<p>  </p> <p>A concise and scalable total synthesis of the therapeutically relevant methyl ester of the prostaglandin D₂ metabolite, tricyclic-PGDM, was accomplished in 8 steps from a known and easily accessed cyclopentene-diol derivative. The route features three key transition metal catalyzed steps. These steps include: a nickel catalyzed Ueno-Stork type dicarbofunctionalization which generates two consecutive stereocenters on the central cyclopentane core, a late-stage palladium-catalyzed carbonylative oxaspirolactonization, and a <em>Z</em>-selective cross metathesis to introduce the <em>Z</em>-butenoate side chain- a motif difficult to introduce through traditional protocols and which caused significant issues in the previous total syntheses of tricyclic-PGDM. Through this route, we have accumulated 75 mg of material for an ¹⁸O tricyclic-PGDM clinical assay which previously suffered from a material shortage. In addition to completing the synthesis, we generalized the <em>Z</em>-selective cross metathesis and nickel catalyzed Ueno-Stork protocols to numerous other substrates further demonstrating the utility of these transformations. </p> <p><br></p> <p>Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from the cofactor SAM to arginine residues on various cytosolic and nuclear proteins. Of the nine members of the PRMT family, PRMT5 has been the most extensively studied and has been shown to regulate processes such as the DNA damage response, cell proliferation, and mRNA translation. Although numerous pathways have been identified that regulate PRMT5 activity, the cytosolic protein MEP50 has been identified as a key regulator in many diseases. PRMT5 and MEP50 interact to form a hetero-octameric complex, which can modulate the activity of PRMT5 for many cellular processes. Two new generations of PRMT5:MEP50 inhibitors were strategically designed and synthesized, which do not suffer from chemical instability like our previously most potent analogues. Our best compounds have IC₅₀ values ranging from 512 to 2.5 nM in LNCaP cells, and were confirmed to target the PRMT5:MEP50 interaction through BiFC analysis.</p>

<p>Natural products, along with natural product derivatives, are known to be at the root of the development of many pharmaceuticals, oftentimes showing unique bioactivity against interesting targets. Specifically, natural products containing furans show activity against a variety of diseases including fungal infections, and cancers. It is hypothesized that unknown natural products containing furans could show more potent or other biological activities. However, it is challenging to discover and isolate these small molecules from cell supernatant. The work described herein showcases the development of a molecular probe that can covalently attach to furan moieties via a [4 + 2] Diels-Alder cycloaddition, making them easily identifiable on liquid chromatography mass spectroscopy (LC-MS). The molecular probe, which undergoes this reaction with a variety of furans, was designed with both a UV-tag and a mass tag to enable easy identification. The probe has been tested with a variety of purified furans, including natural products, methylenomycin furan (MMF) hormones, and MMF derivatives. Moreover, work has begun to test the molecular probe in cell supernatants. </p>

<p>  </p> <p>Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated, with many natural products being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. These elicitors can be natural products, including antibiotics or hormones, or synthetic compounds. The use of small molecule elicitors to induce the production of secondary metabolites has several advantages. First, addition of elicitors to fermentation media can result in increased titers of known natural products. Second, elicitors can enable the discovery of novel natural products typically produced at undetectable levels. