Dicaffeoylquinic acid (DCQA) is a natural polyphenolic compound widely distributed in plants such as coffee beans, which possesses a range of pharmacological activities. Herein, is reported studies undertaken towards the first total synthesis of 3,5-DCQA conjugates. Two synthetic routes were investigated. The first route involves a seven step sequence beginning from quinic acid. The overall yield via this synthetic approach was 30%. The key steps involved in the sequence were a regioselective benzylation of the C-3-hydroxyl group followed by silyl protection of the C-1 and C-4 hydroxyl groups. Deprotection of the benzyl group by hydrogenolysis and opening of the lactone afforded the 3,5-diol. Esterification of the 3,5-diol with 3,4-tert-butyldimethylsilyl caffeoyl chloride afforded the di-ester. Removal of the protecting groups afforded 3,5-DCQA. The second route involved selective protection of the C-3-hydroxyl group with silyl followed by benzylation of the C-1 and C-3 hydroxyl groups. Saponification of the lactone ring followed by benzylation of the carboxylic acid gave the benzyl ester. Silyl deprotection afforded the 3,5-diol. The 3,5-diol was subsequently esterified by refluxing in toluene with commercially available Meldrum’s acid. In the final step, the synthesis of 3,5-DCQA was achieved by a Knoevenagel condensation of 3,4-dihydroxybenzaldehyde and a malonate ester of quinic acid. An efficient method for the synthesis of possible metabolites of quinic acid conjugates was also described. This protocol employs N-(4-methoxyphenyl)-trifluoroacetimidate glucuronyl as the donor. The key reaction in this sequence was the coupling of N-(4-methoxyphenyl)-trifluoroacetimidate glucuronyl with 4-hydroxy-3-methoxy-benzaldehyde.

Flora and fauna from terrestrial and marine environments provide libraries of natural compounds for drug discovery. The last four decades have seen major advances in ocean exploration that have allowed chemists and biologists to explore previously inaccessible and rare marine organisms. The study of under-explored marine organisms can result in the discovery of structurally novel and unusual natural products with drug potential. Prior to 2005, no natural products had been reported from the Fijian red macroalgae Callophycus serratus or Neurymenia fraxinifolia. As a result of the work described in this thesis and others in the same research group, 33 unique brominated meroditerpenes have been isolated and elucidated alpha-pyrone natural products were discovered from N. fraxinifolia, enriching the natural product library for drug development. Several natural products isolated from C. serratus exhibited sub-micromolar inhibition against the human malaria parasite Plasmodium falciparum, including a drug-resistant strain. Derivatization of the natural product bromophycolide A into fluorescent probes allowed the determination of a non-enzymatic mechanism of action against the human malaria parasite P. falciparum. Through a combination of detailed SAR mapping, molecular fluorescent imaging of live cells, UV-vis spectroscopic analyses, and protein affinity techniques, the mechanism of action of bromophycolide A against P. falciparum was determined to involve inhibition of heme crystallization. These studies identify a new class of natural products that target heme detoxification in both drug-sensitive and drug-resistant P. falciparum and suggest an avenue to circumvent drug resistance.

According to the latest facts and figures from the Centers for Disease Control, the prevalence of obesity and Type-2 Diabetes Mellitus (T2DM) across all demographics is escalating worldwide. Among the range of drugs used to treat diabetes there is an interest in treatment options that are derived from more traditional methods, outside of the customarily synthetic and pharmaceutically sourced treatment options. Amid treatment alternatives that are more traditional in origin are a category of compounds called natural products. The natural products of interest are either plant based extracts or specific organic compounds extracted from the natural product. Over the last decade, plant based natural products have been increasingly shown to provide an alternative and supplementary course of treatment for diabetic patients suffering from systemic inflammation and insulin resistance in model systems of metabolic disease. However, information for many natural products is limited to small studies with mixed outcomes and information on human models systems remains limited in scope. In this thesis the most potent therapeutic natural products were evaluated by conducting a thorough literature review and subsequently studying the most promising candidates by characterizing their metabolic effects on human adipocytes. In order to identify the most potent plant based natural products we will use cultured human adipocytes as a model system. Free-fatty acids (FFA) and cytokines including Tumor Necrosis Factor-alpha (TNF-α), have been demonstrated to impair fat metabolism and reduce insulin signaling. Therefore, we will study the protective effect of prominent and potentially beneficial natural compounds, based on comprehensive literature reviews, to assess their effect on FFA metabolism using glycerol release as a measure of lipolysis, cytokine induced lipolysis as a measure of natural product protection against inflammation, and impaired AKT-phosphorylation as a measure of insulin signaling function. Observations gathered concluded that the application of bitter melon, fenugreek, and ginseng plant extracts reduced lipolysis in a dose dependent manner, limited the effects of TNF-α induced lipolysis, and fenugreek and ginseng displayed pro-apoptotic cellular programs.

