Determining the structure and mode of action of microbisporicin, a potent lantibiotic active against multiresistant pathogens

Antibiotics blocking bacterial cell wall assembly (beta-lactams and glycopeptides) are facing a challenge from the progressive spread of resistant pathogens. Lantibiotics are promising candidates to alleviate this problem. Microbisporicin, the most potent antibacterial among known comparable lantibiotics, was discovered during a screening applied to uncommon actinomycetes. It is produced by Microbispora sp. as two similarly active and structurally related polypeptides (A1, 2246-Da and A2, 2230-Da) of 24 amino acids linked by 5 intramolecular thioether bridges. Microbisporicin contains two posttranslational modifications that have never been reported previously in lantibiotics: 5-chloro-trypthopan and mono- (in A2) or bis-hydroxylated (in A1) proline. Consistent with screening criteria, microbisporicin selectively blocks peptidoglycan biosynthesis, causing cytoplasmic UDP-linked precursor accumulation. Considering its spectrum of activity and its efficacy in vivo, microbisporicin represents a promising antibiotic to treat emerging infections.     

α-Diimine Ligand Coordination and C–H Bond Activation in the Reaction of Os3(CO)10(MeCN)2 with 6-R-2,2′-Bipyridine (where R?=?Et, Ph): X-ray Diffraction Structures of the Ortho-Metalated Hydride Clusters HOs3(CO)9(N2C10H6-6-R)

Abstract  

The reactivity of the labile cluster Os₃(CO)₁₀(MeCN)₂ (1) with the monofunctionalized heterocyclic ligands 6-R-2,2′-bipyridine (where R = Et, Ph) has been investigated. The alkyl-substituted heterocycle 6-Et-2,2′-bipyridine reacts with 1 in refluxing CH₂Cl₂ to give an isomeric mixture of HOs₃(CO)₉(N₂C₁₂H₁₁) due to cyclometalation of the side-chain ethyl group (2) and ortho metalation of the unsubstituted bipyridine ring (3). The solid-state structure of the latter cluster, HOs₃(CO)₉(N₂C₁₀H₆-6-Et) (3), has unequivocally established the site of the C-H bond activation in the product. Treatment of 1 with the aryl-substituted ligand 6-Ph-2,2′-bipyridine proceeds similarly with ortho metalation at the ancillary phenyl group and the C-6′ ortho site of the unsubstituted bipyridine ring, as verified by ¹H NMR spectroscopy. The X-ray diffraction structure of the thermodynamically more stable bipyridine-metalated cluster HOs₃(CO)₉(N₂C₁₀H₆-6-Ph) (5) has been determined. The course of these reactions is discussed with respect to our recent study involving the reaction of cluster 1 with the ligand 6-Me-2,2′-bipyridine.     

A Density Functional Theory Study of Optical Rotation in Some Aziridine and Oxirane Derivatives

We present time-dependent density functional theory (TDDFT) calculations of the electronic optical rotation (ORP) for seven oxirane and two aziridine derivatives in the gas phase and in solution and compare the results with the available experimental values. For seven of the studied molecules it is the first time that their optical rotation was studied theoretically and we have therefore investigated the influence of several settings in the TDDFT calculations on the results. This includes the choice of the one-electron basis set, the exchange-correlation functional or the particular polarizable continuum model (PCM). We can confirm that polarized quadruple zeta basis sets augmented with diffuse functions are necessary for converged results and find that the aug-pc-3 basis set is a viable alternative to the frequently employed aug-cc-pVQZ basis set. Based on our study, we cannot recommend the generalized gradient functional KT3 for calculations of the ORP in these compounds, whereas the hybrid functional PBE0 gives results quite similar to the long-range correct CAM−B3LYP functional. Finally, we observe large differences in the solvent effects predicted by the integral equation formalism of PCM and the SMD variant of PCM. For the majority of solute/solvent combinations in this study, we find that the SMD model in combination with the PBE0 functional and the aug-pc-3 basis set gives the best agreement with the experimental values.     

