The Tipper–Strominger Hypothesis and Triggering of Allostery in Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus (MRSA)

The transpeptidases involved in the synthesis of the bacterial cell wall (also known as penicillin-binding proteins, PBPs) have evolved to bind the acyl-d-Ala-d-Ala segment of the stem peptide of the nascent peptidoglycan for the physiologically important cross-linking of the cell wall. The Tipper–Strominger hypothesis stipulates that β-lactam antibiotics mimic the acyl-d-Ala-d-Ala moiety of the stem and, thus, are recognized by the PBPs with bactericidal consequences. We document that this mimicry exists also at the allosteric site of PBP2a of methicillin-resistant Staphylococcus aureus (MRSA). Interactions of different classes of β-lactam antibiotics, as mimics of the acyl-d-Ala-d-Ala moiety at the allosteric site, lead to a conformational change, across a distance of 60 Å to the active site. We directly visualize this change using an environmentally sensitive fluorescent probe affixed to the protein loops that frame the active site. This conformational mobility, documented in real time, allows antibiotic access to the active site of PBP2a. Furthermore, we document that this allosteric trigger enables synergy between two different β-lactam antibiotics, wherein occupancy at the allosteric site by one facilitates occupancy by a second at the transpeptidase catalytic site, thus lowering the minimal-inhibitory concentration. This synergy has important implications for the mitigation of facile emergence of resistance to these antibiotics by MRSA

Synthesis and NMR Characterization of (<i>Z</i>,<i>Z</i>,<i>Z</i>,<i>Z</i>,<i>E</i>,<i>E</i>,ω)-Heptaprenol

We describe a practical, multigram synthesis of (2<i>Z</i>,6<i>Z</i>,10<i>Z</i>,14<i>Z</i>,18<i>E</i>,22<i>E</i>)-3,7,11,15,19,23,27-heptamethyl-2,6,10,14,18,22,26-octacosaheptaen-1-ol [(<i>Z</i>₄,<i>E</i>₂,ω)-heptapren­ol, <b>4</b>] using the nerol-derived sulfone <b>8</b> as the key intermediate. Sulfone <b>8</b> is prepared by the literature route and is converted in five additional steps (18% yield from <b>8</b>) to (<i>Z</i>₄,<i>E</i>₂,ω)-heptaprenol <b>4</b>. The use of Eu­(hfc)₃ as an NMR shift reagent not only enabled confirmation of the structure and stereochemistry of <b>4</b>, but further enabled the structural assignment to a major side product from a failed synthetic connection. The availability by this synthesis of (<i>Z</i>₄,<i>E</i>₂,ω)-heptaprenol <b>4</b> in gram quantities will enable preparative access to key reagents for the study of the biosynthesis of the bacterial cell envelope

Ensemble of Pinanones from the Permanganate Oxidation of Myrtenal

The buffered permanganate oxidation of (−)-myternal, a member of the pinene family, provides the α-hydroxyketone (−)-(1<i>R</i>,3<i>S</i>,5<i>R</i>)-3-hydroxy-6,6-dimethylbicyclo­[3.1.1]­heptan-2-one in preparative yield (65% on a multigram scale). This α-hydroxyketone is oxidized, in a second reaction, to the α,β-diketone (1<i>R</i>,5<i>R</i>)-6,6-dimethylbicyclo­[3.1.1]­heptane-2,3-dione (“PinDione”). As both oxidations are fast, simple, safe, inexpensive, good-yielding, and multigram scalable, these transformations are a preparative expansion of the pinane family

Ensemble of Pinanones from the Permanganate Oxidation of Myrtenal

The buffered permanganate oxidation of (−)-myternal, a member of the pinene family, provides the α-hydroxyketone (−)-(1<i>R</i>,3<i>S</i>,5<i>R</i>)-3-hydroxy-6,6-dimethylbicyclo­[3.1.1]­heptan-2-one in preparative yield (65% on a multigram scale). This α-hydroxyketone is oxidized, in a second reaction, to the α,β-diketone (1<i>R</i>,5<i>R</i>)-6,6-dimethylbicyclo­[3.1.1]­heptane-2,3-dione (“PinDione”). As both oxidations are fast, simple, safe, inexpensive, good-yielding, and multigram scalable, these transformations are a preparative expansion of the pinane family

Regioselective Control of the S_NAr Amination of 5‑Substituted-2,4-Dichloropyrimidines Using Tertiary Amine Nucleophiles