This thesis describes approaches towards the synthesis of 20-deoxybryostatins and 20,20-difluorobryostatins. Towards the 20-deoxybryostatins, a route previously developed within the group was followed with a protecting group change designed to prevent problems encountered with a late-stage deprotection. To this end, (R)-pantolactone was transformed through to dimethyl (4S,6R,8R)-[4-(para-methoxybenzyloxy)-10-hydroxy-6,8-O-isopropylidine-3,3-dimethyl-2-one] phosphonate which was subsequently subjected to a Horner-Wadworth-Emmons condensation with (5R,E)-6-(4-methoxybenzyloxy)-5-triethylsilyloxy-3-(2’-triisopropylsilyloxyethylidene)hexanal. The reactions of the resulting enone through to the advanced intermediate allyl [(4R,6R)-6-((S)-2’-(4-methoxybenzyl)oxy-5-{(2’’S,6’’R)-6’’-(4-methoxybenzyloxymethyl)-4’’-[2-triisopropylsilyloxyeth-(Z)-ylidene]-tetrahydropyran-2’’-yl}-3,3-dimethyl-4’-oxopentyl)-2,2-dimethyl-1,3-dioxan-4-yl]-acetate are described, and this compound corresponds to a C1-C16 northern bryostatin fragment. A synthetic route to 20,20-difluorobryostatins was begun by the synthesis of three 3,3-difluoro-2-hydroxytetrahydro-4H-pyrans which started from an indium-mediated coupling of 3-bromo-3,3-difluoro-1-propene with an aldehyde. A strategy was employed which culminated in the preparation of 3,3-difluoro-2-hydroxy-2-(3-methylbut-1-en-3-yl)-4-(E)-methoxycarbonylmethylenetetrahydro-4H-pyran which corresponds to a C16-C23 20,20-difluorobryostatin southern fragment. Of particular interest is the selectivity observed in the reaction of α,α-difluoroketones with a stabilised Wittig reagent. The indium-mediated coupling was extended to include the first example of the coupling of a 2-alkyl substituted bromide.

Philosophiae Doctor - PhD / The World Health Organisation predicts that by the year 2050, 10 million people could die annually as a result of infections caused by multidrug resistant bacteria. Individuals with compromised immune systems, caused by underlying disease such as HIV, MTB and COVID-19, are at a greater risk. Antibacterial resistance is a global concern that demands the discovery of novel drugs. Natural products, used since ancient times to treat diseases, are the most successful source of new drug candidates with bioactivities including antibiotic, antifungal, anticancer, antiviral, immunosuppressive, anti-inflammatory and biofilm inhibition. Marine bioprospecting has contributed significantly to the discovery of novel bioactive NPs with unique structures and biological activities, superior to that of compounds from terrestrial origin. Marine invertebrate symbionts are particularly promising sources of marine NPs as the competition between microorganisms associated with invertebrates for space and nutrients is the driving force behind the production of antibiotics, which also constitute pharmaceutically relevant natural products.

Philosophiae Doctor - PhD / bioassay guided isolation approach was then used to isolate the high molecular weight antibacterial compound (50kDa-100kDa) from T. actiniarum fermentations. With common protein isolation, purification and detection methods failing to provide insight into the nature of the antibacterial compound, we hypothesized that the active agent is not proteinaceous in nature and may be a high molecular weight exopolysaccharide. Extraction and antibacterial screening of the exopolysaccharide fraction from T. actiniarum showed antibacterial activity as well as lytic activity when subjected to a zymography assay using Pseudomonas putida whole cells as a substrate. Additionally, the biosynthetic pathways for the production of poly-β-1, 6-N-acetyl-glucosamine (PNAG), an exopolysaccharide involved in biofilm formation and chondroitin sulfate, a known and industrially important glycosaminoglycan with antibacterial and anti-inflammatory activity was identified and the mechanism may be novel. Genome mining identified a variety of novel secondary metabolite gene clusters which could potentially encode other novel bioactivities. Therefore a bioassay guided isolation, focused on the small (<3kDa) molecules, was pursued. Secondary metabolites were extracted, fractionated and screened for biofilm inhibition, antibacterial and anticancer activity and activity was observed in all assays. Active fractions were dereplicated by UHPLC-QToF-MS and compounds of interest were isolated using mass guided preparative HPLC. The purity of the isolated compounds was assessed using UHPLC-QToF-MS and NMR and the structure of the target compounds elucidated. Structures that could be determined were the bile acids cholic acid and 3-oxo cholic acid and although not responsible for the observed activities, this is the first report of bile acid production for this genus. This is the first study investigating the bioactive potential of the strain and the first demonstrating that T. actiniarum is a promising source of potentially novel pharmaceutically relevant natural products depicted through both culture-dependent and culture-independent approaches.

