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A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall (peptidoglycan) biosynthesis pathway, is reported. This highly convergent synthesis will enable further studies on bacterial resistance mechanisms and may provide insight toward the development of new chemotherapeutic agents with novel modes of action.

Group I β-lactamases are major resistance determinants to β-lactam antibiotics. Despite intense study, the identity of the catalytic base, the direction of hydrolytic attack, and the functional difference between β-lactam substrates and β-lactam inhibitors remain controversial. To explore these questions, we determined the X-ray crystal structures of several representative β-lactams in their acyl−adduct complexes with the group I β-lactamase AmpC. A complex with the substrate loracarbef and a deacylation-deficient mutant enzyme reveals an ordered water molecule that is consistent with β-face attack of the catalytic water in group I β-lactamases. The ring nitrogen of the substrate is placed to hydrogen-bond with this water in the position it is thought to adopt in the deacylation transition state. In complexes with the inhibitors cloxacillin and moxalactam, conformational restrictions displace the equivalent ring nitrogens, sterically blocking the formation of the presumed deacylation transition-state stru...

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.

Penicillin-binding proteins (PBPs) are the enzymes that are required for the biosynthesis of the bacterial cell wall (10, 11, 23, 31). PBPs catalyze the final steps of the polymerization (transglycosylation) and cross-linking (transpeptidation) of peptidoglycan, an essential component of the bacterial cell wall (10, 11, 23, 31). PBPs are membrane-bound enzymes and targets of β-lactam antibiotics (6, 7, 10, 11, 14, 24, 28–31). The emerging resistance of pathogenic gram-positive bacteria to β-lactam antibiotics is a serious clinical problem (6, 25, 29, 30). Resistance to β-lactam antibiotics in some species such as Streptococcus pneumoniae has occurred by development of altered high-molecular-mass PBPs which have reduced affinity for the antibiotics (6, 7, 10, 11, 14, 24, 25, 29, 30). Extensive molecular and genetic studies have been devoted to the understanding of the mechanisms of bacterial resistance to β-lactam antibiotics (6, 7, 10, 11, 25, 29, 30). PBPs have often been detected by labeling bacterial membrane preparations with 3H-, 14C-, or 125I-labeled penicillin, separating the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and exposing the gels to X-ray films (22, 27, 28). The major limitations of this type of methodology are time intensiveness (days to weeks), accumulation of hazardous materials, and lack of commercial availability. In this study, we describe a new, rapid, sensitive, and nonradioactive method for the detection and study of bacterial PBPs. This method involves the use of BOCILLIN FL (Fig. ​(Fig.1),1), a newly synthesized and commercially available fluorescent penicillin, as a labeling reagent (Molecular Probes, Inc., Eugene, Oreg.). The BOCILLIN FL-labeled PBPs were separated by SDS-PAGE and detected with a FluorImager or with the naked eye under UV light. FIG. 1 Structure of BOCILLIN FL.

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 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 enzyme's 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.

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

Fluorine nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging were examined as noninvasive methods for characterizing antibiotic disposition and pharmacokinetics in vivo. For determination of their utility, a 19 F surface coil was constructed and an m-(trifluoromethyl)-containing penicillin V analogue (LY242072; 1) was synthesized. Various concentrations of 1 were injected intravenously into anesthetized rats whose urethras were occluded. The animals were placed on the surface coil, which was tuned to 19 F, and then into a 4.7-T, 33-cm bore instrument, in which in vivo measurements of 1 were made on urine excreted into the bladder. At sacrifice, the urine was collected, and antibiotic leveis were determined in vitro by both HPLC and high-resolution NMR. The limit of detection of 1 by NMR was 0.7 mg/mL of urine. When compared with standard in vitro quantitative methods using current technology, quantitation by in vivo surface coil NMR is not precise. Magnetic resonance imaging was used to image the bladder at a 35-mm 3 voxel resolution with datum collection times of ˜ 1 h. The 19 F surface coil was used successfully to spectroscopically locate xenobiotic fluorine in the rat thorax. 19 F NMR may offer an opportunity for the noninvasive in vivo detection of the distribution of various classes of therapeutic compounds.

