We report practical methodology for the catalytic, asymmetric synthesis of beta-lactams resulting from the development of a catalyzed reaction of ketenes (or their derived zwitterionic enolates) and imines. The products of these asymmetric reactions can serve as precursors to a number of enzyme inhibitors and drug candidates as well as valuable synthetic intermediates. We present a detailed study of the mechanism of the beta-lactam forming reaction with proton sponge as the stoichiometric base, including kinetics and isotopic labeling studies. Stereochemical models based on molecular mechanics (MM) calculations are also presented to account for the observed stereoregular sense of induction in our reactions and to provide a guidepost for the design of other catalyst systems.

T he antimicrobial activity of phagocytes depends in part on the cells' ability to reduce oxygen to reactive microbicidal oxidants by means of an NADPH oxidase. In resting phagocytes, the NADPH oxidase is in a dormant state, but exposure of the cell to any of a variety of stimuli can activate the enzyme, causing it to release large amounts of O 2 Ϫ by reducing oxygen at the expense of NADPH. The oxidase is activated by the phosphorylation of one of its cytosolic subunits, p47 PHOX , on particular serines (1). In whole cells, the stimulation of an appropriate receptor activates PKC and phosphatidylinositol 3-kinase (PI3-kinase) (2-4). Although NADPH oxidase activation by PKC has been well characterized (5, 6), the association between PI3-kinase and NADPH oxidase activation remains to be established. A possible connection is through the activation of Akt (7), whose role in oxidase activation is suggested by the finding that Akt is activated rapidly when neutrophils are treated with oxidase-activating agents (8) and by the finding that oxidase activation is inhibited by wortmannin (9). The experiments described here show that Akt is able to activate the oxidase by phosphorylating p47 PHOX on serines S304 and S328. Experimental ProceduresMaterials. Active Akt was obtained from Upstate Biotechnology (Lake Placid, NY) or from J.-H.L., whose recombinant material was 95% pure by SDS͞PAGE. [␥-32 P]ATP was purchased from New England Nuclear. Phosphorylated peptides were obtained from Sigma.Isolation and Fractionation of Neutrophils. Neutrophils were obtained from normal subjects by dextran sedimentation and Ficoll͞Hypaque fractionation of freshly drawn citrateanticoagulated blood (10). After treatment on ice for 10-20 min with 5 l of 0.54 M diisopropyl fluorophosphate, the neutrophils, suspended at 10 8 cells per ml in a modified relaxation buffer (0.1 M KCl͞3 mM NaCl͞3.5 mM MgCl 2 ͞10 mM Pipes buffer, pH 7.3), were subjected to nitrogen cavitation. Membranes and cytosol were separated by centrifugation through a Percoll gradient. Aliquots of membrane and cytosol were stored at Ϫ70°C until use.Recombinant Oxidase Components. Recombinant fusion proteins composed of an upstream GST linked to a downstream p47 PHOX , p67 PHOX , or Rac2 were isolated from Escherichia coli transformed with pGEX-6P3 plasmids containing cDNA inserts encoding the downstream proteins. The fusion proteins were isolated by the addition of a prewashed 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) in PBS (133 mM NaCl͞16 mM Na 2 HPO 4 ͞2.7 mM KCl͞1.5 mM KH 2 PO 4 , pH 7.3). The tube was rotated end-over-end for 1 h at 4°C and then microcentrifuged for 3 min at 200 ϫ g to sediment the glutathione-Sepharose beads. The beads were washed four times with 10 bead volumes of PBS, and the bound fusion proteins were eluted by incubating for 30 min at 4°C with three 1-ml washes of 50 mM Tris⅐HCl, pH 8.0͞20 mM glutathione͞0.2 M NaCl. Excess glutathione was removed from the purified recombinant protein by dialysis against relaxation buffer and conce...

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Background: Conditional gene targeting methods were used to investigate the role of melanocortin-3 receptors (MC3Rs). Results: MC3Rs expressed in the brain are not sufficient to defend against diet-induced obesity but can improve metabolic homeostasis. Conclusion: The role of MC3Rs in energy homeostasis involves central and peripheral actions.Significance: This is the first evidence suggesting a role for central and peripheral MC3Rs in energy homeostasis.

BackgroundEstrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer.Patients and methodsTo identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots.ResultsWe identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations.ConclusionsCollectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.