Synthesis of 1-Azatriene Complexes of Tungsten: Metal-Promoted Ring-Opening of Dihydropyridine.

For nearly a century, chemists have explored how transition-metal complexes can affect the physical and chemical properties of linear conjugated polyenes and heteropolyenes. While much has been written about higher hapticity complexes (η4-η6), less is known about the chemistry of their η2 analogues. Herein, we describe a general method for synthesizing 5,6-η2-(1-azatriene) tungsten complexes via a 6π-azaelectrocyclic dihydropyridine ring-opening that is promoted by the π-basic nature of {WTp(NO)(PMe3)}. This study includes detailed spectroscopic and crystallographic data for the η2-dihydropyridine and η2-1-azatriene complexes, both of which were prepared as single regio- and stereoisomers.

Tungsten-anisole complex provides 3,6-substituted cyclohexenes for highly diversified chemical libraries.

Medicinal chemists use vast combinatorial molecular libraries to develop leads for new pharmaceuticals. The syntheses of these compounds typically rely on coupling molecular fragments through atoms with planar (sp2) geometry. These so-called flat molecules often lack the protein binding site specificity needed to be an effective drug. Here, we demonstrate a coupling strategy in which a cyclohexene is used as a linker to connect two diverse molecular fragments while forming two new tetrahedral (sp3) stereocenters. These connections are made with the aid of a tungsten complex that activates anisole toward an unusual double protonation, followed by sequential nucleophilic additions. As a result, either cis- or trans-disubstituted cyclohexenes can be prepared with a range of chemical diversity unparalleled by other dearomatization methods.

The Tungsten-Promoted Synthesis of Piperidyl-Modified erythro-Methylphenidate Derivatives.

Due to its efficacy as a dopamine receptor agonist, methylphenidate (MPH) is of interest as a potential therapeutic for cocaine addiction. While numerous derivatives of MPH have been investigated for their potential medicinal value, functionalization of the piperidine ring has not been explored. The pyridine borane ligand in WTp(NO)(PMe3)(η2-pyBH3) is dearomatized by the metal and can be elaborated to the analogous η2-mesylpyridinium complex. Installing a methyl phenylacetate moiety at the C2' position via a Reformatsky reaction followed by a tandem protonation/nucleophilic addition sequence results in a library of erythro MPH analogues functionalized at the piperidyl C5' position. The functional group is added chemoselectively to C5', cis to the methyl phenylacetate. Repeating this procedure with an enantioenriched source of the tungsten reagent results in enantioenriched MPH derivatives. All identities of the newly reported compounds are supported by comprehensive 2D NMR and HRMS data or crystallographic data.

Genome editing without nucleases confers proliferative advantage to edited hepatocytes and corrects Wilson disease.

Application of classic liver-directed gene replacement strategies is limited in genetic diseases characterized by liver injury due to hepatocyte proliferation, resulting in decline of therapeutic transgene expression and potential genotoxic risk. Wilson disease (WD) is a life-threatening autosomal disorder of copper homeostasis caused by pathogenic variants in copper transporter ATP7B and characterized by toxic copper accumulation, resulting in severe liver and brain diseases. Genome editing holds promise for the treatment of WD; nevertheless, to rescue copper homeostasis, ATP7B function must be restored in at least 25% of the hepatocytes, which surpasses by far genome-editing correction rates. We applied a liver-directed, nuclease-free genome editing approach, based on adeno-associated viral vector-mediated (AAV-mediated) targeted integration of a promoterless mini-ATP7B cDNA into the albumin (Alb) locus. Administration of AAV-Alb-mini-ATP7B in 2 WD mouse models resulted in extensive liver repopulation by genome-edited hepatocytes holding a proliferative advantage over nonedited ones, and ameliorated liver injury and copper metabolism. Furthermore, combination of genome editing with a copper chelator, currently used for WD treatment, achieved greater disease improvement compared with chelation therapy alone. Nuclease-free genome editing provided therapeutic efficacy and may represent a safer and longer-lasting alternative to classic gene replacement strategies for WD.

The double protonation of dihapto-coordinated benzene complexes enables dearomatization using aromatic nucleophiles.

Friedel-Crafts Arylation (the Scholl reaction) is the coupling of two aromatic rings with the aid of a strong Lewis or Brønsted acid. This historically significant C-C bond forming reaction normally leads to aromatic products, often as oligomeric mixtures, dictated by the large stabilization gained upon their rearomatization. The coordination of benzene by a tungsten complex disrupts the natural course of this reaction sequence, allowing for Friedel-Crafts Arylation without rearomatization or oligomerization. Subsequent addition of a nucleophile to the coupled intermediate leads to functionalized cyclohexenes. In this work, we show that by coordinating benzene to tungsten through two carbons (dihapto-coordinate), a rarely observed double protonation of the bound benzene is enabled, allowing its subsequent coupling to a second arene without the need of a precious metal or Lewis acid catalyst.

The Double-Protonation of Dihapto-Coordinated Benzene Complexes: An Enabling Strategy for Dearomatization Using Aromatic Nucleophiles.

Friedel Crafts Arylation (the Scholl reaction) is the coupling of two aromatic rings with the aid of a strong Lewis or Bronsted acid. This historically significant C-C bond forming reaction normally leads to aromatic products, often as oligomeric mixtures, dictated by the large stabilization gained upon their rearomatization. In this preliminary communication, we demonstrate how the pre-coordination of benzene by tungsten disrupts the natural course of this reaction sequence, allowing for Friedel-Crafts Arylation without rearomatization or oligomerization . Subsequent addition of a nucleophile to the coupled intermediate leads to functionalized cyclohexenes. The key feature of this reaction is a tungsten complex bound through two carbons, which enables a rarely observed double protonation of the bound benzene, and subsequent coupling to the second arene without the need of precious metal or Lewis acid catalysts.

