Synthesis of Naturally Occurring Decanolides

Decanolides, 10-membered ring lactones, are examples of natural compounds having biological interest. these molecules have been found to play diverse biological roles in the different organisms from which they are isolated. Decanolides, along with other medium membered rings (ranging from eight to twelve atoms), are difficult to synthesize due to entropic factors (probability of chain ends meeting) and enthalpic factors (transannular strain development in the transition state). It is therefore important to develop and optimize different approaches for the syntheses of these compounds. Phoracantholide I, an odiferous defensive secretion from the metasternal gland of the eucalypt longicorn beetle, Phoracantha synonym, consists of a 10-membered ring lactone. Our synthesis uses Schreiber\u27s ozonolysis methodology to generate a key unsymmetrical aldehyde-ester-alkene intermediate. Ozonolytic cleavage of cyclohexene followed by in situ nucleophilic attach by 4-penten-2-ol generates an a-alkoxy hydroperoxide which can be dehydrated to provide an unsymmetrical aldehyde-ester-alkene intermediate in 42% yield. Finally use of Grubbs RCM catalyst followed by hydrogenation of the crude reaction mixture generates ( _+) -Phoracantholide I in 4 steps and 12% yield overall. Jasmine ketolactone was the first decanolide to be extracted from a plant. It was isolated in 1964 by Demole and Stolle from the oil of Jasmine grandiflorum L. Approaches towards the total synthesis of jasmine ketolactone, a more complex target than phoracantholide I, are also reported suing the same ozonolysis methodology. Herbarumin I is a decanolide which was recently isolated from the fungus Phoma herbarum. It belongs to a family of lactones which have been shown to exhibit significant phytotoxic effects affecting the germination and growth of Amaranthus hypochodriacus seedlings. Our purpose for synthesizing herbarumin I is to test its affects on Arabidopsis thaliana and determine the functional groups responsible for its phytotoxic activity. Experimental procedures for the synthesis of herbarumin I were adapted from Furstner et al

Characterization of AntB, a Promiscuous Acyltransferase Involved in Antimycin Biosynthesis

The <i>in vivo</i> and <i>in vitro</i> characterization of AntB, a dedicated acyltransferase encoded in the antimycin biosynthetic gene cluster, which catalyzes the C-8 acyloxy formation is reported. It is demonstrated that AntB has broad substrate specificity toward both the acyl substrate and the acyl carrier and produces more antimycin analogues with varying C-8 acyloxy moieties

Enzymatic Synthesis of Dilactone Scaffold of Antimycins

Antimycins are a family of natural products possessing outstanding biological activities and unique structures, which have intrigued chemists for over a half century. The antimycin structural skeleton is built on a nine-membered dilactone ring containing one alkyl, one acyloxy, two methyl moieties, and an amide linkage connecting to a 3-formamidosalicylic acid. Although a biosynthetic gene cluster for antimycins was recently identified, the enzymatic logic that governs the synthesis of antimycins has not yet been revealed. In this work, the biosynthetic pathway for antimycins was dissected by both genetic and enzymatic studies for the first time. A minimum set of enzymes needed for generation of the antimycin dilactone scaffold were identified, featuring a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line containing both <i>cis-</i> and <i>trans</i>-acting components. Several antimycin analogues were further produced using <i>in vitro</i> enzymatic total synthesis based on the substrate promiscuity of this NRPS-PKS machinery

Biosynthesis of Amphi-enterobactin Siderophores by <i>Vibrio harveyi</i> BAA-1116: Identification of a Bifunctional Nonribosomal Peptide Synthetase Condensation Domain

The genome of <i>Vibrio harveyi</i> BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (<i>aebA–F</i>) resembling that for entero­bactin, yet entero­bactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative entero­bactin-like biosynthetic gene cluster (<i>aebG</i>). The proximity of this FACL gene to the entero­bactin-like synthetase suggested that <i>V. harveyi</i> may produce amphiphilic entero­bactin-like siderophores. Extraction of the bacterial cell pellet of <i>V. harveyi</i> led to the isolation and structure determination of a suite of eight amphi-entero­bactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxy­benzoyl-l-serine and acyl-l-serine. The FACL knockout mutant, Δ<i>aebG V. harveyi</i>, and the NRPS knockout mutant, Δ<i>aebF V. harveyi</i>, do not produce amphi-entero­bactins. The amphi-entero­bactin biosynthetic machinery was heterologously expressed in <i>Escherichia coli</i> and reconstituted <i>in vitro</i>, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions

Turnerbactin, a Novel Triscatecholate Siderophore from the Shipworm Endosymbiont <i>Teredinibacter turnerae</i> T7901

<div><p>Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont <i>Teredinibacter turnerae</i> T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the <i>entCEBA</i> genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited <i>T. turnerae</i> T7901 cultures. Turnerbactin is a trimer of <i>N</i>-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.</p></div

Human Gut Bacterial Metabolism Drives Th17 Activation and Colitis

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity

Human gut bacterial metabolism drives Th17 activation and colitis.

Bacterial activation of T helper 17 (Th17) cells exacerbates mouse models of autoimmunity, but how human-associated bacteria impact Th17-driven disease remains elusive. We show that human gut Actinobacterium Eggerthella lenta induces intestinal Th17 activation by lifting inhibition of the Th17 transcription factor Rorγt through cell- and antigen-independent mechanisms. E. lenta is enriched in inflammatory bowel disease (IBD) patients and worsens colitis in a Rorc-dependent manner in mice. Th17 activation varies across E. lenta strains, which is attributable to the cardiac glycoside reductase 2 (Cgr2) enzyme. Cgr2 is sufficient to induce interleukin (IL)-17a, a major Th17 cytokine. cgr2+ E. lenta deplete putative steroidal glycosides in pure culture; related compounds are negatively associated with human IBD severity. Finally, leveraging the sensitivity of Cgr2 to dietary arginine, we prevented E. lenta-induced intestinal inflammation in mice. Together, these results support a role for human gut bacterial metabolism in driving Th17-dependent autoimmunity