Allylic Functionalization of 2,5- and 2,4-Cyclo-Hexadiene-1,3-Dicarboxylates

A series of 2,4- and 2,5-cyclohexadiene-1,3-dicarboxylates were functionalized at the allylic position via oxidation (SeO₂, PDC/t-BuOOH) and halogenation (NBS). The regiochemical outcome for different substrates and reactions was studied and the importance of factors such as reaction mechanism, steric hindrance and reaction intermediates stability was discussed.

     

Control of thiolate nucleophilicity and specificity in zinc metalloproteins by hydrogen bonding: lessons from model compound studies

A single hydrogen bond between an amide N-H and a thiolate sulfur in model complexes designed to mimic the binding site of zinc thiolate proteins, is shown to reduce the reactivity of the thiolate toward electrophiles by up to 2 orders of magnitude. In addition a single such bond is also sufficient to achieve nearly 100% regiospecificity of reaction between a strong, and hence inherently indiscriminate, alkylating agent like trimethyl oxonium tetrafluoroborate and a single sulfur in a dithiolate construct. The importance of these results in understanding how two systems such as the zinc fingers of the GATA family and the Escherichia coli DNA repair protein Ada which share the same pseudotetrahedral structure and tetrascysteinyl ligation around the zinc can fulfill such widely divergent (structural vs reactive) roles and how specificity of reaction in multithiolate-containing systems can be achieved is discussed.     

H-bonding interactions and control of thiolate nucleophilicity and specificity in model complexes of zinc metalloproteins

It is shown in model complexes designed to mimic the binding site of zinc-thiolate proteins that a single hydrogen bond between an amide N-H and a Zn-coordinated thiolate reduces its reactivity toward electrophiles by up to 2 orders of magnitude. In addition, we show that a single N-H...S hydrogen bond is sufficient to achieve near 100% regiospecificity of reaction between a strong, and hence inherently indiscriminate, alkylating agent like trimethyloxonium tetraflouroborate and a single sulfur in a dithiolate construct. The importance of these results in understanding how systems such as the zinc fingers of the GATA family and the E. coli DNA repair protein Ada, which share the same pseudotetrahedral structure and tetracysteinyl ligation around the zinc, can fulfill such widely divergent (structural vs reactive) roles and how specificity of reaction in such multi-thiolate containing systems can be achieved is discussed.     

Synthesis and EPR characterization of new models for the one-electron reduced molybdenum site of sulfite oxidase

Deconvoluting the different contributions of thiolate and ene-1,2-dithiolate donors to the underlying electronic structure of the Mo site in sulfite oxidase (SO) has proven to be a difficult task. One way in which these differences might be illuminated is by selectively substituting Se for S in model complexes which possess multiple sulfur donor ligand environments. Here we report the synthesis and structures of new oxo-Mo(V) complexes as effective models for the one-electron reduced active site of SO. We have used the tridentate heteroscorpionate ligand (2-dimethylethanethiol)bis(3,5-dimethylpyrazolyl)methane (L3SH) in order to model the constrained cysteinyl sulfur (S(Cys)) ligand environment observed in the crystal structure of the enzyme, and benzene-1,2-dithiol (bdt) as a mimic of the ene-1,2-dithiolate chelate. [(L3S)MoO(bdt)] and [(L3S)MoO(SPh)(2)] have been structurally characterized by X-ray crystallography, and as such, [(L3S)MoO(bdt)] is only the second known model compound that closely approximates the active site structure of reduced forms of SO. Additionally, benzenethiol (SPh) and benzeneselenol (SePh) have been used to perturb the equatorial ligand environment of [(L3S)MoO(bdt)].) This has provided much needed insight into the electronic structure of the one-electron reduced SO site and has allowed for increased understanding of the individual roles played by these different thiolate donors in the oxidative half-reaction of the enzyme. Interestingly, the EPR spectra of [(L3S)MoO(bdt)], [(L3S)MoO(SPh)(2)], and [(L3S)MoO(SePh)(2)] closely resemble that of both high pH (hpH) and low pH (lpH) SO, except for the fact that the magnitude of g(1) is found to be consistently higher in the model spectra compared to that of the enzyme. It is suggested that this derives from an increase in Mo-S covalency in the models relative to hpH and lpH SO.     