Two natural marine compounds, 3-bromo-4,5-dihydroxybenzaldehyde 2 and 2,3-dibromo-4,5-dihydroxybenzaldehyde 5 together with two novel derivatives, 3-bromo-5-(tert-butyl-dimethyl-silanyloxy)-4-hydroxybenzaldehyde 3 and 1-bromo-2,3-dimethoxy-5-nitrooxy-methylbenzene 9, were synthesized. Compounds 2, 3, and 5 were evaluated for their biological activity towards the inhibition of prostate cancer cell growth using staurosporine a a positive control. All three compounds have shown significant inhibition of prostate cancer cell growth. Compound 9 is yet to be evaluated.

<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>

The thesis entitled “Total synthesis of bio-active macrolide natural products and sulphonamide based ligands in asymmetric catalysis” is divided into two chapters. First chapter of the thesis describes the total synthesis of bio-active macrolide natural products cladospolide A 1, seimatopolide A 2 and synthetic studies towards aetheramides A 3 and B 4 (Figure 1). Figure 1: Bio-active macrolide natural products. Section A of chapter 1 describes the enantiospecific total synthesis of cladospolide A (ent-1). Cladospolide A was isolated from three different sources such as culture filtrate of cladosporium fulvam FI-113, Fungus cladosporium tenuissimum and Fermentation broath of cladosporium sp. FT-0012. Cladospolide A is shown to inhibit the root growth of lettuce seedlings. Enantiospecific total synthesis of cladospolide A ent-1 was accomplished in 9% overall yield in 11 linear steps using D-ribose as a chiral pool precursor. Key reactions in the present approach include olefin cross metathesis and Yamaguchi macrolactonization reactions (Scheme 1). Scheme 1: Total synthesis of cladospolide A (ent-1). Section B of chapter 1 describes the use of furan as a surrogate for the E-but-2-ene-1, 4-dione unit in the total synthesis of seimatopolide A 2. Seimatopolide A 2 was isolated by Heip and co-workers from the fungus Seimatosporium discosioides in 2012 and is shown to activate the γ-subtype peroxysome proliferator-activated receptors (PPAR-γ), which is a pivotal process in the type-2 diabetes. Total synthesis of ent-seimatopolide A was accomplished in 7.8% overall yield in 14 linear steps from furfural. Nagao acetate aldol and Shiina macrolactonization reactions were employed as key reactions for the synthesis of ent-seimatopolide A (ent-2) (Scheme 2). Scheme 2: Stereoselective total synthesis of seimatopolide A (ent-2). In section C of Chapter 1, studies towards the synthesis of aetheramides A 3 and B 4 are described. Aetheramides A 3 and B 4 are isolated by Müller’s group in 2012 from the novel myxobacterial genus “Aetherobacter”. Aetheramides are cyclic depsipeptides, which are shown to inhibit the HIV-I infection with IC50 values of ∼0.015 μM and cytostatic activity against human colon carcinoma (HCT-116) cells with IC50 values of 0.11 μM. Stereochemistry at two chiral centers present in the molecules is unassigned. The first approach (Scheme 3) relied on macrolactonization as the key step while the second approach (Scheme 4) relied on RCM to accomplish the macrolactonization. The required precursors were synthesized from elaboration of chiral furyl carbinol, while synthesis of the RCM precursor was accomplished employing the aldol reaction. Scheme 3: Macrolactonization strategy for synthesis of 3 from chiral furyl carbinol. Scheme 4: RCM strategy for synthesis of 3 from chiral furyl carbinol. The successful synthesis of the macrolactone core of aetheramide A 1 is accomplished by employing the ring closing metathesis reaction to construct the C18-C19 bond. RCM precursor has been synthesized by the amidation of the amine derived from R-mandelic acid, while the acid fragment is synthesized from allyl trityl ether (Scheme 5). Scheme 5: RCM strategy for synthesis of 3 from R-mandelic acid. Second chapter of the thesis describes the synthesis and application of novel sulfinamide ligands in asymmetric catalysis. In section A of chapter 2, chiral 2-pyridylsulfinamides are shown to be effective catalysts in the alkylation of aryl and alkyl aldehydes with diethylzinc providing the corresponding alcohols in excellent enantioselectivity. It was found that the chirality present at sulfur in the ligand is pivotal for the asymmetric induction (Scheme 6). Scheme 6: Asymmetric alkylation of benzaldehyde with some of the 2-Pyridyl sulfinamide catalysts. Second section of chapter 2 describes the synthesis and application of C2-symmetric bis-sulfinamides in Rh (I) catalyzed