Larry C. Blaszczak

Genome of the Bacterium Streptococcus pneumoniae Strain R6

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Single Molecule Dynamics of Lysozyme Processing Distinguishes Linear and Cross-linked Peptidoglycan Substrates

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzymes time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate.

Fluorotamoxifen. A Caveat on the Generality of Electrophilic Destannylation

Abstract A one-step synthesis of fluorotamoxifen from tamnoxifen is described. Attempts to achieve synthesis of the title compound by fluorodestannylation or transmetallation were unsuccessful.

Using Steric Hindrance to Design New Inhibitors of Class C β-Lactamases

beta-lactamases confer resistance to beta-lactam antibiotics such as penicillins and cephalosporins. However, beta-lactams that form an acyl-intermediate with the enzyme but subsequently are hindered from forming a catalytically competent conformation seem to be inhibitors of beta-lactamases. This inhibition may be imparted by specific groups on the ubiquitous R(1) side chain of beta-lactams, such as the 2-amino-4-thiazolyl methoxyimino (ATMO) group common among third-generation cephalosporins. Using steric hindrance of deacylation as a design guide, penicillin and carbacephem substrates were converted into effective beta-lactamase inhibitors and antiresistance antibiotics. To investigate the structural bases of inhibition, the crystal structures of the acyl-adducts of the penicillin substrate amoxicillin and the new analogous inhibitor ATMO-penicillin were determined. ATMO-penicillin binds in a catalytically incompetent conformation resembling that adopted by third-generation cephalosporins, demonstrating the transferability of such sterically hindered groups in inhibitor design.

Allyltin reagents. A general approach to carbon-carbon bond formation applied to penicillin derived azetidinones - I.

Abstract Allyltributyltin has been shown to be a useful reagent for allyl transfer to penicillin derived azetidinones. The radical mechanism of the reaction has been demonstrated and its stereochemical consequences recorded. The process opens new possibilities for the semi-synthesis of novel bicyclic β-lactam antibiotics from penicillin or cephalosporin.

Radical Rearrangement: A Strategy for Conversion of Cephalosporin to 1-Carba(dethia)cephalosporin

Abstract A radical rearrangement approach to semisynthesis of the carbacephem class of β-lactam antibiotics is reported. The crucial bond construction in assembly of the carbacephem framework was accomplished by intramolecular C–C bond formation between an azetidin-2-one-4-yl and a pendant diene ester. This reactive intermediate was generated by fragmentation of a cephem derived radical followed by loss of sulfur dioxide. The radical precursor was prepared in 63% yield over four steps and the subsequent rearrangement reaction provided carbacephem products in a single step at 23% yield.

Synthesis of new 3-thiosubstituted carbacephem antibiotics and their activity against penicillin resistant Streptococcus pneumoniae

The synthesis of a series of 3-thiosubstituted carbacephem derivatives is described. The compounds were assayed against penicillin susceptible, intermediate and resistant strains of Streptococcus pneumoniae. Several analogs displayed potent in vitro activity against these organisms.

The use of a Mercury-Penicillin V Derivative to Localize Penicillin-Binding Proteins in Escherichia coli

Precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in a wild-type strain and two PBP overproducing mutants of Escherichia coli JM101 was investigated by high resolution electron microscopy (EM). A novel mercury-penicillin V (Hg-pen V) derivative was used as a heavy-metal labeled, electron dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth-phase. Results obtained from susceptibility tests, lysis experiments and PBP affinity assays revealed few differences between strains and between the antimicrobial activities of Hg-pen V and the parent compound, penicillin V. When cells were grown in the presence of Hg-pen V, Hg-pen V-PBP complexes could readily be visualized by EM of unstained whole mounts as distinct randomly situated electron dense particles. Thin sections of Hg-pen V-treated bacteria revealed similar electron dense particles located predominantly on the plasma membrane and seldom in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space thereby demonstrating the high resolution of the Hg-pen V probe. In addition, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron dense particles. The presence of electron dense particles in cells of the wild-type strain, demonstrated that our probe could identify PBPs in a naturally-occuring strain without inducing PBP overproduction.