The bacterium Rhodococcus is a potential source for novel antimicrobial metabolites. Recently, the Rhodococcus strain MTM3W5.2 was isolated from a soil sample collected from Morristown, East Tennessee and was found to produce an inhibitor molecule that is active against similar Rhodococcus species. The aim of this research is to extract, purify, and characterize the active compound. The compound was obtained from both agar and broth cultures of strain MTM3W5.2 and purified by primary fractionation of crude extract on a Sephadex LH-20 column, followed by semi-preparative reversed phase column chromatography. Final purification was achieved using multiple rounds of an analytical C18 HPLC column. Based on the results obtained from UV-Vis, FT-IR, and HR-MS, the molecule is a polyketide with a molecular formula of C52H78O13 and an exact mass of 911.5490 amu. The partial structure of this compound has been determined using 1D and 2D NMR spectroscopy.

Due to increases in antibiotic resistance stemming from the overuse of commercial antibiotics, the need to discover novel antibacterial compounds is becoming more urgent. A species of Rhodococcus, MTM3W5.2, has been discovered and was found to produce a metabolite with inhibitory activity against closely related species. The aim of this study is to elucidate the structure of the inhibitory metabolite by isolating and purifying it; then characterizing it using spectroscopic techniques. The compound was isolated from MTM3W5.2 RM broth cultures using n-butanol extraction, which yielded an active crude extract. The crude extract was then subjected to fractionation using a Sephedex LH-20 column with a 100% methanol solvent. The inhibitory activity of the fractions was tested through disk diffusion assay using Rhodococcus erythropolis as an indicator. Further preparation was completed using preparative reverse-phase high-performance liquid chromatography. Advanced purification was conducted by multiple rounds of analytical reverse-phase HPLC. Throughout the study the HPLC fractions were characterized and stability was monitored using UV-Visible spectroscopy. Two pure samples at 58.xx and 72.xx minutes from HPLC collections were selected for further structural identification and are currently being studied using spectroscopic techniques, most notably 2D NMR

Many commercialized medicinal compounds are analogs of chemicals isolated from sources found in nature (also called natural products). However, the natural sources of these chemicals, such as plants, fungi, or insects, only offer small quantities of these bioactive agents. Thus, it is typically desirable to find ways to synthesize these products and their analogs in large quantities using cost-effective methods that also minimize the impact on the environment. It is also important to develop strategies that expedite the process of modifying the natural products, which allows medicinal chemists to determine which functional groups are enhancing or deleterious to the bioactivity. In the Brewer lab, I have investigated organic reactions and methodologies with this aim - to find ways to efficiently break and form carbon-carbon bonds, and to utilize these reactions in the total synthesis of structurally related natural products. The total synthesis of natural products is often used to showcase a methodology's utility by applying it in a more complex structure. The Lewis acid-promoted fragmentation of γ-silyloxy-β-hydroxy-α-diazo esters to provide tethered aldehyde ynoates was discovered and developed in the Brewer lab. This methodology was extended to bicyclic systems, in which the ring-fusion bond fragmented as a way to afford 10-membered ring ynones and ynolides, which are traditionally challenging to synthesize. This work will exhibit how the fragmentation reaction that provided 10-membered ynolides has the potential to lend itself to the synthesis of several structurally related, bioactive natural products via a divergent total synthesis strategy. In addition, this dissertation will describe our discovery that modifying the diazo carbonyl precursor to a β-hydroxy-α-diazo ketone changes the course of the Lewis acid-promoted reaction. Rather than a fragmentation sequence, the compound is converted to a vinyl cation, which undergoes a rearrangement then a C-H insertion of a second vinyl cation intermediate. This transition metal-free rearrangement/C-H insertion reaction provided cyclopentenone products. The migratory aptitudes of non-equivalent substituents in the cationic rearrangement step will also be discussed. Finally, the disparate reactivities of vinyl cations derived from diazo ketone, diazo ester, and diazo amide precursors will be detailed from an experimental and computational perspective. The results underscore the fact that this rearrangement and C-H insertion reaction may eventually be an effective way to prepare complex cyclopentyl-containing structures, which are common motifs in biologically active natural products.

Cancer is a serious family of disease that continues to cripple and claim those afflicted. For the last several decades, America has invested in a national program to alleviate cancer and cancer related suffering, ultimately seeking a cure. As part of this goal, the National Cancer Institute (NCI) has spent significant effort scouring the globe with the hope of finding naturally occurring compounds that can successfully combat cancer. Presently, this effort has uncovered many natural products with chemotherapeutic potential and many of the lead agents used in the fight against cancer are either natural products themselves or are compounds inspired by a natural product. This work describes one family of natural products uncovered by the NCI that is being explored for chemotherapeutic applications, namely the schweinfurthins. The schweinfurthins were isolated by the NCI; however the natural source, Macaranga schweinfurthii, did not provide these compounds in ample quantity to permit further study. The paucity of natural material indicated that a chemical synthesis of these compounds would be the most reliable method to provide meaningful amounts of schweinfurthins. The present work describes the chemical synthesis of four of the most potent schweinfurthins, describes the synthesis of numerous structural analogues, and details advances to the field of cascade cyclizations which makes their synthesis possible.

