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Reimer, D, Hughes CC.  2017.  Thiol-based probe for electrophilic natural products reveals that most of the ammosamides are artifacts. Journal of Natural Products. 80:126-133.   10.1021/acs.jnadprod.6b00773   AbstractWebsite

To date, 16 members of the ammosamide family of natural products have been discovered, and except for ammosamide D each of these metabolites is characterized by an unusual chlorinated pyrrolo[4,3,2-de]quinoline skeleton. Several ammosamides have been shown to inhibit quinone reductase 2, a flavoenzyme responsible for quelling toxic oxidative species in cells or for killing cancer cells outright. Treatment of the extract from an ammosamide-producing culture (Streptomyces strain CNR-698) with a thiol-based reagent designed to label electrophilic natural products produced an ammosamide C-thiol adduct. This observation led us to hypothesize, and then demonstrate through experimentation, that all of the other ammosamides are derived from ammosamide C via nonenzymatic processes involving exposure to nucleophiles, air, and light. Like many established electrophilic natural products, reaction with the thiol probe suggests that ammosamide C is itself an electrophilic natural product. Although ammosamide C did not show substantial cytotoxicity against cancer cells, its activity against a marine Bacillus bacterial strain may reflect its ecological role.

Castro-Falcon, G, Hahn D, Reimer D, Hughes CC.  2016.  Thiol probes to detect electrophilic natural products based on their mechanism of action. Acs Chemical Biology. 11:2328-2336.   10.1021/acschembio.5b00924   AbstractWebsite

New methods are urgently needed to find novel natural products as structural leads for the development of new drugs against emerging diseases such as cancer and multiresistant bacterial infections. Here we introduce a reactivity-guided drug discovery approach for electrophilic natural products, a therapeutically relevant class of natural products that covalently modify their cellular targets, in crude extracts. Using carefully designed halogenated aromatic reagents, the process furnishes derivatives that are UV-active and highly conspicuous via mass spectrometry by virtue of an isotopically unique bromine or chlorine tag. In addition to the identification of high-value metabolites, the process facilitates the difficult task of structure elucidation by providing derivatives that are primed for X-ray crystallographic analysis. We show that a cysteine probe efficiently and chemoselectively labels enone-, beta-lactam-, and beta-lactone-based electrophilic natural products (parthenolide, andrographolide, wortmannin, penicillin G, salinosporamide), while a thiophenol probe preferentially labels epoxide-based electrophilic natural products (triptolide, epoxomicin, eponemycin, cyclomarin, salinamide). Using the optimized method, we were able to detect and isolate the epoxide-bearing natural product tirandalydigin from Salinispora and thereby link an orphan gene cluster to its gene product.

Richter, TKS, Hughes CC, Moore BS.  2015.  Sioxanthin, a novel glycosylated carotenoid, reveals an unusual subclustered biosynthetic pathway. Environmental Microbiology. 17:2158-2171.   10.1111/1462-2920.12669   AbstractWebsite

Members of the marine actinomycete genus Salinispora constitutively produce a characteristic orange pigment during vegetative growth. Contrary to the understanding of widespread carotenoid biosynthesis pathways in bacteria, Salinispora carotenoid biosynthesis genes are not confined to a single cluster. Instead, bioinformatic and genetic investigations confirm that four regions of the Salinispora tropicaCNB-440 genome, consisting of two gene clusters and two independent genes, contribute to the in vivo production of a single carotenoid. This compound, namely (2S)-1-(-D-glucopyranosyloxy)-3,4-didehydro-1,2-dihydro-phi,-caroten-2-ol, is novel and has been given the trivial name sioxanthin'. Sioxanthin is a C-40-carotenoid, glycosylated on one end of the molecule and containing an aryl moiety on the opposite end. Glycosylation is unusual among actinomycete carotenoids, and sioxanthin joins a rare group of carotenoids with polar and non-polar head groups. Gene sequence homology predicts that the sioxanthin biosynthetic pathway is present in all of the Salinispora as well as other members of the family Micromonosporaceae. Additionally, this study's investigations of clustering of carotenoid biosynthetic genes in heterotrophic bacteria show that a non-clustered genome arrangement is more common than previously suggested, with nearly half of the investigated genomes showing a non-clustered architecture.

