Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (␤-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via ␤-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery.The emergence of bacterial strains resistant to antibiotic therapy is a major public health threat (1). Of particular concern is Staphylococcus aureus, because this Gram-positive pathogen is the leading cause of infections in the bloodstream, lower respiratory tract, skin, and soft tissue in the United States (2). S. aureus strains exhibiting resistance against multiple antibiotics, such as methicillin-resistant S. aureus, are isolated in 30 -60% of community and Ͼ80% of hospital infections with this pathogen (3). Vancomycin or other glycopeptides are considered last-resort therapies for methicillin-resistant S. aureus; however, S. aureus strains with intermediate or full resistance to vancomycin can cause infections for which antimicrobial treatment may no longer be effective (4).Surface proteins of Gram-positive bacteria play important roles during pathogenesis (5). Sortases anchor these polypeptides to the bacterial cell wall envelope (6). For example, S. aureus sortase A recognizes proteins destined for the cell surface via an LPXTG motif in their C-terminal sorting signal (7). Following cleavage between the threonine and the glycine residues, an acyl-enzyme intermediate captures cleaved substrate at the active site thiol of sortase (8). Nucleophilic attack of the amino group of the peptidoglycan precursor lipid II (at the thioester intermediate resolves the acyl enzyme and forms an amide bond between the C-terminal threonine of surface protein and pentaglycine crossbridges (9). Lipid II-linked polypeptide is subsequently incorporated into the cell wall envelope of staphylococci (10). The final product of this pathway, protein linked to cell wall pentaglycine cross-bridges, is displayed on the bacterial surface and enables interactions between the pathogen and tissues of its host.Surface protein anchoring to the cell wall envelope is thought to be an essential strategy for bacterial survival during infection, because mutants lacking genes for one or more sortase enzymes are attenuated in virulence (11). Inhibition of sortases by small molecules may therefore function as a therapeutic strategy for bacterial infections. ...

The rate constants for the reactions of phenol with the hydroxyl radical (OH*) in water have been measured from room temperature to 380 degrees C using electron pulse radiolysis and transient absorption spectroscopy. The reaction scheme designed to fit the data shows the importance of an equilibrium, giving back reactants (OH* radical and phenol) from the dihydroxycyclohexadienyl radical formed by their reaction, and the non-negligible contribution of the hydroxycyclohexadienyl radical absorption from H* atom addition. The accuracy of the reaction scheme and the reaction rate constants determined from it have been determined by the analysis of two different experiments, one under pure N2O atmosphere and the second under a mixture a N2O and O2. We report reaction rates for the H* and OH* radical addition to phenol, the formation of phenoxyl, the second-order recombination, the reaction of dihydroxycyclohexadienyl with O2, and the decay of the peroxyl adduct. Nearly all of the reaction rates deviate strongly from Arrhenius behavior.

Yields for H2, H(.) atom, and hydrated electron production in beta/gamma radiolysis of water have been measured from room temperature up to 400 degrees C on a 250 bar isobar, and also as a function of pressure (density) at 380 and 400 degrees C. Radiolysis was carried out using a beam of 2-3 MeV electrons from a van de Graaff accelerator, and detection was by mass spectrometer analysis of gases sparged from the irradiated water. N2O was used as a specific scavenger for hydrated electrons giving N2 as product. Ethanol-d(6) was used to scavenge H(.) atoms, giving HD as a stable product. It is found that the hydrated electron yield decreases and the H(.) atom yield increases dramatically at lower densities in supercritical water, and the overall escape yield increases. The yield of molecular H2 increases with temperature and does not tend toward zero at low density, indicating that it is formed promptly rather than in spur recombination. A minimum in both the radical and H2 yields is observed around 0.4 kg/dm(3) density in supercritical water.

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Cross-check ExperimentsThe HD yield at room temperature (Table 1) is very low compared to earlier measurements of H • atom yield, and we were not certain that a 0.02 m concentration of ethanol-d6 gives enough scavenging power (5.4×10 4 s -1 at room temperature) to compete efficiently for H • atoms with other second order reactions or impurities. As a test we performed a series of experiments with 0.01 m (normal) ethanol aqueous solutions with 2.5×10 -3 m N 2 O. Figure S1 shows the radiation yields of H 2 in 0.01 m ethanol compared to the HD+H 2 yield in 0.02 m ethanol-d6. Both yields compare well from 100 o C up to 300 o C, justifying the small concentration of ethanol-d6 used for the H • atom yield determination.