This research focuses on the understanding and engineering of nonribosomal peptide biosynthetic pathways in Streptomyces coelicolor CH999, Escherichia coli BAP1 and Saccharomyces cerevisiae BJ5464-NpgA. The biosynthetic systems of indigoidine from bacteria and beauvericin/bassianolide from fungi were studied in this research. The production of these valuble products was significantly increased by enhancing their synthetic pathway with metabolic engineering approaches. Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. Indigo is a dark blue crystalline powder and has been known for more than 4,000 years. It is commonly used to dye cotton yarn for the production of denim cloth to make blue jeans but the chemical synthesis of indigo requires harsh conditions and use of a strong base. Indigoidine is a new natural blue dye that is vi assembled from two molecules of L-glutamine under the catalysis of indigoidine synthetase. We identified a novel indigoidine synthetic gene from the genome of Streptomyces chromofuscus ATCC 49982. The successful heterologous expression of Sc-indC in E. coli BAP1 give us a pretty good yield of indigoidine under the optimized conditions. The production of this blue dye was then further improved by introducing two additional genes, sc-indB and glnA, into the biosynthetic pathway. Beauvericins and bassianolide are anticancer natural products from fungi and are assembled by corresponding iterative nonribosomal peptide synthetases. The beauvericin (BbBEAS) and bassianolide (BbBSLS) synthetases were successfully reconstituted in S. cerevisiae BJ5464-NpgA, leading to the production of beauvericins and bassianolide, respectively. The production of beauvericins was significantly improved by co-expression of BbBEAS and ketoisovalerate reductase (KIVR). To better understand the synthetic strategy of fungal iterative NRPs, the module/domain of BbBSLS and BbBEAS were dissected and reconstituted in S. cerevisiae. The result shows the intermodular linker is essential for the reconstitution of the separate modules and the domain swapping results indicated the fungal iterative NRPSs use a liner biosynthetic route which is different than bacterial iterative NRPs. The in vitro reactions of C2 and C3 with monomer/dimer/trimerN-acetylcysteamines demonstrated that C2 forms the amide bond and C3 catalyses the synthesis of the ester bond. Beauvericin could be reconstituted in vitro through co-reaction of C2(BbBEAS) and C3(BbBEAS) with D-Hiv-SNAC and N-Me-L-Phe- SNAC. This work also provides an unprecedented tool for engineering fungal iterative NRPSs to yield ‘unnatural’ cyclooligomer depsipeptides with varied chain lengths.

Lactacystin is a microbial metabolite isolated by Omura that exhibits neurotrophic activity in neuroblastoma cell lines. Lactacystin and especially its β-lactone analog are the first examples of non-polypeptide small molecules capable of specifically inhibiting the 20S proteasome. Various asymmetric total syntheses of lactacystin and its analogs have been reported. The total synthesis of clasto -lactacystin β-lactone is achieved using L-serine methyl ester as the starting material and the sole source of stereochemical induction. The success of this synthesis hinges on two featured transformations. The first key step involves formation of the γ -lactam core via rhodium (II) catalyzed intramolecular C-H insertion of the α-diazo-α-(phenylsulfonyl)acetamide intermediate. The methodology for this transformation has been developed and applied to the synthesis of highly functionalized stereogenic γ-lactams from natural α-amino acids. Three control elements that govern γ-lactam formation are described. This step is highlighted by the xvi simultaneous creation of two stereogenic centers of the γ-lactam core. The second key step involves the late stage aldol coupling for quaternary carbon formation and installation of the hydroxyisobutyl group. In all previously reported syntheses, this is the very first aspect which is addressed. The stereochemical outcome of this step is directed by the chiral environment of the enolate itself. Various attempts to achieve selectivity are explored and reported. Completion of the synthesis of clasto-lactacystin β-lactone requires 17 steps with an overall yield of 10%. Some general attempts for optimizing the synthetic scheme are discussed as well as the future direction of this research.