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of “silent” natural products. In this work, we sought to expand on the method of chemical induction by utilizing oxytetracycline at a sub-MIC concentration to induce secondary metabolite production in Streptomyces. We have shown that translation-inhibiting antibiotics, specifically oxytetracycline, have a profound effect on the production of coeliomycin P1, actinorhodin, and calcium-dependent antibiotics (CDAs) in S. coelicolor and S. lividans. The expression of actinorhodin in S. lividans under these conditions is unique, unlike its counterpart, S. coelicolor, which can produce actinorhodin under standard conditions. In addition to the increased production of known secondary metabolites, we have also demonstrated the induction of BGCs in several other strains of Streptomyces, which were observed via LC-MS. </p> <p>In addition to exploring antibiotics as elicitors we have explored the traditional approach of natural product discovery by taking an bioactivity guided approach. Several strain that we isolated from soil collect of Hawaii were screened for activity against several pathogenic strains primarily looking for which strain will inhibit the growth of a. baumannii, which is an intriguing target because the rate of resistance to common antibacterial medication is rising and it’s membrane composition is vastly different compared to other gram negative bacterium like E.coli. From this preliminary screening 1 strain (Streptomyces sp. CS62) out of the 8 that tested exhibited the desired biological activity. The supernatant of Streptomyces sp .CS62 was processed and screen by LC-MS to gain insight on the type of molecules that Streptomyces CS62 could produce. Upon our initial screening process none of the masses observed in the mass spec were matched to knowns. However, after 2D NMR analysis and genomic analysis it was unveiled that Streptomyces sp. CS62 produces factumycin a known antibacterial agent that targets A.baumannii .This unfourtunate turn of events illustrates the issues with natural product discovery and the need to improve natural product databases.</p> <p>In conjunction to discovering a novel producer of factumycin we are also investigating the production of antifungal compounds from Staphylococcus lugdunensis  a commensal strain that modulates the microbiome by producing lugdunin. The supernatant collected of Staphylococcus lugdunensis  is exclusively being test against Candida auris due to the immense health risk it possess to society because of its innate resistance to many antifungal drugs and its ability to rapidly gain resistance to other classes of antifungals.</p> <p>In addition to exploring the influence of antibiotics on secondary metabolite production and using bioactivity as a guide to discovering antibiotics. We are evaluating the soils collected from unique environments as potential sources for novel natural products. Specifically, we are evaluating the biosynthetic potential of bacteria from ore-forming environments, specifically fluorspar and topaz mines. Soils from ore-forming environments tend have low pH, high saline content, low water holding capacity, and poor nutrient availability. Therefore, ore-forming environments pose a hostile environment for life. To date, no one has explored the natural product potential, or the bacterial diversity, exhibited in these harsh environments. To assess the bacterial diversity, bacteria were isolated from various ore-forming environments using a procedure that is selective for actinobacteria. Following bacterial isolation, genomic DNA was isolated and 16s rRNA gene sequencing was performed to gauge the type of bacteria that were isolated. To stimulate secondary metabolite production, bacteria were then subjected to 7 different media conditions. The supernatant collected from these media conditions were tested against ESKAPE pathogens utilizing the CTSI broth microdilution assay. LC-MS MS analysis was performed for samples exhibiting biological activity. GNPS molecular networking was then utilized to determine potential molecules present in each sample.  Through this process we were able to identify one strain, which we named Streptomyces sp. S1A that exhibited a board range of biological activity (anticancer and antibacterial) and possess a wide array of biosynthetic gene clusters ranging complex macrolides (PKS and NRPS) to terpenes. </p> <p>In summary this multifaced approach to natural product discovery may lead to the discovery of novel antibiotics, enable us to increase production of known or unknown antibiotics through chemical induction, and the characterization of metabolites from Streptomyces sp. S1A will shed insight on the biochemical potential of organisms that inhabit ore-forming environments </p>