conjugate addition of PhB(OH)2 to enones. Chirality present at sulphur in sulfonamide as well as symmetry present in the ligand plays crucial role in the outcome of the reaction (Scheme 7). Scheme 7: Asymmetric arylation of enones using C2-symmetric bis-sulfinamide/olefin ligands. The thesis entitled “Total synthesis of bio-active macrolide natural products and sulphonamide based ligands in asymmetric catalysis” is divided into two chapters. First chapter of the thesis describes the total synthesis of bio-active macrolide natural products cladospolide A 1, seimatopolide A 2 and synthetic studies towards aetheramides A 3 and B 4 (Figure 1). Figure 1: Bio-active macrolide natural products. Section A of chapter 1 describes the enantiospecific total synthesis of cladospolide A (ent-1). Cladospolide A was isolated from three different sources such as culture filtrate of cladosporium fulvam FI-113, Fungus cladosporium tenuissimum and Fermentation broath of cladosporium sp. FT-0012. Cladospolide A is shown to inhibit the root growth of lettuce seedlings. Enantiospecific total synthesis of cladospolide A ent-1 was accomplished in 9% overall yield in 11 linear steps using D-ribose as a chiral pool precursor. Key reactions in the present approach include olefin cross metathesis and Yamaguchi macrolactonization reactions (Scheme 1). Scheme 1: Total synthesis of cladospolide A (ent-1). Section B of chapter 1 describes the use of furan as a surrogate for the E-but-2-ene-1, 4-dione unit in the total synthesis of seimatopolide A 2. Seimatopolide A 2 was isolated by Heip and co-workers from the fungus Seimatosporium discosioides in 2012 and is shown to activate the γ-subtype peroxysome proliferator-activated receptors (PPAR-γ), which is a pivotal process in the type-2 diabetes. Total synthesis of ent-seimatopolide A was accomplished in 7.8% overall yield in 14 linear steps from furfural. Nagao acetate aldol and Shiina macrolactonization reactions were employed as key reactions for the synthesis of ent-seimatopolide A (ent-2) (Scheme 2). Scheme 2: Stereoselective total synthesis of seimatopolide A (ent-2). In section C of Chapter 1, studies towards the synthesis of aetheramides A 3 and B 4 are described. Aetheramides A 3 and B 4 are isolated by Müller’s group in 2012 from the novel myxobacterial genus “Aetherobacter”. Aetheramides are cyclic depsipeptides, which are shown to inhibit the HIV-I infection with IC50 values of ∼0.015 μM and cytostatic activity against human colon carcinoma (HCT-116) cells with IC50 values of 0.11 μM. Stereochemistry at two chiral centers present in the molecules is unassigned. The first approach (Scheme 3) relied on macrolactonization as the key step while the second approach (Scheme 4) relied on RCM to accomplish the macrolactonization. The required precursors were synthesized from elaboration of chiral furyl carbinol, while synthesis of the RCM precursor was accomplished employing the aldol reaction. Scheme 3: Macrolactonization strategy for synthesis of 3 from chiral furyl carbinol. Scheme 4: RCM strategy for synthesis of 3 from chiral furyl carbinol. The successful synthesis of the macrolactone core of aetheramide A 1 is accomplished by employing the ring closing metathesis reaction to construct the C18-C19 bond. RCM precursor has been synthesized by the amidation of the amine derived from R-mandelic acid, while the acid fragment is synthesized from allyl trityl ether (Scheme 5). Scheme 5: RCM strategy for synthesis of 3 from R-mandelic acid. Second chapter of the thesis describes the synthesis and application of novel sulfinamide ligands in asymmetric catalysis. In section A of chapter 2, chiral 2-pyridylsulfinamides are shown to be effective catalysts in the alkylation of aryl and alkyl aldehydes with diethylzinc providing the corresponding alcohols in excellent enantioselectivity. It was found that the chirality present at sulfur in the ligand is pivotal for the asymmetric induction (Scheme 6). Scheme 6: Asymmetric alkylation of benzaldehyde with some of the 2-Pyridyl sulfinamide catalysts. Second section of chapter 2 describes the synthesis and application of C2-symmetric bis-sulfinamides in Rh (I) catalyzed conjugate addition of PhB(OH)2 to enones. Chirality present at sulphur in sulfonamide as well as symmetry present in the ligand plays crucial role in the outcome of the reaction (Scheme 7). Scheme 7: Asymmetric arylation of enones using C2-symmetric bis-sulfinamide/olefin ligands.

published_or_final_version / Chemistry / Master / Master of Philosophy