The solvent-free (Cp*IrCl2)2 catalyzed N-alkylation of urea with benzyl alcohol has been studied. A variety of reaction conditions were studied and optimized to produce a high yield (82%) of N,N-dibenzylurea. A series of substituted benzyl alcohols were examined at the optimal reaction conditions. However, the preparation of substituted benzyl urea derivatives using conditions optimized for benzyl alcohol gave poor yields or intractable mixtures.

Over 4,000 flavonoids have been identified, and among these, many of them are known to possess cardioprotective, anti-inflammatory, antimicrobial, and antitumor effects. However, most of these properties have yet to be fully understood. In this study, extracts from Gnaphalium gracile, thought to possess a mixture of flavonoids, have been tested for cytotoxic activity on pancreatic (MiaPaca, Panc28), colon (HCT-116, Caco-2), and prostate (PC3, LNCaP), cancer cell lines. Polar extracts from the leaves of G. gracile have the most cytotoxic effect on these cancer cell lines, particularly the prostate cancer cell lines PC3 and LNCaP. Evidence suggests the extracts have antineoplastic effects on these cancer cells lines possibly due to differentiation status on pancreatic and colon cancer, but not prostate cancer. Cytotoxic activity is not dependent on tumorigenic potential. Further research is needed to identify the bioactive compounds within these extracts.

New antibiotics with novel targets or mechanisms of action are needed to counter the steady emergence of bacterial pathogens that are resistant to antibiotics used in the clinic. MraY, a promising novel target for antibiotic development, initiates the lipid cycle for the biosynthesis of peptidoglycan cell wall, which is essential for the survival of most, if-not-all, bacteria. MraY is an enzyme that catalyzes the transfer and attachment of phospho-MurNAc-pentapeptide to a lipid carrier, undecaprenylphosphate. Muraymycins are recently discovered lipopeptidyl nucleoside antibiotics that exhibit remarkable antibiotic activity against Gram-positive as well as Gram-negative bacteria by inhibiting MraY. We conducted a thorough examination of the metabolic profile of Streptomyces sp. strain NRRL 30473, a known producer of muraymycins. Eight muraymycins were isolated and characterized by a suite of spectroscopic methods, including three new members of muraymycin family named B8, B9 and C5. Muraymycins B8 and B9, which differ from other muraymycins by having an elongated fatty acid side chain, showed potent antibacterial activity against Escherichia coli ∆tolC mutant and pM IC50 against Staphylococcus aureus MraY. Muraymycin C5, which is characterized by an N-acetyl modification of the disaccharide’s primary amine, greatly reduced its antibacterial activity, which possibly indicates this modification is used for self-resistance. In addition to the discovery of new muraymycins, eleven enzymes from the biosynthetic pathway were functionally assigned and characterized in vitro. Six enzymes involved in the biosynthesis of amino ribofuranosylated uronic acid moiety of muraymycin were characterized: Mur16, a non-heme, Fe(II)-dependent α-ketoglutarate: UMP dioxygenase; Mur17, an L-threonine: uridine-5′-aldehyde transaldolase; Mur20, an L-methionine:1-aminotransferase; Mur26, a low specificity pyrimidine nucleoside phosphorylase; Mur18, a primary amine-requiring nucleotidylyltransferase; Mur19, a 5-amino-5-deoxyribosyltransferase. A one-pot enzyme reaction was utilized to produce this disaccharide moiety and its 2′′-deoxy analogue. Two muraymycin-modifying enzymes that confer self-resistance were functionally assigned and characterized: Mur28, a TmrB-like ATP-dependent muraymycin phosphotransferase, and Mur29, a muraymycin nucleotidyltransferase. Notably, Mur28 preferentially phosphorylates the intermediate, aminoribofuranosylated uronic acid, in the muraymycin biosynthetic pathway to produce a cryptic phosphorylated-dissacharide intermediate. Mur23 and Mur24 were assigned as two enzymes that modify the cryptic phosphorylated intermediate by attachment of an aminopropyl group. Mur24 catalyzes the incorporation of butyric acid into the phosphorylated-disaccharide. Following the incorporation, Mur23 catalyzes a PLP-dependent decarboxylation. Finally, Mur15, which belongs to the cupin family, is functionally assigned as a non-heme, Fe(II)-dependent α-ketoglutarate dioxygenase that catalyzes the β-hydroxylation of a leucine moiety in muraymycin D1 to form muraymycin C1. Mur15 can also hydroxylate the γ-position of leucine moiety to muraymycins with fatty acid chain in β-position.