<p dir="ltr">Phosphatidylinositol phosphates (PIPs) and soluble inositol phosphates (IPs) serve as critical secondary messenger molecules that regulate cellular processes. The INPP5 family of phosphatases play an essential role in regulating levels of PIP-5’ and IP-5’ molecules. Src homology 2-containing-inositol phosphatases (SHIP), are a subgroup of the INPP5 family that consists of two members, SHIP1 and SHIP2. Both SHIP proteins have been identified to hydrolyze PI(3,4,5)P3 into PI(3,4)P2. Interestingly, the dysregulation of PI(3,4,5)P3 and SHIP proteins have been observed in multiple diseases, such as cancer, diabetes, and neurodegenerative disease. Recently, SHIP1 was identified as a potential risk factor for the development of Late-onset Alzheimer’s Disease (LOAD). Furthermore, knockdown and inhibition of SHIP1 using small-molecule inhibitors were shown to reduce phenotypes associated with LOAD. Taking these studies together suggests SHIP1 to be a potential therapeutic target for the treatment of LOAD.</p><p dir="ltr"><br></p><p dir="ltr">Despite SHIP1’s therapeutic potential, the development of specific small-molecule inhibitors that target SHIP1 has been challenging. One explanation for this challenge is that very little is known about the overall structure and function of SHIP1. In this thesis I will discuss in detail how we generated multiple SHIP1 constructs to improve our understanding of SHIP1’s overall structure and function in an <i>in vitro </i>setting.</p><p><br></p><p dir="ltr">Efficient protein production is essential for studying enzyme structure and function. The choice of expression system can impact protein yield and stability. The E. coli (BL21) and Baculovirus expression systems are two commonly used systems for protein production. While E. coli is cost-effective and can yield a large amount of protein, the Baculovirus system offers advantages in terms of protein folding and post-translational modifications. Using both systems to generate SHIP1 protein, we demonstrate that the Baculovirus system significantly enhances SHIP1 solubility for all generated constructs, making it the preferable choice for investigating the structure and function of SHIP1.</p><p><br></p><p dir="ltr">SHIP1, a 133 kDa protein, which comprises five established domains: an N-terminal Src Homolgy 2 (SH2) domain, 2.) a pleckstrin homology-related (PH) domain, 3.) an inositol phosphatase catalytic (Ptase) domain, 4.) a C2 domain, and 5.) a C-terminal domain containing proline-rich regions (PXXP) and tyrosine phosphorylated (NPXY) motifs. Despite their regulatory roles in phosphatase activity, protein-protein interactions, and membrane association, limited information is available about their structures and how they contribute SHIP1’s biochemical functions. In this study, we utilized baculovirus-expressed SHIP1 constructs to investigate the impact of each domain on macromolecular structure. Interestingly, a previously unrecognized domain within SHIP1 that directly impacts the enzyme's oligomeric state was identified. This work highlights that SHIP1's individual domains can significantly impact its overall structure and function, providing valuable insights for the development of potential therapeutics in the treatment of LOAD.</p><p><br></p><p dir="ltr">Accurate determination of phosphatase kinetics is vital for understanding the enzymatic activity and its potential involvement in disease. Using our baculovirus generated SHIP1 constructs, we employed in-vitro assays, including the malachite green (MG) and the 2-amino-6-mercapto-7-methylpurine riboside (MESG) coupled enzyme assays, to gain insight into SHIP1 kinetics. Results from the MG assay shows that SHIP1 can hydrolyze the PI(3,4,5)P3 diC8 substrate more efficiently than I(1,3,4,5)P4. Additionally, SHIP1’s PH domain was observed to increase the turnover of PI(3,4,5)P3 diC8. Furthermore, dimerization of SHIP1 was not observed to alter SHIP1 kinetics in any way. Lastly, no major differences in I(1,3,4,5)P4 kinetics were observed with the addition of SHIP1’s N-terminus. These results offer the first comprehensive biochemical characterization of SHIP1 across its substrates and N-terminal domains.</p><p><br></p><p dir="ltr">The development of potent and specific small-molecule inhibitors that target SHIP1 remains challenging. One potential cause for this challenge is that no structures of SHIP1 have been solved in complex with active compounds, making structure-based drug design impossible. In this study, we developed a covalent compound, <b>TAD-58547</b>, from a previously published fragment-based screen that was conducted on SHIP1’s Ptase and C2 domain. <b>TAD-58547 </b>was shown to effectively inhibited SHIP1's Ptase and C2 domains at modest potency. Using X-ray crystallography, this compound was observed to form a covalent interaction with a cysteine residue near the Phosphatase-C2 