A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall (peptidoglycan) biosynthesis pathway, is reported. This highly convergent synthesis will enable further studies on bacterial resistance mechanisms and may provide insight toward the development of new chemotherapeutic agents with novel modes of action.

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.

Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP) assays. The disadvantages of most forms of labeled penicillin G are instability on storage and the long exposure times usually required for autoradiography or fluorography of electrophoretic gels. We investigated the utility of radioiodinated penicillin V as an alternative reagent. Radioiodination of p-(trimethylstannyl)penicillin V with [125I]Na, using a modification of the chloramine-T method, is simple, high yielding, and site specific. We demonstrated the general equivalence of commercially obtained [3H]penicillin G and locally synthesized [125I]penicillin V (IPV) in their recognition of bacterial PBPs. Profiles of PBPs in membranes from Bacteroides fragilis, Escherichia coli, Providencia rettgeri, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Enterococcus faecium labeled with IPV or [3H]penicillin G were virtually identical. Use of IPV as the imaging agent in competition experiments for determination of the affinities of various beta-lactam antibiotics for the PBPs of E. coli yielded results similar to those obtained in experiments with [3H]penicillin G. Dried electrophoretic gels from typical PBP experiments, using IPV at 37.3 Ci/mmol and 30 micrograms/ml, exposed X-ray film in 8 to 24 h. The stability of IPV on storage at 4 degrees C was inversely proportional to specific activity. At 37.3 Ci/mmol and 60 micrograms/ml, IPV retained useful activity for at least 60 days at 4 degrees C. IPV represents a practical and stable reagent for rapid PBP assays.

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.

A series of non-covalent inhibitors of the serine protease dipeptidyl peptidase IV (DPP-IV) were found to adopt a U-shaped binding conformation in X-ray co-crystallization studies. Remarkably, Tyr547 undergoes a 70 degrees side-chain rotation to accommodate the inhibitor and allows access to a previously unexposed area of the protein backbone for hydrogen bonding. (c) 2007 Elsevier Ltd. All rights reserved.

A novel mild access to the β-carboline skeleton is described. The reaction is a Bischler-Napieralski-type cyclocondensation, promoted by (PhO) 3 P.Cl 2 , which is performed in dichloromethane at -30 °C. The products are easily obtained in good yields and do not require further chromatographic purification.

[reaction: see text] The (PhO)(3)P.Cl(2) reagent, prepared in situ by titrating a solution of triphenyl phosphite with chlorine, is used to convert N-monosubstituted amides into their corresponding amines. The reaction, if compared to other traditional methods, shows the advantage of very mild conditions and low temperature (-30 degrees C--rt).

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.

[reaction: see text]. A synthesis of stereochemically pure and orthogonally protected N-acetyl-(2-deoxy-2-aminoglucopyranosyl)-beta-[1,4]-N-acetylmuramyl (NAG-NAM) monopeptide (2), the glycopeptide core of lipid II and of the bacterial cell wall repeating unit, is reported.

[19312-27-5] C10H22Sn2 (MW 379.66) InChI = 1S/C4H4.6CH3.2Sn/c1-3-4-2;;;;;;;;/h1-2H2;6*1H3;; InChIKey = XARXNRBKDLHCGG-UHFFFAOYSA-N (synthon for the 2,3-dianion of 1,3-butadiene) Physical Data: low-melting solid, bp 65–66 °C/0.8 mmHg (distillation); stable indefinitely at −20 °C under nitrogen Preparative Methods: 1 treatment of either 2,3-dichloro-1,3-butadiene or commercially available 1,4-Dichloro-2-butyne with 2 equiv Trimethylstannyllithium in THF affords 1,4-bis(trimethylstannyl)-2-butyne as the kinetic product. This alkyne can be isomerized to 2,3-bis(trimethylstannyl)-1,3-butadiene by a catalytic amount of either Methyllithium or trimethylstannyllithium and 2 equiv of HMPA in THF (eq 1). Therefore either dihalide can serve as precursor to either stannane. A one-pot synthesis can be accomplished in 70% yield. It may be advantageous to prepare the trimethylstannyllithium reagent with lithium wire containing 1% sodium.2 (1) Handling, Storage, and Precautions: organotin compounds should be considered toxic and should be handled with appropriate care.