Type II polyketide synthase (PKS) produced natural products have proven to be an excellent source of pharmacologically relevant molecules due to their rich biological activities and chemical scaffolds. Type II-PKS manufactured polyketides share similar polycyclic aromatic backbones leaving their diversity to stem from various chemical additions and alterations facilitated by post-PKS tailoring enzymes. Evidence suggests that post-PKS tailoring enzymes form complexes in order to facilitate the highly orchestrated process of biosynthesis. Thus, protein-protein interactions between these enzymes must play crucial roles in their structures and functions. Despite the importance of these interactions little has been done to study them. In the mithramycin (MTM) biosynthetic pathway the Baeyer−Villiger monooxygenase (BVMO) MtmOIV and the ketoreductase MtmW form one such enzyme pair that catalyze the final two steps en route to the final product. MtmOIV oxidatively cleaves the fourth ring of the mithramycin intermediate premithramycin B (PreB) via a Baeyer−Villiger reaction, generating MTM’s characteristic tricyclic aglycone core and highly functionalized pentyl side chain at position 3. This Baeyer−Villiger reaction precedes spontaneous lactone ring opening, decarboxylation, and the final step of MTM biosynthesis, a reduction of the 4′- keto group catalyzed by the ketoreductase MtmW. Another example of co-dependent post-PKS tailoring enzymes from the gilvocarcin biosynthetic pathway is composed of GilM and GilR. These two enzymes form an unusual synergistic tailoring enzyme pair that does not function sequentially. GilM exhibits dual functionality by catalyzing the reduction of a quinone intermediate to a hydroquinone and stabilizes O-methylation and hemiacetal formation. GilM mediates its reductive catalysis through the aid of GilR that provides its covalently bound FADH(2) for the GilM reaction, through which FAD is regenerated for the next catalytic cycle. A few steps later, following glycosylation related events unique to each gilvocarcin derivative, GilR dehydrogenates the hemiacetal moiety created by GilM to establish the formation of a lactone and the final gilvocarcin chromophore. To achieve a better understanding of post-type II PKS tailoring enzymes and their protein-proteininteractions for the benefit of future combinatorial biosynthetic efforts two specific aims were devised. Specific aim 1 was to investigate the structure of MtmOIV and the role of active site residues in its catalytic mechanism. Specific aim 2 was to integrate the function of GilM and its protein-protein interactionswith GilR that lead to their synergistic activity and sharing of GilR’s bicovalently bound FAD moiety.

Several nucleoside natural product antibiotics from Streptomyces sp. and actinomycetes have recently been shown to target bacterial peptidoglycan cell wall biosynthesis by inhibiting the bacterial translocase I (MraY). The biosynthetic gene clusters for A-90289, liposidomycins and caprazamycins revealed a protein with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD). This enzyme (LipL) is a mononuclear, non-heme, Fe(II) dependent α-keto glutarate (α-KG) :uridine monophosphate (UMP) dioxygenase responsible for the net dephosphorylation and two electron oxidation of UMP to uridine-5’-aldehyde. The postulated reaction coordinates involving the activation of the C-5’ center in UMP and the corresponding formation of uridine-5’-aldehyde are modeled on extensive spectroscopic and structural characterizations of TauD. In this dissertation, the postulated radical mechanism for LipL involving the formation of an unstable hydroxylated intermediate is investigated via the characterization of a key product obtained from the reaction of LipL (and its homolog Cpr19) with a synthetically modified surrogate substrate where the bridging phosphoester oxygen in UMP is replaced with a 5’ C-P bond. We further validate our hypothesis by analyzing the reactions of both LipL and Cpr19 with specifically 2H1 – labeled UMP substrate and confirming the expected products via mass spectrometry. In addition, we explore substrate promiscuity of the enzymes and utilize a set of site specific mutants of Cpr19 as means of gaining better insight into the active site residues. Predictive models for Cpr19 and LipL structures are developed by the combination of experimental results and chemical logic.