domain interface. Intriguingly, the inhibitor's potency was observed to be reduced in the presence of the SH2 domain. In addition to testing <b>TAD-58547</b> against our SHIP1 constructs, we investigated the effect of SHIP1’s N-terminus on the potency of a literature compound, <b>TAD-58616</b>. This compound was shown to inhibit all our tested constructs at low µM concentrations. Furthermore, using x-ray crystallography <b>TAD-58616 </b>was solved in complex with SHIP1’s Ptase and C2 domain. Intriguingly, density for <b>TAD-58616 </b>was shown to interact with a site previously identified from the fragment-based screen. While we initially determined this site to be a result of crystal packing, fragments bound to this site may have the potential to inhibit SHIP1. The work presented in this study reinforced the importance of testing inhibitors against physiological relevant forms of SHIP1, when developing potential therapeutics.</p><p><br></p><p dir="ltr">Lastly, new evidence has suggested that the binding of phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) and immunoreceptor tyrosine-based inhibitory motifs (p-ITIM) to SHIP1’s N-terminal SH2 domain is essential for its “Anchorage and Activation” at the plasma membrane (PM). With this model it is believed that SHIP1’s SH2 domain, places the phosphatase into an auto-inhibited state. Upon binding to immune receptor proteins and adaptor proteins that contain ITAM/ITIM sequences, SHIP1 becomes un-auto-inhibited, allowing it to efficiently hydrolyze PI(3,4,5)P3 embedded in the PM. While this model does support the notion that SHIP1 activity is mediated by its PM localization, our biophysical and biochemical characterization add another level of complexity to this regulatory event. Taking all these results together, we propose a novel model for SHIP1 called “Anchorage and Assist” and suggest innovative therapeutic strategies for targeting SHIP1.</p><p><br></p><p dir="ltr">In conclusion, this thesis highlights the importance of choosing suitable expression systems for efficient protein production. Additionally, it offers insight into SHIP1's regulatory mechanisms through the discovery of a novel domain impacting its oligomeric state. Furthermore, the accurate determination of SHIP1 kinetics enhances our understanding of this phosphatase and its potential implications in disease. Also, the identification and crystallization of a novel and previously determined inhibitor scaffolds in complex with SHIP1 increases our ongoing efforts to develop a small-molecule inhibitor that specifically targets SHIP1. Lastly, using recently published data, detailing SHIP1 PM localization and activation, we proposed a new model for SHIP1 activity and suggest novel therapeutic strategies for targeting SHIP1.</p>

<p>  </p> <p>The access of ring junction functionalized 5,6-hydrindanone systems has been elusive in the realm of synthetic methodology, and the functionalization of a pre-built ring system rarely explored. These 5,6-hydridanone systems are prevalent in a variety of terpenoid ring systems, especially that of steroidal molecules. Previous synthetic methods to reach these systems using a Diels-Alder cycloaddition proved to be difficult and lacked labile functional groups that would be useful for substitution after the cycloaddition. The design of the α-nitrile cyclopentenone dienophile allows for both post-cyclization adduct functionalization, as well as lowering the energy barrier of the cycloaddition itself. In this work, it is shown that the Lewis acid promoted Diels-Alder reaction with α-nitrile β-methyl cyclopentenone dienophile can be performed under standard temperatures and pressures unlike previously established methods.1 This reaction can generate four chiral centers in a single synthetic step when the starting materials are prochiral. After the generation of 5,6-hydrindanone systems, radical cleavage of the nitrile functionality also allowed for electrophile trapping at the ring junction. This radical cleavage and electrophile trapping pathway allows for functionalization of a quaternary carbon at the ring junction, a method that should be fruitful in the generation of difficult to synthesize steroidal and other terpenoid molecules.