This thesis describes the chemical investigations of several ascidian species collected from the Great Barrier Reef, Queensland, Australia. The thesis is divided into two separate components, Part A focuses on the isolation and structure elucidation of 11 previously undescribed ascidian metabolites. All structures were assigned using a combination of spectroscopic and/or chemical methods. Part B relates to the isolation and chemical conversion of a natural product to a combinatorial template. The natural product template was subsequently used in the generation of a solution-phase combinatorial chemistry library. A further two combinatorial libraries were generated from a synthesised model compound that was related to the natural product template. Part A. Investigation of Aplidium longithorax collected from the Swains Reefs resulted in the isolation of two new para-substituted cyclofarnesylated quinone derived compounds, longithorones J (30) and K (31). The former compound had its absolute stereochemistry determined by the advanced Mosher method. From an Aplidium longithorax collected from Heron Island, two new cyclofarnesylated hydroquinone compounds, longithorols C (46) and D (47) and a novel macrocyclic chromenol, longithorol E (48) were isolated. Longithorol C (46) had its absolute stereochemistry determined by the advanced Mosher method. Chemical investigation of the deep-purple colonial ascidian, Didemnum chartaceum collected from Swains Reefs led to the isolation of five new lamellarin alkaloids, which included the 20-sulfated derivatives of lamellarins B (94), C (95) and L (96), the 8-sulfated derivative of lamellarin G (97) and the non-sulfated compound, lamellarin Z (98). The known lamellarins A (63), B (80), C (64), E (65), G (67), and L (71) plus the triacetate derivatives of lamellarin D (82) and N (83) were also isolated. An aberration in the integration of signals in the 1H NMR spectra of the 20-sulfated derivatives (94-96) led to NMR relaxation studies. T1 values were calculated for all protons in the sulfated lamellarins (94-97) and their corresponding non-sulfated derivatives (80, 64, 71, 67). The protons ortho to the sulfate group in compounds (94-97) had T1 values up to five times larger than the corresponding protons in their non-sulfated derivatives (80, 64, 71, 67). A specimen of Eudistoma anaematum collected from Heron Island was shown to contain a new b-carboline alkaloid, eudistomin V (130), in addition to the two known metabolites, eudistomin H (105) and I (106). Part B. The known natural products, 1,3-diphenethylurea (29), 1,3-dimethylxanthine (30), 1,3-dimethylisoguanine (31) and the salts of tambjamine C (16), E (18) and F (19) were isolated from the ascidian, Sigillina signifera collected in Blue Lagoon, Lizard Island. Base hydrolysis on mixtures of the salts of tambjamine C (16), E (18) and F (19) resulted in the production of 4-methoxy-2,2-bipyrrole-5-carbaldehyde (26). This natural product template (26) was used in the generation of an enamine combinatorial chemistry library (98, 103-111) using solution-phase parallel synthesis. The biaryl compound, 4-(2-thienyl)-1H-pyrrole-2-carbaldehyde (59) was successfully synthesised using Suzuki-Miyaura coupling conditions and subsequently used as a template in the generation of an amine (67, 77, 80-87) and imine (78, 92-95) combinatorial library using solution-phase parallel synthesis.

No description available.

Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.

Biosynthetic natural products are invaluable resources that have been gleaned from the environment for generations, and they play an essential role in drug development. Natural product biosynthesis also possesses the latent ability to affect biological systems adversely. This work implements recent advances in genomic, proteomic and microbiological technologies to understand further biosynthetic molecules that may influence progression of human disease. Azinomycin A and B are antitumor metabolites isolated from the terrestrial bacterium Streptomyces sahachiroi. The azinomycins possess an unusual aziridine [1,2-a] pyrrolidine ring that reacts in concert with an epoxide moiety to produce DNA interstrand cross-links. Genomic sequencing of S. sahachiroi revealed a putative azinomycin resistance protein (AziR). Overexpression of AziR in heterologous hosts demonstrated the protein increases cell viability and decreases DNA damage response in the presence of azinomycin. Fluorescence titration indicated AziR functions as an azinomycin binding protein. An understanding of azinomycin resistance is important for future engineering and drug delivery strategies. Additionally, the S. sahachiroi draft genome obtained via 454 pyrosequencing and Illumina sequencing revealed several silent secondary metabolic pathways that may provide new natural products with biomedical application. β-lactoglobulin (BLG), the most abundant whey protein in bovine milk, has been observed to promote the self-condensation of retinal and similar α,β-unsaturated aldehydes. BLG is a possible non-genetic instigator of cycloretinal and A2E accumulation in the macula, a condition associated with age-related macular degeneration. BLG-mediated terpenal condensation has been optimized for in vitro study with the retinal mimic citral. In rabbits fed retinal and BLG or skim milk, cycloretinal formation was detected in the blood by 1H-NMR, and SDS-PAGE analysis indicated BLG was present in blood serum, suggesting the protein survives ingestion and retains catalytic activity. Mass spectrometry and site-directed mutagenesis provided mechanistic insight toward this unusual moonlighting behavior. The experiments described in this dissertation serve to further natural product biosynthesis discovery and elucidation as they relate to consequences for human health. Efforts to solve azinomycin biosynthesis via enzymatic reconstitution, characterize compounds produced by orphan gene clusters within S. sahachiroi, and obtain a clear mechanism for BLG-promoted cycloterpenal formation are immediate goals within the respective projects.