[77425-85-5] C16H38SiSn (MW 377.34) InChI = 1S/C4H11Si.3C4H9.Sn/c1-5(2,3)4;3*1-3-4-2;/h1H2,2-4H3;3*1,3-4H2,2H3; InChIKey = IOKUELHTFOPLKK-UHFFFAOYSA-N (precursor to trimethylsilylmethyllithium; methylenation of aldehydes and ketones) Alternate Name: tributyl(trimethylsilylmethyl)tin. Physical Data: bp (distillation) 146 °C/2.5 mmHg. Preparative Methods: is conveniently prepared by the action of Tri-n-butylstannyllithium on (Chloromethyl)trimethylsilane in THF/hexane at 0 °C.1 Alternatively, Trimethylsilylmethylmagnesium Chloride can be treated with Tri-n-butylchlorostannane in refluxing ether.2 Excellent yields (>90%) may be realized by either method. Handling, Storage, and Precautions: organotin compounds should be considered toxic and should be handled with appropriate care. Use in a fume hood.

[62762-48-5] C6H16S2Sn (MW 271.00) InChI = 1S/C3H7S2.3CH3.Sn/c1-4-3-5-2;;;;/h3H,1-2H3;3*1H3; InChIKey = PZBJSYAHWOROJB-UHFFFAOYSA-N (precursor to acyl anion equivalent with 1,4-addition selectivity; dianion equivalent1) Physical Data: bp (distillation) 93–95 °C/7 mmHg; n20D 1.5548. Preparative Methods: commercially available bis(methylthio)methane is deprotonated by n-Butyllithium in THF/hexane at −60 to 0 °C. The resulting lithio derivative is quenched at −40 °C with 1 equiv of Chlorotrimethylstannane. An aqueous workup followed by vacuum distillation affords the bis(methylthio)(trimethylstannyl)methane reagent in 82% yield.2 The analogous Bis(methylthio)(trimethylsilyl)methane can be prepared by a similar procedure.3 Handling, Storage, and Precautions: organotin compounds should be considered toxic and should be handled with appropriate care. Use in a fume hood.

The mecA-27r gene, which encodes PBP2a-27r, was modified by site-specific mutagenesis, resulting in replacement of the N-terminal membrane anchor with a short chelating peptide (CP-PBP2a-27r). CP-PBP2a-27r retained the same binding affinity for beta-lactam antibiotics as the wild-type enzyme. Approximately 95% pure CP-PBP2a-27r was recovered in a single step by use of chelating-peptide-immobilized metal ion affinity chromatography.

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.

Benzothiophenes 3 and 4, derived from the acrylophenone antiestrogen trioxifene (2), are characterized by high estrogen receptor (ER) affinity and low residual estrogenicity compared to tamoxifen (1a). In order to characterize further the growth suppression mechanism for these structural types we have prepared structural variants of 2 bearing hydroxy groups positioned to maximize ER affinity. Thus, dihydronaphthalenes 5 and 6 and benzofluorenes 7 and 8 were prepared and studied in MCF-7 human breast cancer cells, in comparison with 3 and 4. All compounds were powerful suppressants of cell growth, with 50% inhibition ranging from 4.5 to 160 nM. Greatest potency was seen with diphenols 6 and 8. These compounds had intracellular ER affinities ranging from 0.2 to 4.1% of that of estradiol, suggestive of a potential for partial agonist effects. Simultaneous exposure of cells to 0.1 microM concentrations of estradiol and 3 or 4 did not affect the degree of growth inhibition seen with 0.1 microM 3 or 4 alone. Partial reversal of inhibition occurred when 0.1 microM 5-8 were each accompanied by 0.1 microM estradiol. Under these conditions complete reversal of growth inhibition has been found with 1a, 1b, and other triarylethylenes. Calmodulin, a putative target for triarylethylenes, and which is antagonized by 1a, was shown to interact weakly with 7 and 8 and not at all with 3-6. These results suggest that MCF-7 cell growth suppression by 3-8 may be due to interaction with unidentified receptors besides ER and extend earlier findings indicating that events occurring after interaction of these compounds with ER differ from those of triarylethylene antiestrogens.

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.

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

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.