Hsalsme), the salicylaldehyde 2-N-methyl-3-thiosemicarbazone (Hsaltsc), the methylpyruvate Schiff base of S-methyldithiocarbazate (Hmpsme) and the quinoline-2-methoxycarboxaldehyde Schiff base of S-methyldithiocarbazate (quinolsme); sac− = the saccharinate anion) have been synthesized and characterized by IR, electronic and magnetic susceptibility measurements. Magnetic and spectroscopic data support a distorted square-planar structure for the [M(ONS/NNS)(sac)] complexes (ONS/NNS = salsme, saltsc, mpsme or quinolsme). The structures of Hsalsme, [Cu(salsme)sac] and [Cu(quinolsme)sac] have been determined by X-ray diffraction. The complex, [Cu(salsme)sac] has a distorted square-planar structure with the Schiff base acting as a uninegatively charged ONS tridentate chelating agent coordinating the copper(II) ion via the phenolic oxygen, the azomethine nitrogen and the thione sulfur atoms, the fourth coordination position being occupied by an N-bonded saccharinate anion. The reaction of [Cu(sac)4(H2O)2]·2H2O with the 2-quinolinecarboxaldehyde Schiff base of S-methyldithiocarbazate (Hqsme) in boiling methanol does not lead to the formation of the expected mixed-ligand complex, [Cu(qsme)(sac)], but an unusual substitution of the hydrogen atom attached to the azomethine carbon by a methoxy group occurs with the concomitant formation of the complex [Cu(quinolsme)(sac)] of a new ligand (quinolsme = anionic form of the quinolone-2-methoxycarboxaldehyde Schiff base of S-methyldithiocarbazate). An X-ray crystal structure determination reveals that the Schiff base Hquinolsme is coordinated to the copper(II) ion in its deprotonated thiolate form as an NNS tridentate chelating agent and the fourth coordination position of the square-planar copper(II) centre is occupied by an N-bonded saccharinate anion.

We developed a series of second-generation di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands to improve the efficacy and safety profile of these potential antitumor agents. Two novel DpT analogues, Dp4e4mT and DpC, exhibited pronounced and selective activity against human lung cancer xenografts in vivo via the intravenous and oral routes. Importantly, these analogues did not induce the cardiotoxicity observed at high nonoptimal doses of the first-generation DpT analogue, Dp44mT. The Cu(II) complexes of these ligands exhibited potent antiproliferative activity having redox potentials in a range accessible to biological reductants. The activity of the copper complexes of Dp4e4mT and DpC against lung cancer cells was synergistic in combination with gemcitabine or cisplatin. It was demonstrated by EPR spectroscopy that dimeric copper compounds of the type [CuLCl](2), identified crystallographically, dissociate in solution to give monomeric 1:1 Cu:ligand complexes. These monomers represent the biologically active form of the complex.

Crystal structures of two isomeric norcantharidin derivatives (3R,3aR,4S,7R,7aS)-3-(allyloxy)hexahydro-4,7-epoxyisobenzofuran-1(3H)-one (7b), and (3S,3aR,4S,7R,7aS)-3-((E)-but-2-en-1-yloxy)hexahydro-4,7-epoxyisobenzofuran-1(3H)-one (8a) have been determined. In both instances the equivalent enantiomer was also obtained. The crystal structures of these compounds clarify the stereochemistry inferred only by NMR analysis before. Two norcantharidin analogues have been separated into their two possible diastereomers and the structural identity of each compound has been confirmed from their corresponding crystal structures and NMR.

The Fe(III) abstraction from Fe(III)/DFO and Fe(III)/EDTA complex systems by thiosemicarbazone ligands derived from 2-acetylpyridine has been studied from a kinetico-mechanistic perspective at relevant pH conditions and at varying temperatures and buffer solutions. The reactions have been found to be extremely dependent on the dominant E/Z isomeric form of the TSC ligands present in the reaction medium. Consequently the isomerisation processes occurring on the free ligands have also been monitored under equivalent conditions. The isomerisation process is found to be acid dependent, despite the absence of protonation under the conditions used, and presumably proceeds via an azo-type tautomer of the ligand. In all cases the existence of outer-sphere interaction processes has been established, both promoting the reactions and producing dead-end complexes. The better oriented forms of the ligands (EZ thiolate) have been found to react faster with the [Fe(HDFO)](+) complex, although for mono-N(4) substituted thiosemicarbazones the process is retarded by the formation of a dead-end outer-sphere complex. A comparison with the abstraction of Fe(III) from [Fe(EDTA)(H(2)O)](-) has also been conducted with significant differences in the kinetic features that implicate keystone outer-sphere interactions which dominate reactivity, even with isomeric forms that are not the best suited for direct complexation.

Iron chelators of the 2'-benzoylpyridine thiosemicarbazone (BpT) class show substantial potential as anticancer agents. To explore structure-activity relationships, new BpT analogues were designed that incorporated halogen substituents on the noncoordinating phenyl group (XBpTs). These XBpT ligands exhibited potent antiproliferative activity with some analogues exceeding that of the parent BpT compound. Importantly, there was an appreciable therapeutic index in vitro, as mortal cells were significantly less affected by these chelators relative to neoplastic cells. The addition of a halogen led to a halogen-specific increase in the redox potential of XBpT-Fe complexes. Probing for chelator-induced intracellular reactive oxygen species (ROS) with the fluorescent probe, 2',7'-dichlorofluorescein, revealed a 1.5-4.7-fold increase in fluorescence upon incorporation of Cl, Br, or I to the parent analogues. Furthermore, an important structure-activity relationship was deduced where the addition of halogens led to a positive correlation between intracellular ROS generation and antiproliferative activity in the more hydrophilic BpT parent compounds.