</p> <p>In the work on synthetic cell penetrating peptides, camptothecin whilst a notably effective topoisomerase I inhibitor, has never quite reached it’s potential as a therapeutic due to its poor solubility in living systems. Previously, cationic amphiphilic polyproline helices (CAPH) molecules from the Chmielewski lab have been hydrophobically functionalized through O-alkylation of hydroxyprolines at specific regions within the peptide to generate a hydrophobic face. The combination of the cationic faces and the hydrophobic face have made the CAPH molecules notably cell penetrant and tunable. With camptothecin’s notable insolubility in water, it may serve as valuable surrogate to the hydrophobic groups on CAPH molecules and allowing it to be delivered intracellularly. Using an endogenously cleavable linker, we have worked towards a CPP that acts as a drug delivery vehicle. Acting as a replacement of the hydrophobic residue of a CAPH molecule, camptothecin will be chaperoned into the cell and should be released through the action of intracellular esterases.</p>

<p>  </p> <p>A historically important reaction, the Ueno-Stork reaction promotes, through the use of toxic organotin species, the cyclization of a haloacetal onto an alkene generating bicyclic acetals. This reaction has been used over the years in several total syntheses of biologically relevant natural products, especially the prostaglandin class of natural products. Herein, will be described the development of a novel nickel-catalyzed Ueno-Stork cyclization reaction, which no toxic organotin and radical promoters are used, and instead a greener, operationally friendly, and non-toxic earth abundant nickel catalyst is applied. Optimization studies, substrate scope, scalability, relative stereochemistry of the bicyclic acetals, as well as derivatization of the products were studied. Furthermore, the newly developed reaction was applied on the total synthesis of tricyclic-PGDM Methyl ester, a prostaglandin D2 metabolite of important clinical relevance that currently suffers from material supply issues.</p> <p>Cyclopropanol ring opening reactions have different reactivity modes. Either a metal homoenolate species or a b-keto radical species can be formed after ring opening depending on the reaction conditions applied. More specifically, hydroxycyclopropanols have been studied to access several important motifs present in an array of natural products and medicinally important molecules. The Dai group has used this strategy to access several motifs through intramolecular trapping of the homoenolate species with and without the presence of carbon monoxide to generate oxaspirolactones, THF/THP-fused bicyclic lactones, and disubstituted THF/THP heterocycles. Herein, it will be discussed the application of similar concepts to access new classes of heterocycles 4-ketovalerolactones and 3-furanones. The optimization of two reaction conditions to selectively synthesize each product starting from the same starting material was studied. Furthermore, the substrate scope, scale-up, and derivatization studies of each motif will be disclosed. </p>

HIV-1 protease inhibitor drugs are important components of current antiretroviral therapy (cART). The cART treatment regimens dramatically improved life expectancy and mortality of patients with HIV-1 infection and AIDS. However, new and improved protease inhibitor drugs are essential for future treatment options. To this end, syntheses of optically active (3a<i>S</i>,4<i>S</i>,7a<i>R</i>)-hexahydro-4<i>H</i>-furo[2,3-<i>b</i>]pyran-4-ol, (3a<i>R</i>,4<i>R</i>,7a<i>S</i>)-hexahydro-4<i>H</i>-furo[2,3-b]pyran-4-ol, and (3<i>R</i>,3a<i>S</i>,6a<i>R</i>)-hexahydrofuro[2,3-6]furan-3-ol have been accomplished. These stereochemically defined heterocyclic derivatives are important high-affinity P2 ligands for a variety of highly potent HIV-1 protease inhibitors. The key steps for the synthesis hexehydrofuropyranol involve an efficient Paternò-Büchi [2+2] photocycloaddition, catalytic hydrogenation, acid-catalyzed cyclization to form the racemic ligand alcohol, and enzymatic resolution with immobilized Amano Lipase PS-30. Optically active ligand alcohols were obtained with high enantiomeric purity. Enantiomer (-)-ligand alcohol has been converted to potent HIV-1 protease inhibitors. <div><br></div><div>(3<i>R</i>,3a<i>S</i>,6a<i>R</i>)-Hexahydrofuro[2,3-<i>b</i>]furan-3-ol(<i>bis</i>-tetrahydrofuran) is a key subunit of darunavir, an FDA approved HIV-1 protease inhibitor drug which is widely used for the treatment of HIV/AIDS patients. This stereochemically defined bicyclic heterocycle is also embedded in a variety of highly potent HIV-1 protease inhibitors. The synthesis of optically active <i>bis</i>-tetrahydrofuran was achieved in optically pure form utilizing commercially