Exploring Gram-negative bacteria as a source of biologically active natural products and application to the undergraduate research experience
Christine M. Theodore
The University of Tampa
Compounds from macro marine organisms are thought to be produced by microbial symbionts. Recent literature reviews have shown significant parallels between compounds isolated from macro organisms and non-photosynthetic Gram-negative bacteria (NPGNB). Though these structural parallels exist, little attention has been focused on these NPGNBs. Marine-derived NPGNBs represent an untapped well of potential microbial-produced compounds. As a proof of concept, sampling of California nearshore sediments gave rise to the discovery of a morphologically interesting Gram-negative bacterium. In addition to four known cyclic depsipeptides (kailiuns B-E) and two new analogues were also isolated and structurally analyzed (kailiuins G and H). Currently, these methods are being adapted to accommodate undergraduate researchers and sampling of Florida Gulf-Coast sediments.
Total Synthesis of (-)-Martinellic Acid Enabled by Enantioselective Copper-Catalyzed Quinoline Alkynylation
Mukesh Pappoppula, Flavio S. P. Cardoso, B. Owen Garrett, and Aaron Aponick*
University of Florida
(-)-Martinellic acid, and (+)-martinelline were isolated from martinella iquitosensis roots found in South America by Merck laboratories in 1995. These alkaloids were among the first potent, naturally occurring (non-peptide) bradykinin (BK) receptor antagonists (μm) to be reported. Also they exhibit anti-α-adrenic activity (nm), anti-muscarinic and moderately effective antibiotic against both Gram-positive and Gram-negative bacteria. The first total synthesis of (-)-martinellic acid was reported in 2001 by Ma et al.
A highly enantioselective alkynylation of quinolinium salts was developed using the chiral imidazole-based biaryl P,N-ligand StackPhos and copper bromide to establish the absolute stereochemistry of the martinella core from a 4-substituted quinoline. The three-component reaction between a quinoline, a terminal alkyne, and ethyl chloroformate afforded the desired products in high yields with excellent enantioselectivities (up to 98% ee). The development of this method and its application to the enantioselective total synthesis of (-)-martinellic acid will be described.
TOWARDS TERPENOID SCAFFOLDS VIA MULTI-FUNCTIONALIZATION OF kNOEVENAGEL ADDUCTS
Peter Vertesaljai, Matthew Williams, and Alexander J. Grenning
Department of Chemistry, University of Florida, Gainesville FL 32611-7200, USA
Knoevenagel adducts obtained from inexpensive ketones and malonic acid derivatives readily undergo selective transformations under mild conditions which makes them attractive building blocks. Our research focuses on the utility of Knoevenagel adducts in scalable, divergent syntheses towards terpenoid natural products and their analogs. A sequence of alkylation/[3,3]-sigmatropic rearrangement/α-alkylation/ring-closing metathesis was devised to construct a library of angular polycyclic carbon frameworks. Diversity is introduced by proper choice of starting materials as well as late stage functional group interconversion.
Another study based on selective functionalization allowed for the synthesis of highly substituted perhydroindoles. Experiments were aimed at outlining the scope and understanding the reactivity of such structures.
THE MECHANISM OF BREVETOXIN INDUCED OXIDATIVE STRESS AND EFFECT OF ANTIOXIDANT.
Anupama Tuladhar; Kathleen Rein*
Department of Chemistry and Biochemistry, Florida International University, Miami, Fl, 33199.
Karenia brevis, a red tide dinoflagellate, commonly blooms in the Gulf of Mexico and west coast of Florida. It is associated with massive fish and marine mammal poisoning and is known to produce neurotoxins known as brevetoxins (PbTx). Marine mammals and human cell lines exposed to red tide bloom have also shown signs of oxidative stress associated with PbTx exposure. These indicators include increased Reactive Oxygen Species (ROS) and DNA damage. Our group has established that PbTx-2 (but not PbTx-3) is an inhibitor of mammalian thioredoxin reductase (TrxR). The role of TrxR is the maintenance of redox homeostasis by reduction of thioredoxin and elimination of ROS via peroxiredoxins. Our hypothesis is that the selenocysteine of mammalian TrxR adds to PbTx-2 which have α,β-unsaturated bond in a Michael addition. Adducts between selenocysteine and PbTx-2 as well as cysteine and PbTx-2 have been easily prepared and characterized by FTICR-MS. Compromising the selenocysteine of TrxR results in the formation of a SecTRAP (Selenium Compromised Thioredoxin Reductase-derived Apoptotic Proteins), a pro-oxidant known to induce cell death. Our efforts to confirm the formation of an adduct between TrxR and PbTx-2 will be described. The effect of PbTx-2 induced oxidative stress is studied in the human lymphoblast cells and antioxidant such as such as vitamin C and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX) are used to mitigate the oxidative stress. This research thus provides the mechanism of inhibition of TrxR and potential antioxidant treatment for PbTx exposed humans and marine mammals.