Synthesis, characterization, electrochemistry, electronic structure, and isomerization of mononuclear oxo-molybdenum(V) complexes: the serine gate hypothesis in the function of DMSO reductases

Crystal structures of DMSO reductases isolated from two different sources and the crystal structure of related trimethylamine-N-oxide reductase indicate that the angle between the terminal oxo atom on the molybdenum and the serinato oxygen varies significantly. To understand the significance of this angular variation, we have synthesized two isomeric compounds of the heteroscorpionato ligand (L1OH) (cis- and trans-(L1O)Mo(V)OCl(2)), where the phenolic oxygen mimics the serinato oxygen donor. Density functional and semiempirical calculations indicate that the trans isomer is more stable than the cis. The lower stability of the cis isomer can be attributed to two factors. First, a strong antibonding interaction between the phenolic oxygen with molybdenum d(xy) orbital raises the energy of this orbital. Second, the strong trans influence of the terminal oxo group in the trans isomer places the phenol ring, and hence the bulky tertiary butyl group, in a less sterically hindered position. In solution, the cis isomer spontaneously converts to the thermodynamically favorable trans isomer. This geometric transformation follows a first-order process, with an enthalpy of activation of 20 kcal/mol and an entropy of activation of -9 cal/mol K. Computational analysis at the semiempirical level supports a twist mechanism as the most favorable pathway for the geometric transformation. The twist mechanism is further supported by detailed mass spectral data collected in the presence of excess tetraalkylammonium salts. Both the cis and trans isomers exhibit well-defined one-electron couples due to the reduction of molybdenum(V) to molybdenum(IV), with the cis isomer being more difficult to reduce. Both isomers also exhibit oxidative couples because of the oxidation of molybdenum(V) to molybdenum(VI), with the cis isomer being easier to oxidize. This electrochemical behavior is consistent with a higher-energy redox orbital in the cis isomer, which has been observed computationally. Collectively, this investigation demonstrates that by changing the O(t)-Mo-O(p) angle, the reduction potential can be modulated. This geometrically controlled modulation may play a gating role in the electron-transfer process during the regeneration steps in the catalytic cycle.     

Isomerization and oxygen atom transfer reactivity in oxo-Mo complexes of relevance to molybdoenzymes

Both dioxo Mo(VI) and mono-oxo Mo(V) complexes of a sterically restrictive N2O heteroscorpionate ligand are found to exist as cis and trans isomers. The thermodynamically stable isomer differs for the two oxidation states, but in each case, we have isolated the kinetically labile isomer and followed its isomerization to the thermodynamically stable form. The Mo(VI) complex is more stable in the cis geometry and isomerizes more than 6 times faster than the Mo(V) complex, which prefers the trans geometry. In OAT reactions with PPh3, the trans isomer of the dioxo-Mo(VI) reacts approximately 20 times faster than the cis isomer. Thus, there are both oxidation state and donor atom dependent differences in isomeric stability and reactivity that could have significant functional implications for molybdoenzymes such as DMSO reductase.     

A family of dioxo-molybdenum(VI) complexes of N2X heteroscorpionate ligands of relevance to molybdoenzymes

Four new Mo(VI)-dioxo complexes of a family of N2X heteroscorpionate ligands are reported which, together with data already available for (TpR)-, provide a unique example of a comprehensive set of isostructural, isoelectronic complexes differing only in one biologically relevant donor atom. A study of these complexes allows for a direct comparison of structural, spectroscopic, and oxygen atom transfer reactivity properties of the Mo(VI)-dioxo center (of relevance to various families of molybdoenzymes) as a function of donor atom identity.