The absolute stereochemistry of the steroidal saponins bethosides B and C was previously assigned as (22R,25R) on the basis of work that employed Horeau's method. Our studies of helosides A and B created doubt about both the original assignment and consequently our conclusion that relied upon it. The absolute configurations of bethosides B and C are revised to (22S,25R) following X-ray crystallographic analysis of their aglycone. Synthesis and full spectral characterization of both the 22R and 22S aglycones is reported to facilitate future stereochemical assignments in this series of saponins.

The bicyclic hexaamine "cage" ligand Me(8)tricosaneN(6) (1,5,5,9,13,13,20,20-octamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane) is capable of encapsulating octahedral metal ions, yet its expanded cavity allows the complexed metal to adopt a variety of geometries comprising either hexadentate or pentadentate coordination of the ligand. When complexed to Cu(II) the lability of the metal results in a dynamic equilibrium in solution between hexadentate- and pentadentate-coordinated complexes of Me(8)tricosaneN(6). Both [Cu(Me(8)tricosaneN(6))](ClO(4))(2) (6-coordinate) and [Cu(Me(8)tricosaneN(6))](S(2)O(6)) (5-coordinate) have been characterized structurally. In weak acid (pH 1) a singly protonated complex [Cu(HMe(8)tricosaneN(6))](3+) has been isolated that finds the ligand binding as a pentadentate with the uncoordinated amine being protonated. vis-NIR and electron paramagnetic resonance (EPR) spectroscopy show that the predominant solution structure of [Cu(Me(8)tricosaneN(6))](2+) at neutral pH comprises a five-coordinate, square pyramidal complex. Cyclic voltammetry of the square pyramidal [Cu(Me(8)tricosaneN(6))](2+) complex reveals a reversible Cu(II/I) couple. All of these structural, spectroscopic, and electrochemical features contrast with the smaller cavity and well studied "sarcophagine" (sar, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane) Cu(II) complexes which are invariably hexadentate coordinated in neutral solution and cannot stabilize a Cu(I) form.

N6-methyl mucronatine (C8H12N5O) has been isolated from a dictyoceratid sponge collected in South East Queensland. The solid state structure of the new metabolite (I) was confirmed by X-ray crystallography, while an NMR study in d 4-MeOH reveals the presence of a minor tautomer identified as (II). The crystal structure of the purine N10-methyl mucronatine (C8H12N5O) is reported, isolated from a dictyoceratid sponge collected in South East Queensland, Australia

Cancer is one of the leading causes of death worldwide and there is an increasing need for novel anti-tumor therapeutics with greater selectivity and potency. A new strategy in the treatment of cancer has focused on targeting an essential cell metabolite, iron (Fe). Iron is vital for cell growth and metabolism, forming a crucial component of the active site of ribonucleotide reductase (RR), the rate-limiting enzyme in DNA synthesis. Cancer cells in particular require large amounts of Fe to proliferate, making them more susceptible to the Fe deficiency caused by Fe chelators. Beginning with primordial siderophores, Fe chelators have since evolved to a new generation of potent and efficient anti-cancer agents. Recently, investigations have led to the generation of novel di-2-pyridylketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands that demonstrate marked and selective anti-tumor activity both in vitro and in vivo against a wide spectrum of tumors. The mechanism of action of these novel ligands includes alterations in the expression of key regulatory molecules as well as the generation of redox active Fe complexes. Interestingly, non-synthetic Fe chelators including silybin and curcumin, both of which are derived from plants, also have vast potential in the treatment of cancer. This review explores the development of novel Fe chelators for the treatment of cancer and their mechanisms of action.

The potentially pentadentate chelate 2,6-diacetylpyridine-bis(N-methyl-S-methyldithiocarbazate) (Nmedapsme) has been synthesized and structurally characterized by X-ray diffraction. Its reactions with nickel(II) salts did not lead to pentadentate coordinated ligand complexes but ternary complexes of general formula, [Ni(Nmedapsme)(nmesme)L]X·H2O (L = Br−, I−; X = I−, BF4−) where Nmedapsme binds as a tridentate and nmesme = N-methyl-S-methyldithiocarbazate. The related ternary nickel(II) complexes of formula, Ni(Nmedapsme)(nmetsc)Br2 has also been prepared and characterized. X-ray crystal structures of [Ni(Nmedapsme)(nmesme)I]I·H2O and [Ni(Nmedapsme)(nmesme)Br]BF4·H2O revealed that, in these complexes, the Nmedapsme ligand acts as a tridentate NNN donor while the distal S-donors are not coordinated. The bidentate (NS) ligand, nmesme coordinates to the nickel(II) ion via the amino nitrogen and the thione sulfur atoms, the sixth coordination site is occupied by an anion. In both complexes, the nickel(II) ion adopts a distorted octahedral configuration. The complex [Cu(nmesme)2(ONO2)]NO3 was obtained from an unsuccessful attempt to complex copper(II) with Nmedapsme. Hydrolysis of the parent Schiff base Nmedapsme occurred during complexation. An X-ray crystallographic structure analysis shows that the complex, [Cu(nmesme)2(ONO2)]NO3 has an approximately square–pyramidal geometry with the two nmesme ligands coordinated to the copper(II) ion as NS bidentate chelating agents via the amino nitrogen and thione sulfur atoms and the fifth coordination position of copper(II) is occupied by a monodentate nitrate ligand.