<div> Carbon monoxide is a useful carbon linchpin to construct complex molecules of natural products by stitching different pieces of target molecules together. Recently, our group reported a novel and efficient palladium-catalyzed spirolactonization by Dr. Dexter Davis to construct oxaspirolacones from esters or lactones. As an essential motif, oxaspirolactone structures in natural products exhibit diverse and exciting structures and biological activities. The first part of this thesis mainly describes the total synthesis of stemoamide alkaloids in the stemona family and the application of our palladium-catalyzed spirolactonization, which was developed by our group to complete total synthesis of bisdehydroneostemoninine and bisdehydrostemoninine with Prof. Kaiqing Ma. The total synthesis features a one-pot ring-closing cross-metathesis, Lewis acid-mediated Friedel-Crafts reaction and lactonization, and accomplished bisdehydrostemonine in 15 steps. The total synthesis of stemoamide, tuberostemoamide, and sessilifoliamide A were finished, and the critical step features an mCPBA oxidation to convert pyrrole to lactam in one step without destructing other functional groups. </div><div> In the second part of this thesis, we developed a novel and efficient palladium-catalyzed cascade amino-carbonylative lactonization to streamline the synthesis of dihydropyrrole-fused furanones in collaboration with Prof. Seleem’s lab for biological activities. Using this method, we quickly expanded this method to construct different ring structures, such as β-lactone and dihydropyrrole-fused pyrrolone. This method was applied to the total synthesis study towards stemofoline alkaloids. Our palladium-catalyzed spirolactonizaiton was also used in this total synthesis study for target molecules. </div><div><br></div>

<p>The hamigeran diterpenoid is a family of natural products with diverse structures and biological activities. Most of the syntheses focus on hamigerans with [6–6–5] tricyclic core, but synthetic efforts toward the more challenging [6–7–5] tricyclic hamigerans are very limited. Herein, our studies in synthesizing the [6–7–5] tricyclic hamigerans are disclosed.  Through a benzyne-β-ketoester annulative ring expansion and a Nazarov reaction, an approach toward the [6–7–5] tricyclic carbon skeleton of the hamigeran natural products was developed. A Ni-catalyzed conjugate methyl addition or a Corey–Chaykovsky reaction installed the all-carbon quaternary center, and a Suzuki cross coupling followed by reduction introduced the isopropyl group. However, the reduction of challenging tetra-substituted double bond or the regio-selective cyclopropane opening was not successful despite multiple conditions were tried. A revised synthetic strategy was proposed and resulted in a convergent total synthesis of (±)-hamigeran M, enabled by five C–H functionalization reactions and proceeding in 11 steps in 3.9% overall yield. The C–H functionalizations include a hydroxy-directed C–H borylation, one C–H metalation-1,2-addition, one C–H metalation-Negishi coupling, a late-stage oxazole-directed C–H borylation-oxidation, and one electrophilic bromination. Further elaboration of the intermediates obtained here has delivered an advanced polysubstrituted precursor towards multiple other hamigerans.</p>

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.