Alvarez-Mico, X, Jensen PR, Fenical W, Hughes CC.  2013.  Chlorizidine, a Cytotoxic 5H-Pyrrolo 2,1-a isoindol-5-one-Containing Alkaloid from a Marine Streptomyces sp. Organic Letters. 15:988-991.   10.1021/ol303374e   AbstractWebsite

Cultivation of an obligate marine Streptomyces strain has provided the cytotoxic natural product chlorizidine A. X-ray crystallographic analysis revealed that the metabolite is composed of a chlorinated 2,3-dihydropyrrolizine ring attached to a chlorinated 5H-pyrrolo[2,1-a]isoindol-5-one. The carbon stereocenter in the dihydropyrrolizine is S-configured. Remarkably, the 5H-pyrrolo[2,1-a]isoindol-5-one moiety has no precedence in the field of natural products. The presence of this ring system, which was demonstrated to undergo facile nucleophilic substitution reactions at the activated carbonyl group, is essential to the molecule's cytotoxicity against HCT-116 human colon cancer cells.

Yang, MH, Liu Z, Hughes C, Stern AM, Wang H, Zhong ZT, Kan B, Fenical W, Zhu J.  2013.  Bile salt-induced intermolecular disulfide bond formation activates Vibrio cholerae virulence. Proceedings of the National Academy of Sciences of the United States of America. 110:2348-2353.   10.1073/pnas.1218039110   AbstractWebsite

To be successful pathogens, bacteria must often restrict the expression of virulence genes to host environments. This requires a physical or chemical marker of the host environment as well as a cognate bacterial system for sensing the presence of a host to appropriately time the activation of virulence. However, there have been remarkably few such signal-sensor pairs identified, and the molecular mechanisms for host-sensing are virtually unknown. By directly applying a reporter strain of Vibrio cholerae, the causative agent of cholera, to a thin layer chromatography (TLC) plate containing mouse intestinal extracts, we found two host signals that activate virulence gene transcription. One of these was revealed to be the bile salt taurocholate. We then show that a set of bile salts cause dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulfide bonds between cysteine (C)-207 residues in its periplasmic domain. Various genetic and biochemical analyses led us to propose a model in which the other cysteine in the periplasmic domain, C218, forms an inhibitory intramolecular disulfide bond with C207 that must be isomerized to form the active C207-C207 intermolecular bond. We then found bile salt-dependent effects of these cysteine mutations on survival in vivo, correlating to our in vitro model. Our results are a demonstration of a mechanism for direct activation of the V. cholerae virulence cascade by a host signal molecule. They further provide a paradigm for recognition of the host environment in pathogenic bacteria through periplasmic cysteine oxidation.

Yamanaka, K, Ryan KS, Gulder TAM, Hughes CC, Moore BS.  2012.  Flavoenzyme-Catalyzed Atropo-Selective N,C-Bipyrrole Homocoupling in Marinopyrrole Biosynthesis. Journal of the American Chemical Society. 134:12434-12437.   10.1021/ja305670f   AbstractWebsite

Axially chiral biaryl compounds are frequently encountered in nature where they exhibit diverse biological properties. Many are biphenols that have C-C or C-O linkages installed by cytochrome P450 oxygenases that control the regio- and stereoselectivity of the intermolecular coupling reaction. In contrast, bipyrrole-coupling enzymology has not been observed. Marinopyrroles, produced by a marine-derived streptomycete, are the first 1,3'-bipyrrole natural products. On the basis of marinopyrrole's unusual bipyrrole structure, we explored its atropo-selective biosynthesis in Streptomyces sp. CNQ-418 in order to elucidate the N,C-bipyrrole homocoupling enzymology. Through a series of genetic experiments involving the discovery and heterologous expression of marinopyrrole biosynthesis genes, we report that two flavin-dependent halogenases catalyze the unprecedented homocoupling reaction.