Dosing a synbiotic of human milk oligosaccharides and B. infantis leads to reversible engraftment in healthy adult microbiomes without antibiotics.

Predictable and sustainable engraftment of live biotherapeutic products into the human gut microbiome is being explored as a promising way to modulate the human gut microbiome. We utilize a synbiotic approach pairing the infant gut microbe Bifidobacterium longum subspecies infantis (B. infantis) and human milk oligosaccharides (HMO). B. infantis, which is typically absent in adults, engrafts into healthy adult microbiomes in an HMO-dependent manner at a relative abundance of up to 25% of the bacterial population without antibiotic pretreatment or adverse effects. Corresponding changes in metabolites are detected. Germ-free mice transplanted with dysbiotic human microbiomes also successfully engraft with B. infantis in an HMO-dependent manner, and the synbiotic augments butyrate levels both in this in vivo model and in in vitro cocultures of the synbiotic with specific Firmicutes species. Finally, the synbiotic inhibits the growth of enteropathogens in vitro. Our findings point to a potential safe mechanism for ameliorating dysbioses characteristic of numerous human diseases.

Phenyl Sulfones: A Route to a Diverse Family of Trisubstituted Cyclohexenes from Three Independent Nucleophilic Additions.

A novel process is described for the synthesis of di- and trisubstituted cyclohexenes from an arene. These compounds are prepared from three independent nucleophilic addition reactions to a phenyl sulfone (PhSO2R; R = Me, Ph, and NC4H8) dihapto-coordinated to the tungsten complex {WTp(NO)(PMe3)}(Tp = trispyrazolylborate). Such a coordination renders the dearomatized aryl ring susceptible to protonation at a carbon ortho to the sulfone group. The resulting arenium species readily reacts with the first nucleophile to form a dihapto-coordinated sulfonylated diene complex. This complex can again be protonated, and the subsequent nucleophilic addition forms a trisubstituted cyclohexene species bearing a sulfonyl group at an allylic position. Loss of the sulfinate anion forms a π-allyl species, to which a third nucleophile can be added. The trisubstituted cyclohexene can then be oxidatively decomplexed, either before or after substitution of the sulfonyl group. Nucleophiles employed include masked enolates, cyanide, amines, amides, and hydride, with all three additions occurring to the same face of the ring, anti to the metal. Of the 12 novel functionalized cyclohexenes prepared as examples of this methodology, nine compounds meet five independent criteria for evaluating drug likeliness. Structural assignments are supported with nine crystal structures, density functional theory studies, and full 2D NMR analysis.

Hydroamination of Dihapto-Coordinated Benzene and Diene Complexes of Tungsten: Fundamental Studies and the Synthesis of γ-Lycorane.

Reactions are described for complexes of the form WTp(NO)(PMe3)(η2-arene) and various amines, where the arene is benzene or benzene with an electron-withdrawing substituent (CF3, SO2Ph, SO2Me). The arene complex is first protonated to form an η2-arenium species, which then selectively adds the amine. The resulting η2-5-amino-1,3-cyclohexadiene complexes can then be subjected to the same sequence with a second nucleophile to form 3-aminocyclohexene complexes, where up to three stereocenters originate from the arene carbons. Alternatively, 1,3-cyclohexadiene complexes containing an ester group at the 5 position (also prepared from an arene) can be treated with acid followed by an amine to form trisubstituted 3-aminocyclohexenes. When the amine is primary, ring closure can occur to form a cis-fused bicyclic γ-lactam. Highly functionalized cyclohexenes can be liberated from the tungsten through oxidative decomplexation. The potential utility of this methodology is demonstrated in the synthesis of the alkaloid γ-lycorane. An enantioenriched synthesis of a lactam precursor to γ-lycorane is also described. This compound is prepared from an enantioenriched version of the tungsten benzene complex. Regio- and stereochemical assignments for the reported compounds are supported by detailed 2D-NMR analysis and 13 molecular structure determinations (SC-XRD).

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus.

The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool.IMPORTANCE The SaeRS two-component system is a master transcriptional activator of virulence factor production in response to the host environment in S. aureus, and strains lacking FA kinase have severely attenuated SaeRS-dependent virulence factor transcription. FA kinase is required for the activation of exogenous FAs, and it plays a role in cellular lipid homeostasis by recycling cellular FAs into the phospholipid biosynthetic pathway. Activation of the sensor kinase, SaeS, is mediated by its membrane anchor domain, and the FAs which accumulate in FA kinase knockout strains are potent inhibitors of SaeS-dependent signaling. This work identifies FAs as physiological effectors for the SaeRS system and reveals a connection between cellular lipid homeostasis and the regulation of virulence factor transcription. FA kinase is widely distributed in Gram-positive bacteria, suggesting similar roles for FA kinase in these organisms.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes.

On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei.

BACKGROUND: Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle. RESULTS: Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA. CONCLUSIONS: Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host.

The intersection of anthropology and medicine as portrayed in Paul Farmer's photo album.

Paul Farmer, physician, anthropologist, and author, spoke at the 2009 Society for Medical Anthropology Conference at Yale University in September.