ANTI-MRSA NATURAL PRODUCTS FROM AN EPIGENETIC MODIFIED FLORIDIAN MANGROVE-ASSOCIATED FUNGUS
Sylvia Soldatou1, 2, Renee Fleeman3, Lindsey N. Shaw3, Bill J. Baker1, 2
1 School of Chemistry, National University of Ireland, Galway, Ireland
2 Department of Chemistry, Center for Drug Discovery and Innovation (CDDI), University of South Florida, Tampa, USA
3 Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, USA
The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are responsible for the majority of nosocomial infections and are resistant to most antibacterial agents. We focus on mangrove-associated microorganisms which have been found to be a great source of chemically diverse and biologically active metabolites. Moreover, we are interested in enhancing the bioactivity of these secondary metabolites by epigenetic regulation of the fungi using HDAC and DNMT inhibitors.
During the course of this work, a suite of new and known compounds with anti-MRSA activity have been uncovered from a Floridian red mangrove-associated fungus. Using an enhanced rice medium, the fungus was cultured under the regulation of an HDAC inhibitor. After a 21-day incubation period the fungal culture was extracted in organic solvents. Following the anti-MRSA activity, the EtOAc partition was active against MRSA at 5 µg/ml and was subjected to MPLC. All bioactive MPLC fractions were purified through several rounds of HPLC which led to the isolation of at least four new and know compounds, exhibiting moderate activity against MRSA. The structures of the pure compounds which belong to the benzopyrone and benzofuran families were elucidated by 1D and 2D NMR spectroscopy.
Biosynthesis of deep-sea marine natural products: genes, enzymes, and pathways
Harbor Branch Oceanographic Institute, Florida Atlantic University
5600 US 1 North, Fort Pierce, FL, 34946
Among marine organisms, sponges are the most prolific producers of marine natural products (MNPs) which are recently becoming an attractive source of drug discovery. One probable reason is the huge amount of diverse microorganisms symbiotically living inside a sponge provide an extremely rich reservoir of natural product biosynthetic genes. However, the supply of a potent MNP is often the major issue banning it from further clinical studies. My laboratory focuses on the biosynthesis of MNPs from deep-sea sponges to identify biosynthetic origin and pathways and ultimately to develop sustainable supplies for them.
Leiodermatolide (LDM), isolated from the sponge Leiodermatium sp. (sampled at ~400 m), is a trans-AT (acyl transferase) type-I polyketide, in which AT domains are encoded by separate genes, instead of generally integrated into the architecture of polyketide synthases (PKSs). LDM is a potent mitosis-targeting agent and shows strong in vitro inhibitory activity against cancer cell proliferation. Notably, LDM is potent against the leukemia cell line HEL92.1.7 with a GI50 of 1.0 nM; this cell line contains the Pgp efflux transporter which is a major cause of drug resistance in chemotherapy. The in vivo antitumor activity of LDM was recently confirmed in a mouse model of metastatic pancreatic cancer. LDM arrests cell division at the G2/M phase. Notably, LDM employs a distinct mode of action that fundamentally differs from current anti-mitotic drugs including taxanes and vinca alkaloids. LDM doesn’t bind to tubulin, nor does it induce or inhibit tubulin polymerization in vitro. LDM also causes an immediate block of microtubule elongation.
A fosmid library of LDM-producing sponge Leiodermatium was constructed. The library is being screened by primers specifically targeting the highly conversed KS (ketosynthase) domains of trans-AT type I PKSs and other specific structural features. A set of fosmids were obtained. Cloning, sequencing, and restriction analysis indicated that those fosmids share common sequences but each with a unique restriction pattern. A 24-kb DNA sequence was identified, which are likely involved in the biosynthesis of a trans-AT type I polyketide. Domains such as KS, ACP (acyl carrier protein), KR (keto-reductase) and DH (dehydrogenase) were identified, but lacing AT domains. Sequencing of additional fosmids is in the process.