Haste, NM, Hughes CC, Tran DN, Fenical W, Jensen PR, Nizet V, Hensler ME.  2011.  Pharmacological properties of the marine natural product marinopyrrole a against methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 55:3305-3312.   10.1128/aac.01211-10   AbstractWebsite

The ongoing spread of methicillin-resistant Staphylococcus aureus (MRSA) strains in hospital and community settings presents a great challenge to public health and illustrates the urgency of discovering new antibiotics. Marinopyrrole A is a member of a structurally novel class of compounds identified from a species of marine-derived streptomycetes with evidence of antistaphylococcal activity. We show that marinopyrrole A has potent concentration-dependent bactericidal activity against clinically relevant hospital-and community-acquired MRSA strains, a prolonged postantibiotic effect superior to that of the current first-line agents vancomycin and linezolid, and a favorable resistance profile. Marinopyrrole A showed limited toxicity to mammalian cell lines (at >20x MIC). However, its antibiotic activity against MRSA was effectively neutralized by 20% human serum. A variety of marinopyrrole analogs were isolated from culture or synthetically produced to try to overcome the inhibitory effect of serum. While many of these compounds retained potent bactericidal effect against MRSA, their activities were also inhibited by serum. Marinopyrrole A has significant affinity for plastic and may therefore have potential as a potent anti-MRSA agent in cutaneous, intracatheter, or antibiotic-lock applications.

Hughes, CC, Kauffman CA, Jensen PR, Fenical W.  2010.  Structures, reactivities, and antibiotic properties of the marinopyrroles A-F. Journal of Organic Chemistry. 75:3240-3250.   10.1021/jo1002054   AbstractWebsite

Cultivation of actinomycete strain CNQ-418, retrieved from a deep ocean sediment sample off the coast of La Jolla, CA, has provided marinopyrroles A F. Sharing just 98% 16S rRNA gene sequence identity with S. sannurensis, the strain likely represents a new Streptomyces species. The metabolites contain an unusual 1,3'-bipyrrole core decorated with several chlorine and bromine substituents and possess marked antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The congested N,C-biaryl bond establishes an axis of chirality that, for marinopyrroles A-E, is configurationally stable at room temperature. Moreover, the natural products are fashioned strictly in the M-configuration. The Paal-Knorr condensation was adapted for the synthesis of the 1,3'-bipyrrole core. Halogenation of this material with N-bromosuccinimide cleanly furnished the 4,4',5,5'-tetrahalogenated core that characterizes this class of marine-derived metabolites.

Hughes, CC, Fenical W.  2010.  Total synthesis of the ammosamides. Journal of the American Chemical Society. 132:2528-2529.   10.1021/ja9106572   AbstractWebsite

The ammosamides A-C are chlorinated pyrrolo[4,3,2-de]quinoline metabolites isolated from the marine-derived Streptomyces strain CNR-698. The natural products, which possess a dense array of heteroatoms, were synthesized in 17-19 steps from 4-chloroisatin. That the Five nitrogen atoms were introduced at the appropriate time and in a suitable oxidation state was key to the success of the total synthesis. Compared to synthetic deschloro ammosamide B, natural ammosamide B is much less susceptible to oxidative degradation.

Hughes, CC, Fenical W.  2010.  Antibacterials from the sea. Chemistry-a European Journal. 16:12512-12525.   10.1002/chem.201001279   AbstractWebsite

The ocean contains a host of macroscopic life in a great microbial soup. Unlike the terrestrial environment, an aqueous environment provides perpetual propinquity and blurs spatial distinctions. Marine organisms are under a persistent threat of infection by resident pathogenic microbes including bacteria, and in response they have engineered complex organic compounds with antibacterial activity from a diverse set of biological precursors. The diluting effect of the ocean drives the construction of potent molecules that are stable to harsh salty conditions. Members of each class of metabolite- ribosomal and non-ribosomal peptides, alkaloids, polyketides, and terpenes-have been shown to exhibit antibacterial activity. The sophistication and diversity of these metabolites points to the ingenuity and flexibility of biosynthetic processes in Nature. Compared with their terrestrial counterparts, antibacterial marine natural products have received much less attention. Thus, a concerted effort to discover new antibacterials from marine sources has the potential to contribute significantly to the treatment of the ever increasing drug-resistant infectious diseases.

Pangerl, M, Hughes CC, Trauner D.  2010.  Total Synthesis of Newbouldine via Reductive N–N Bond Formation. Tetrahedron. 66:6626-6631. Abstract

The first total synthesis of newbouldine has been achieved employing a new, reductive N–N bond forming reaction. The asymmetric synthesis confirms that the natural product is a racemate.