The S_NAr reaction of 2,4-dichloropyrimidines, further substituted with an electron-withdrawing substituent at C-5, has selectivity for substitution at C-4. Here we report that tertiary amine nucleophiles show excellent C-2 selectivity. In situ N-dealkylation of an intermediate gives the product that formally corresponds to the reaction of a secondary amine nucleophile at C-2. This reaction is practical (fast under simple reaction conditions, with good generality for tertiary amine structure and moderate to excellent yields) and significantly expands access to pyrimidine structures

Catalytic Spectrum of the Penicillin-Binding Protein 4 of <i>Pseudomonas aeruginosa</i>, a Nexus for the Induction of β‑Lactam Antibiotic Resistance

<i>Pseudomonas aeruginosa</i> is an opportunistic Gram-negative bacterial pathogen. A primary contributor to its ability to resist β-lactam antibiotics is the expression, following detection of the β-lactam, of the AmpC β-lactamase. As AmpC expression is directly linked to the recycling of the peptidoglycan of the bacterial cell wall, an important question is the identity of the signaling molecule(s) in this relationship. One mechanism used by clinical strains to elevate AmpC expression is loss of function of penicillin-binding protein 4 (PBP4). As the mechanism of the β-lactams is PBP inactivation, this result implies that the loss of the catalytic function of PBP4 ultimately leads to induction of antibiotic resistance. PBP4 is a bifunctional enzyme having both dd-carboxypeptidase and endopeptidase activities. Substrates for both the dd-carboxypeptidase and the 4,3-endopeptidase activities were prepared by multistep synthesis, and their turnover competence with respect to PBP4 was evaluated. The endopeptidase activity is specific to hydrolysis of 4,3-cross-linked peptidoglycan. PBP4 catalyzes both reactions equally well. When <i>P. aeruginosa</i> is grown in the presence of a strong inducer of AmpC, the quantities of both the stem pentapeptide (the substrate for the dd-carboxypeptidase activity) and the 4,3-cross-linked peptidoglycan (the substrate for the 4,3-endopeptidase activity) increase. In the presence of β-lactam antibiotics these altered cell-wall segments enter into the muropeptide recycling pathway, the conduit connecting the sensing event in the periplasm and the unleashing of resistance mechanisms in the cytoplasm

Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the β‑Lactam Antibiotics

The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium <i>Paraburkholderia acidophila</i> and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a cobiosynthesized β-lactam antibiotic. β-Lactams inhibit the penicillin-binding proteins, enzymes also critical to cell-wall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penicillin-binding protein (by β-lactams) activities results in deformation of the septal cell wall, observed microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbiological studies. These studies identify potentiation by the bulgecin at subminimum inhibitory concentrations of the β-lactam against three pathogenic Gram-negative bacteria and establish for the first time that this potentiation results in a significant increase in the bactericidal efficacy of a clinical β-lactam

Use of Silver Carbonate in the Wittig Reaction

An efficient synthesis of olefins by the coupling of stabilized, semistabilized, and nonstabilized phosphorus ylides with various carbonyl compounds in the presence of silver carbonate is reported. Wittig olefination of aromatic, heteroaromatic, and aliphatic aldehydes (yields >63%) and a ketone (yield 42%) are demonstrated. These reactions proceed overnight at room temperature, under weakly basic conditions, and as such extend the applicability of the Wittig reaction to base-sensitive reactants

Use of Silver Carbonate in the Wittig Reaction

An efficient synthesis of olefins by the coupling of stabilized, semistabilized, and nonstabilized phosphorus ylides with various carbonyl compounds in the presence of silver carbonate is reported. Wittig olefination of aromatic, heteroaromatic, and aliphatic aldehydes (yields >63%) and a ketone (yield 42%) are demonstrated. These reactions proceed overnight at room temperature, under weakly basic conditions, and as such extend the applicability of the Wittig reaction to base-sensitive reactants

Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the β‑Lactam Antibiotics

The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium <i>Paraburkholderia acidophila</i> and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a cobiosynthesized β-lactam antibiotic. β-Lactams inhibit the penicillin-binding proteins, enzymes also critical to cell-wall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penicillin-binding protein (by β-lactams) activities results in deformation of the septal cell wall, observed microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbiological studies. These studies identify potentiation by the bulgecin at subminimum inhibitory concentrations of the β-lactam against three pathogenic Gram-negative bacteria and establish for the first time that this potentiation results in a significant increase in the bactericidal efficacy of a clinical β-lactam