Nicotine and nornicotine are the two main alkaloids that accumulate in Nicotiana tabacum L. (tobacco), and nornicotine is the N-demethylation metabolite of nicotine. Nicotine is synthesized in the root, and probably primarily in the root tip. Both nicotine and nornicotine exist as two isomers that differ from each other by the orientation of H atom at the C-2' position on the pyrrolidine ring. (S)-nicotine is the dominant form in tobacco leaf and the enantiomer fraction of nicotine (EFnic), the fraction of (R)-enantiomer over the total nicotine, is approximately 0.002. Despite considerable efforts to elucidate nicotine and nornicotine related metabolism, a comprehensive understanding of the factors responsible for regulating the variable EF for nornicotine (0.04 to 0.75 ) relative to nicotine has been lacking. The objectives of these investigations were to understand the mechanisms behind the discrepancy. There are three nicotine demethylases reported to be active in tobacco. In vitro recombinant CYP82E4, CYP82E5v2 and CYP82E10 demethylated (R)-nicotine three, ten and ten-fold faster than (S)-nicotine, respectively, and no racemization was observed in either nicotine or nornicotine during demethylation. To confirm these in vitro results, the accumulation and demethylation of nicotine enantiomers throughout the growth cycle and curing process were investigated. Scion stock grafts were used to separate the contributions of roots (source) from leaves (sink) to the final accumulation of nicotine and nornicotine in leaf. The results indicate that nicotine consists of 4% of the R enantiomer (0.04 EFnic) when synthesized. However, (R)-nicotine is selectively demethylated by CYP82E4, CYP82E5 and CYP82E10, resulting in an approximate 0.01 EFnic and 0.60 EFnnic in the root. After most of (R)-nicotine is demethylated in root, nicotine and nornicotine are translocated to leaf, where nicotine is further demethylated. Depending on the CYP82E4 activity, an EFnnic of 0.04 to 0.60 is produced and only 0.2% of the remaining nicotine in the leaf is (R)-configuration.

The relationship between the endocannabinoid system in the renal medulla and the long-term regulation of blood pressure is not well understood. To investigate the possible role of the endocannabinoid system in renomedullary interstitial cells, mouse medullary interstitial cells (MMICs) were obtained, cultured and characterized for their responses to treatment with a selective inhibitor of fatty acid amide hydrolase (FAAH), PF-3845. Treatment of MMICs with PF-3845 increased cytoplasmic lipid granules detected by Sudan Black B staining and multilamellar bodies identified by transmission electron microscopy. HPLC analyses of lipid extracts of MMIC culture medium revealed a 205nm-absorbing peak that showed responsiveness to PF-3845 treatment. The biologic activities of the PF-3845-induced product (PIP) isolated by HPLC were investigated in anesthetized, normotensive surgically-instrumented mice. Intramedullary and intravenous infusion of PIP at low dose rates (0.5-1 AU/10 min) stimulated diuresis and natriuresis, whereas at higher doses, these parameters returned toward baseline but mean arterial pressure (MAP) was lowered. Whereas intravenous bolus doses of PIP stimulated diuresis, GFR and medullary blood flow (MBF) and reduced or had no effect on MAP, an intraperitoneal bolus injection of PIP reduced MAP, increased MBF, and had no effect on urinary parameters. Genetic or pharmacological ablation of the cannabinoid type 1 receptors in mice completely abolished the diuretic and vasodepressor properties of intramedullary infused PIP, suggesting that the PF-3845-induced product requires the presence of CB1 receptors in order to elicit its renal effects. In a radioactive competition binding assay, using Chinese hamster ovary cells expressing CB1 receptors, PIP successfully displaced the CB1 selective inverse agonist [3H] SR141716A, revealing that the lipid extract was able to compete for binding to CB1 receptors. Finally, we investigated the tubular location of diuretic activity that the PF-3845-induced lipid fraction exhibits. In a renal function in vivo experiment, we pre-treated anesthetized mice with an intramedullary infusion of one of four well-known diuretics. This procedure was followed by an intramedullary infusion of PIP (1AU). Only inhibition of the proximal tubule sodium reabsorption diminished the diuretic activity of the PF-3845-induced product, suggesting that the lipid fraction requires a physiologically intact proximal tubular reabsorption mechanism for it to produce diuresis. These data support a model whereby PF-3845 treatment of MMICs results in increased secretion of a neutral lipid which acts directly to promote diuresis and natriuresis and indirectly through metabolites to produce vasodepression. Efforts to identify the structure of the PF-3845-induced lipid and its relationship to the previously proposed renomedullary antihypertensive lipids are ongoing.