available and inexpensive 1,2-<i>O</i>-isopropylidene-α-D-xylofuranose or 1,2-O-isopropylidene-α-D-glucofuranose as the starting material. The key steps involve a highly stereoselective substrate-controlled hydrogenation of ethyl 2-(dihydrofuran-3(2H)-ylidene)acetate, a Lewis acid-catalyzed anomeric reduction of a 1,2-<i>O</i>-isopropylidene-protected glycofuranoside, and a Baeyer-Villiger oxidation of a tetrahydrofuranyl-2-aldehyde derivative. Optically active (3<i>R</i>,3a<i>S</i>,6a<i>R</i>)-hexahydrofuro[2,3-<i>b</i>]furan-3-ol ligand was converted to darunavir efficiently. Furthermore, both furopyranol and bis-tetrahydrofuran ligand alcohols have been converted into a variety of potent HIV-1 protease inhibitors including inhibitors containing P2'-boronic acid ligands.<br></div><div><br></div><div>Diabetes mellitus is a chronic, progressive metabolic disorder that seriously threatens human health worldwide, particularly in developing countries. The prevalence of diabetes has been increasing steadily, especially in developing countries. Carambolaflavone A is a natural flavonoid isolated from the leaves of starfruit tree, <i>Averrhoacarambola</i>, in 2005. Carambolaflavone A possesses a <i>C</i>-aryl glycosidic linkage. Carambolaflavone A exhibited significant antihyperglycemic properties. More detailed biological studies reveal that it can lower acute blood glucose. The biology and chemistry of carambolaflavone A attracted our interest in synthesis and further design of interesting structural variants. A convergent total synthesis of carambolaflavone A has been accomplished. The synthesis highlights a bismuth triflate-catalyzed stereoselective C-aryl glycosylation of flavan and an appropriately protected D-fucose derivative as the key step. The glycosylation partners were synthesized from commercially available (±)-naringenin and D-(+)-galactose, respectively. An oxidative bromination and elimination reaction sequence was utilized to construct the flavone. The natural product is obtained in 10 steps (longest linear sequence) from D-(+)-galactose.<br></div>

<p>  </p> <p>Organic synthesis is the application of one or more reactions to the preparation of a particular target molecule, and can pertain to a single-step transformation or to a number of sequential chemical steps depicted by a scheme overall. The selection of a reaction or series of reactions while considering chemo-, regio-, and stereoselectivities in addition to protecting group strategies & redox manipulations highlights the complexity in designing & executing a synthetic plan while making a judgement about what is the most effective and efficient plan to synthesize any given chemical compound among numerous available options. To this end, chemical synthesis is the unifying theme of this thesis & was utilized and strategically applied to construct increasingly complex and diverse molecular architectures. </p> <p>Being the precise science that organic chemistry is, this discipline extends into many areas such as technology, biology & medicine, and even into the fine arts since it fosters unparalleled creativity and imagination in its practice. Research foci in chemical synthesis can encompass both the discovery and development of powerful reactions and the invention of strategies for the construction of defined target molecules, natural or man-made, more or less complex. Studies in the former area, synthetic methodology, fuel and enable studies in the latter area, target molecule and total synthesis campaigns, where the latter area offers a testing ground for the former. Consequently, the bulk of this research work is in organic methodology and will be covered in greater depth during chapters 2 and 3 where strategies, optimizations, & analyses are elaborated upon in light of searching & navigating the vast body of chemical literature in an effort to broaden and strengthen one's laboratory expertise as a synthetic chemist. Lastly, chapter 4 focuses not on traditional synthesis but on organic structure analysis relying on various techniques such as nuclear magnetic resonance (NMR), infrared (IR), ultraviolet-visible (UV-Vis) spectroscopy in combination with mass spectrometry (MS) and/or X-ray crystallography to hypothesize and confirm established structures, specifically phenolic oligomers. An ability to use spectroscopic data to evaluate organic structures by combining practical experience with fundamental knowledge will serve as a hallmark skill in one’s ability to problem-solve as an organic chemist.</p>