Hughes, CC, Yang YL, Liu WT, Dorrestein PC, La Clair JJ, Fenical W.  2009.  Marinopyrrole A target elucidation by acyl dye transfer. Journal of the American Chemical Society. 131:12094-+.   10.1021/ja903149u   AbstractWebsite

The targeting of marinopyrrole A to actin was identified using a fluorescent dye transfer strategy. The process began by appending a carboxylic acid terminal tag to a phenol in the natural product. The resulting probe was then studied in live cells to verify that it maintained activity comparable to marinopyrrole A. Two-color fluorescence microscopy confirmed that both unlabeled and labeled materials share comparable uptake and subcellular Localization in HCT-116 cells. Subsequent immunoprecipitation studies identified actin as a putative target in HCT-116 cells, a result that was validated by mass spectral, affinity, and activity analyses on purified samples of actin. Further data analyses indicated that the dye in the marinopyrrole probe was selectively transferred to a single residue K,15, an event that did not occur with related acyl phenols and reactive labels. In this study, the combination of cell, protein, and amino acid analysis arose from a single sample of material, thereby, suggesting a means to streamline and reduce material requirements involved in mode of action studies.

Nett, M, Guider TAM, Kale AJ, Hughes CC, Moore BS.  2009.  Function-Oriented Biosynthesis of beta-Lactone Proteasome Inhibitors in Salinispora tropica. Journal of Medicinal Chemistry. 52:6163-6167.   Doi 10.1021/Jm901098m   AbstractWebsite

The natural proteasome inhibitor salinosporamide A from the marine bacterium Salinispora tropica is a promising drug candidate for the treatment of multiple myeloma and mantle cell lymphoma. Using a comprehensive approach that combined chemical synthesis with metabolic engineering, we generated a series of salinosporamide analogues with altered proteasome binding affinity. One of the engineered compounds is equipotent to salinosporamide A in inhibition of the chymotrypsin-like activity of. the proteasome yet exhibits superior activity in the cell-based HCT-116 assay.

Hughes, CC, MacMillan JB, Gaudencio SP, Fenical W, La Clair JJ.  2009.  Ammosamides A and B target myosin. Angewandte Chemie-International Edition. 48:728-732.   10.1002/anie.200804107   AbstractWebsite

The protein target of the ammosamides, cytotoxic natural products from a marine-derived actinomycete, has been elucidated. An immunoaffinity fluorescent tag was used to construct a fluorescent molecular probe (see structure). First, uptake and localization of the probe in cells was visualized with fluorescence microscopy. The probe was then used to co-immunoprecipitate proteins that bound to the ammosamide core. Myosin was thus identified as a target.

Hughes, CC, MacMillan JB, Gaudencio SR, Jensen PR, Fenical W.  2009.  The ammosamides: structures of cell cycle modulators from a marine-derived Streptomyces species. Angewandte Chemie-International Edition. 48:725-727.   10.1002/anie.200804890   AbstractWebsite

From the ocean: Ammosamides A (1) and B (2) were isolated from a marine-derived Streptomyces species collected in the Bahamas. The structures of these chlorinated tricyclic pyrroloquinoline alkaloids were solved using X-ray crystallographic techniques. Ammosamide A (1) was shown to contain an unusual thio-γ-lactam ring. Both metabolites show specific nanomolar cytotoxicity against selected cancer cell lines.

Hughes, CC, Prieto-Davo A, Jensen PR, Fenical W.  2008.  The marinopyrroles, antibiotics of an unprecedented structure class from a marine Streptomyces sp.. Organic Letters. 10:629-631.   10.1021/ol702952n   AbstractWebsite

Cultivation of an obligate marine Streptomyces strain has furnished the marinopyrroles A and B, densely halogenated, axially chiral metabolites that contain an uncommon bispyrrole structure. X-ray analysis of marinopyrrole B showed that the natural product exists as an atropoenantiomer with the M-configuration. Though configurationally stable at room temperature, M(-)-marinopyrrole A can be racemized at elevated temperatures to yield the non-natural P-(+)-atropo-enantiomer. The marinopyrroles possess potent antibiotic activities against methicillin-resistant Staphylococcus aureus.

