Isolation, X-ray Structures, and Electronic Spectra of Reactive Intermediates in Friedel−Crafts Acylations

Reactive intermediates in the Friedel−Crafts acylation of aromatic donors are scrutinized upon their successful isolation and X-ray crystallography at very low temperatures. Detailed analyses of the X-ray parameters for the [1:1] complexes of different aliphatic and aromatic-acid chlorides with the Lewis acids antimony pentafluoride and pentachloride, gallium trichloride, titanium and zirconium tetrachlorides provide unexpected insight into the activation mechanism for the formation of the critical acylium carbocations. Likewise, the X-ray-structure examinations of aliphatic and aromatic acylium electrophiles also isolated as crystalline salts point to the origins of their electrophilic reactivity. Although the Wheland intermediates (as acylium adducts to arene donors) could not be isolated in crystalline form owing to their exceedingly short lifetimes, transient (UV−vis) spectra of benzenium adducts of acylium carbocations with hexamethylbenzene can be measured and directly related to Wheland intermediates with other cationic electrophiles that have been structurally established via X-ray studies

Isolation and X-ray Structures of Labile Benzoic- and Acetic-Acidium Carbocations

New carbocationic salts (via O-protonation of substituted benzoic acids) are prepared for the first time by controlled hydration of the corresponding benzoylium salts and isolated in pure crystalline form. Precise X-ray structural analyses reveal the rather unexpected (electronic) structure of the carboxylic-acidium functionality

Crystallographic Distinction between “Contact” and “Separated” Ion Pairs: Structural Effects on Electronic/ESR Spectra of Alkali-Metal Nitrobenzenides

The classic nitrobenzene anion-radical (NB-• or nitrobenzenide) is isolated for the first time as pure crystalline alkali-metal salts. The deliberate use of the supporting ligands 18-crown-6 and [2.2.2]cryptand allows the selective formation of contact ion pairs designated as (crown)M+NB-•, where M+ = K+, Rb+, and Cs+, as well as the separated ion pair K(cryptand)+NB-•both series of which are structurally characterized by precise low-temperature X-ray crystallography, ESR analysis, and UV−vis spectroscopy. The unusually delocalized structure of NB-• in the separated ion pair follows from the drastically shortened N−C bond and marked quinonoidal distortion of the benzenoid ring to signify complete (95%) electronic conjugation with the nitro substituent. On the other hand, the formation of contact ion pairs results in the substantial decrease of electronic conjugation in inverse order with cation size (K+ \u3e Rb+) owing to increased localization of negative charge from partial (NO2) bonding to the alkali-metal cation. Such a loss in electronic conjugation (or reverse charge transfer) may be counterintuitive, but it is in agreement with the distribution of odd-electron spin electron density from the ESR data and with the hypsochromic shift of the characteristic absorption band in the electronic spectra. Most importantly, this crystallographic study underscores the importance of ion-pair structure on the intrinsic property (and thus reactivity) of the component ions - as focused here on the nitrobenzenide anion

Structural Effects of Carbon Monoxide Coordination to Carbon Centers. π and σ Bindings in Aliphatic Acyl \u3cem\u3eversus\u3c/em\u3e Aromatic Aroyl Cations

The binding of carbon monoxide to carbon centers has been examined with two series of aromatic and aliphatic oxocarbonium ions that are successfully isolated as crystalline and highly reactive (hygroscopic) aroylium and acylium salts with poorly coordinating counteranions. X-Ray crystallographic analyses at −150 °C afford precise structural parameters for the characteristic linear carbonyl bond (rCO) and the bond to the carbon centers (rCα). The correlations of these structural parameters evaluated for alkyl (Me, Et and i-Pr) and aryl (p-Me, 2,4,6-trimethyl, p-MeO and p-fluorophenyl) oxocarbonium ions with the corresponding carbonyl stretching frequencies in the solid-state (reflectance) IR spectra yield valuable insight into the binding mode of carbon monoxide. Most noteworthy is the synergic (π–σ) bonding in aroylium structures in contrast to the mainly σ bonding in acylium structures that are organic mimics for carbon monoxide bonding in classical and nonclassical metal carbonyls, respectively

Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis </it>is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of <it>B. anthracis </it>(Ames) alanine racemase (Alr_<it>Bax</it>), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr_{<it>Bax </it>}structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.</p> <p>Results</p> <p><it>B. anthracis </it>has two open reading frames encoding for putative alanine racemases. We show that only one, <it>dal1</it>, is able to complement a D-alanine auxotrophic strain of <it>E. coli</it>. Purified Dal1, which we term Alr_<it>Bax</it>, is shown to be a dimer in solution by dynamic light scattering and has a V_maxfor racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified Alr_{<it>Bax </it>}is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, Alr_{<it>Bax </it>}makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.</p> <p>Conclusion</p> <p>The chloride ion in Alr_{<it>Bax </it>}is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two Alr_{<it>Bax </it>}structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.</p

The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years.Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against <it>S. pneumoniae </it>and other bacterial pathogens.</p> <p>Results</p> <p>Here we report the crystal structure of alanine racemase from <it>S. pneumoniae </it>(Alr_SP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3₁21 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that Alr_SPshares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway.</p> <p>Conclusions</p> <p>We have solved the structure of Alr_SP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.</p

Purification and preliminary crystallization of alanine racemase from Streptococcus pneumoniae

<p>Abstract</p> <p>Background</p> <p>Over the past fifteen years, antibiotic resistance in the Gram-positive opportunistic human pathogen <it>Streptococcus pneumoniae </it>has significantly increased. Clinical isolates from patients with community-acquired pneumonia or otitis media often display resistance to two or more antibiotics. Given the need for new therapeutics, we intend to investigate enzymes of cell wall biosynthesis as novel drug targets. Alanine racemase, a ubiquitous enzyme among bacteria and absent in humans, provides the essential cell wall precursor, D-alanine, which forms part of the tetrapeptide crosslinking the peptidoglycan layer.</p> <p>Results</p> <p>The alanine racemases gene from <it>S. pneumoniae </it>(<it>alr</it>_<it>SP</it>) was amplified by PCR and cloned and expressed in <it>Escherichia coli</it>. The 367 amino acid, 39854 Da dimeric enzyme was purified to electrophoretic homogeneity and preliminary crystals were obtained. Racemic activity was demonstrated through complementation of an <it>alr </it>auxotroph of <it>E. coli </it>growing on L-alanine. In an alanine racemases photometric assay, specific activities of 87.0 and 84.8 U mg^-1were determined for the conversion of D- to L-alanine and L- to D-alanine, respectively.</p> <p>Conclusion</p> <p>We have isolated and characterized the alanine racemase gene from the opportunistic human pathogen <it>S. pneumoniae</it>. The enzyme shows sufficient homology with other alanine racemases to allow its integration into our ongoing structure-based drug design project.</p

Evolutionary fates within a microbial population highlight an essential role for protein folding during natural selection

Physicochemical properties of molecules can be linked directly to evolutionary fates of a population in a quantitative and predictive manner.Reversible- and irreversible-folding pathways must be accounted for to accurately determine in vitro kinetic parameters (KM and kcat) at temperatures or conditions in which a significant fraction of free enzyme is unfolded.In vivo population dynamics can be reproduced using in vitro physicochemical measurements within a model that imposes an activity threshold above which there is no added fitness benefit

Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids

Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calciumdependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides