Browsing by Subject "Natural products"

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    Plant phenylpropanoid biosynthesis in Escherichia coli: engineering novel pathways and tools
    (2014-10) Bloch, Sarah E.
    Plant phenylpropanoid natural products are important in the discovery of safe and effective therapeutics. Most plant natural products cannot be economically mass produced via extraction from plant tissue or chemical synthesis. In recent decades, engineering microbes to carry out the biosynthesis of plant natural products has emerged as a powerful technology. The goal of this thesis was to expand the capabilities of microbial biosynthesis of plant phenylpropanoids in Escherichia coli through exploring novel biosynthetic pathways and metabolic engineering tools. I first explored the biosynthesis of valuable lignans in E. coli, establishing random oxidative radical coupling through overexpression of a laccase and attempting to show stereoselective coupling by a dirigent protein. I also designed and built a biosynthetic pathway for rosmarinic acid, a valuable hydroxycinnamic acid ester, showed pathway bottlenecks and limitations, and identified future optimization strategies. I have also begun a project to better understand cargo protein encapsulation within bacterial microcompartments and to develop their utility as a means of spatially organizing metabolic pathways. This work has contributed significantly to the field of microbial metabolic engineering and has laid the groundwork for future economically viable production platforms.
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    Strategies for the synthesis of bioactive compounds: biomimetic approaches to the Salinosporamides and the hexadehydro-Diels-Alder reaction
    (2013-08) Willoughby, Patrick
    Part I: The salinosporamides are a subset of polyketide-derived marine natural products that have as their key structural feature a fused γ-lactam/β-lactone moiety. These molecules are known to inhibit the 20S proteasome (20SP) by utilizing the β-lactone reactivity, a promising target for cancer therapeutics. We propose that salinosporamide A may be formed biosynthetically by a non-enzymatic bis-cyclization event involving an uncatalyzed intramolecular ketene-ketone [2+2] cycloaddition of an amidoketene precursor. We also propose a transition state that places the C2 substituent pseudo-equatorial, allowing for enhanced stereoselectivity during bis-cyclization. Enolate formation from a thioester followed by internal acylation and concurrent loss of thiolate would form a 5(4H)-oxazolone. It is known that 5(4H)-oxazolones readily tautomerize to 4-hydroxyoxazoles, which are in equilibrium with their mesoionic Münchnone forms. Valence bond isomerism interconverts Münchnones to amidoketenes. Spontaneous ketene-ketone [2+2] cycloaddition would siphon the precursors through such a tautomeric manifold to give the natural product. Progress toward the synthesis of the biosynthetic precursors to salinosporamide A is disclosed in Chapters 3-5.Part II: The second part of this Thesis describes our recent, serendipitous encounter with the hexadehydro-Diels-Alder (HDDA) reaction. The HDDA reaction is a mode of the Diels-Alder reaction that involves [4+2] cycloaddition between a diyne and an alkyne to form aryne reactive intermediate. In Part II we show the scope of substrates that are capable of generating an aryne via the HDDA reaction. Additionally, we show that HDDA-mediated generation of arynes allows for the discovery of new modes of aryne trapping, for example, the desaturation of hydrocarbons.
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    Synthesis and Evaluation of Transvalencin Analogues and Adenylation Inhibitors as Antitubercular Agents and Chemical Probes
    (2013-06) Nelson, Kathryn Marie
    >Mycobacterium tuberculosis (Mtb), the etiological agent of pulmonary tuberculosis, is the leading cause of death due to an infectious disease worldwide. Due to a lack of new drug development, poor fidelity to currently available therapeutics, and repeated exposure to therapeutics, Mtb has become multidrug resistant, extensively drug resistant, and even totally drug resistant in some patients. With nearly 9 million deaths and 1.4 million new cases reported by the World Health Organization (WHO) in 2011, new therapeutics that act by novel mechanisms of action are desperately needed to fight this global health threat. Herein we describe our efforts to develop new antitubercular agents by attacking the bacteria's need for iron. This approach involves the inhibition of the biosynthetic pathway to produce siderophores, small molecule iron chelators responsible for acquiring iron in limiting conditions, such as a human host. A prototypical inhibitor of the initiating enzyme in this pathway, MbtA, had previously been developed by our lab, and was used to develop a small set of analogues for in vivo evaluation. We employed Sprague-Dawley rats to evaluate the oral bioavailability of our compounds, revealing that the pKa of the linker nitrogen of the scaffold had a large effect on compound permeability.In addition, we studied the mechanism of action of our parent inhibitor, Sal-AMS, through the development of a photoaffinity probe to label and pull down proteins for target identification. A probe containing a benzophenone moiety for photo-crosslinking and a small alkyne handle for attachment of an imaging or enrichment tag was successfully synthesized. This probe was successful in identifying the intended enzyme of interest (MbtA) as a binding partner, but did not yield any additional hits, suggesting Sal-AMS is a highly specific inhibitor. We also studied a natural product, transvalencin Z, that had been reported as selective against mycobacteria. This compound was very similar in structure to the mycobactins from Mtb, suggesting that it might interfere with iron acquisition or homeostasis. We successfully synthesized the 4 possible diastereomers of the reported structure in an attempt to define the absolute stereochemistry of the natural product, but were unable to match spectroscopic data to the literature report. We attempted to confirm the true stereochemistry through activity testing, but again found our negative results to be in stark contrast to those reported by the discovery group. Finally, we aided a collaborator in the development and synthesis of a probe against DhbE in Bacillus subtilis. Dr. Jun Yin of the University of Chicago was studying the substrate specificity of adenylation domains, and chose an enzyme highly homologous to our target MbtA. We designed a probe that incorporated a similar inhibitor, DHB-AMS, and a long flexible linker with a biotin attached for Dr. Yin's unique yeast cell display assay. Dr. Yin was able to utilize these probes to successfully identify mutant adenylation enzymes with altered specificity towards nonnative substrates. This technique is an exciting new way to potentially access analogues of natural products through manipulation of the biosynthetic machinery, instead of through the organic chemist. These studies have continued to advance our understanding of a new mechanism of action against Mtb, and have brought us one step closer to a preclinical candidate.