Miller, AK, Hughes CC, Kennedy-Smith JJ, Gradl SN, Trauner D.  2006.  Total Synthesis of (−)-Heptemerone B and (−)-Guanacastepene E. Journal of the American Chemical Society. 128:17057-17062.: American Chemical Society Abstract

A concise, stereoselective, and convergent total synthesis of the unnatural enantiomer of the neodolastane diterpenoid heptemerone B has been completed. Saponification of (?)-heptemerone afforded (?)-guanacastepene E. The absolute stereochemistry of (?)-heptemerone B was thus established as 5-(S), the same as (?)-guanacastepene E. The longest linear sequence of the synthesis comprises 17 (18) steps from simple known starting materials. Our general synthetic approach integrates a diverse set of reactions, including an intramolecular Heck reaction to create one quaternary stereocenter and a cuprate conjugate addition for the establishment of the other. The central seven-membered ring was closed with an uncommon electrochemical oxidation, whereas the five-membered ring was formed through ring-closing metathesis. The absolute configuration of the two key building blocks was established through an asymmetric reduction and an asymmetric ene reaction.

Bowie, AL, Hughes CC, Trauner D.  2005.  Concise Synthesis of (±)-Rhazinilam through Direct Coupling. Organic Letters. 7:5207-5209.: American Chemical Society Abstract

A concise synthesis of rhazinilam through direct, palladium-catalyzed, intramolecular coupling is described.

Endicott, CA, Strauss HL, Hughes CC, Trauner D.  2005.  Infrared Hole Burning and Conformational Change in a Borane−Ammonia Complex. The Journal of Physical Chemistry A. 109:7714-7717.: American Chemical Society   10.1021/jp051969i   AbstractWebsite

The N?D stretching region in the infrared spectrum of the ammonia complex of tris-(2-methoxymethyl-phenol)-borane containing one D atom has been examined. The N?D bands have been hole burned, and the resulting spectra reveal the reorientation kinetics of the ammonia. The ammonia is hydrogen bonded with the bond distances and reorientation barrier typical of other compounds. The N?D stretching frequencies are higher than those of comparison compounds.

Hughes, CC, Miller AK, Trauner D.  2005.  An Electrochemical Approach to the Guanacastepenes. Organic Letters. 7:3425-3428.: American Chemical Society   10.1021/ol047387l   AbstractWebsite

An asymmetric approach toward the [6?7?5] ring system of the guanacastepenes is described.

Hughes, CC, Trauner D.  2004.  Palladium-Catalyzed Couplings to Nucleophilic Heteroarenes: the Total Synthesis of (−)-Frondosin B. Tetrahedron. 60:9675-9686. Abstract

The total synthesis of (−)-frondosin B, the enantiomer of naturally-occuring (+)-frondosin B, is described, wherein a palladium-catalyzed cyclization is used to establish the tetracyclic ring system of the natural product.

Hughes, CC, Kennedy-Smith JJ, Trauner D.  2003.  Synthetic Studies toward the Guanacastepenes. Organic Letters. 5:4113-4115.: American Chemical Society   10.1021/ol035559t   AbstractWebsite

An asymmetric approach toward the [6?7] ring system of the guanacastepenes is described.

Hughes, CC, Scharn D, Mulzer J, Trauner D.  2002.  Borane−Ammonia Complexes Stabilized by Hydrogen Bonding. Organic Letters. 4:4109-4111.: American Chemical Society   10.1021/ol026865u   AbstractWebsite

Novel boron?ammonia complexes, wherein an NH3 molecule is tightly bound through all four of its atoms, have been prepared and studied. The solid-state structure of ortho MOM-phenyllithium is reported.

Hughes, CC, Trauner D.  2002.  The Total Synthesis of (−)-Amathaspiramide F. Angewandte Chemie International Edition. 114:4738-4741.: WILEY-VCH Verlag   10.1002/1521-3757(20021202)114:23<4738::AID-ANGE4738>3.0.CO;2-C   AbstractWebsite


Hughes, CC, Trauner D.  2002.  Concise Total Synthesis of (−)-Frondosin B Using a Novel Palladium-Catalyzed Cyclization. Angewandte Chemie International Edition. 41:1569-1572.: WILEY-VCH Verlag GmbH   10.1002/1521-3773(20020503)41:9<1569::AID-ANIE1569>3.0.CO;2-8   AbstractWebsite