Chapter one details my work associated with the photorearrangement of [8]-pyridinophane N-oxide and its application in to an eight-step asymmetric synthesis of (+)-marineosin A. The route proceeds by condensing fragments of reversed polarity relative to conventional prodiginine constructions. The resultant unstable chromophore is disrupted by a unique cycloisomerization promoted at a tailored manganese surface providing a premarineosin and subsequently marineosin A in a particularly concise manner. A pyridinophane N-oxide photorearrangement in flow and structural isomers of premarineosin are discussed, as is the reassignment of marineosin stereochemistry. The route gives access to the natural product as well as diastereomers, congeners and analogs that are currently inaccessible by other means. Following completion of this route, I began studying the mechanism of the pyridinophane N-oxide photorearrangement using time course studies with support from DFT calculations. Based on the data and the isolation of two previously unknown heterocyclophanes, I outlined a unified mechanistic scheme that explains competing processes under varying photochemical conditions. Chapter two describes my work characterizing of prodiginine–microRNA binding interactions. I developed a fluorescence-based binding assay to determine the binding strength of prodiginines to an oncogenic microRNA precursor. After identifying the small molecule’s binding site, I fused it to a readily crystallizable riboswitch with the aim of obtaining a co-crystal structure. I also explored several other crystallization constructs and developed a structure-affinity relationship by synthesizing and assaying novel prodiginine compounds. From this I identified a lead compound which is one of the strongest microRNA binders discovered to date.

Chapter 1 provides an introduction to the biological significance of natural product prodiginines such as streptorubin, roseophilin, and the marineosins. This includes a brief discussion of structural features and biological mechanisms of action. The prodiginine of interest in our synthetic studies concerned marineosin A. This chapter then discusses how we used our previously published route to scale up the synthesis of marineosin A, challenges encountered, and how we were able to accomplish one of our goals of accessing novel synthetic analogs. We then segue into mechanistic studies concerning an atypical acid-promoted migration of a hydroxy-propyl sidechain that affords a new unprecedented class of synthetic rearranged prodiginines. We then postulate a potential operative mechanism based on data obtained experimentally and computationally. Chapter 2, we show that unprotected peptides react with commercial octafluorocyclopentene (OFCP) to afford fluorinated macrocycles via successive ipso substitutions of vinyl fluoride. We quantify relative reaction rates and establish sequence variations having combinations of cysteine, tyrosine, histidine, and serine residues. Compounds bearing permutations of these functional groups are shown to react rapidly at room temperature to give stable, isolable products. We also introduce our initial discoveries involving polysubstitution cascades with OFCP. Chapter 3, we further probe and diversify our polysubstitution cascades of linear unprotected peptides with OFCP to generate complex fluorinated polycycles. The reactions occur in a single flask at 0–25 �C and require no catalysts or heavy metals. OFCP can directly polycyclize linear sequences using native functionality, or fluorospiroheterocyclic intermediates can be intercepted with exogenous nucleophiles. The latter tactic generates molecular hybrids composed of peptides, sugars, lipids, and heterocyclic components. Building on this strategy we also develop novel small-molecules as inserts to afford orthogonal cyclizations and diversify our synthetic outcomes. This platform can create stereoisomers of both single- and double-looped macrocycles. Calculations indicate that the latter can mimic diverse protein surface loops. Subsets of the molecules have low energy conformers that may shield their polar surface area through intramolecular hydrogen bonding. A significant fraction of OFCP-derived macrocycles tested show moderate to high passive permeability in parallel artificial membrane permeability assays (PAMPA).

Ghrelin O-acyl Transferase (GOAT) is a recently discovered member of the membrane bound O-acyl transferase (MBOAT) family of enzymes. GOAT is uniquely able to catalyze the transfer of an n-octanoyl group to its substrate, the peptide hormone ghrelin. Acylated ghrelin is insulinostatic and can promote feeding behavior in higher mammals. Pharmacological inhibition of GOAT has emerged as an attractive means to modulate the ghrelin signaling pathway. GOAT is the only humanenzyme known to promote octanoylation. Ghrelin is the only substrate of GOAT, and ghrelin is the only known octanoylated hormone. Further, ghrelin and GOAT are produced principally in the digestive tract. Unlike ligands of the ghrelin receptor, a GOAT inhibitor would not need to penetrate the blood brain barrier.

Herein, we detail our efforts to develop potent, in vivo active small molecule GOAT inhibitors by a peptidomimetic approach. Towards this goal we outline experimental procedures for assaying GOAT activity in vitro, and address unique challenges overcome during this work. Our medicinal chemistry has been driven by hypotheses concerning the catalytic mechanism and binding mode of GOAT and its co-substrates ghrelin and octanoyl-CoA. We unify our understanding of structure-activity relationships for a new class of GOAT inhibitors, and examine the progression of inhibitor structure which has led to these conclusions. Multiple synthetic routes appropriate for small and large scale syntheses of GOAT inhibitors are presented.

Top performing inhibitors exhibit high nanomolar potency in vitro, and suppress circulating acyl-ghrelin in-vivo for up to three hours in a mouse. We have extensively characterized the in vivo and in vitro pharmacokinetic properties of these materials. These inhibitors lay a strong foundation for further medicinal chemistry refinement, and provide a powerful new means of interrogating ghrelin biology and assessing the therapeutic potential of GOAT inhibition.

Many actively pursued pharmacological targets are difficult to drug using conventional small molecule therapeutics because they lack conventional binding sites. These so called ‘undruggable’ targets typically interact with other proteins via shallow, solvent exposed interfaces. Peptidomimetic macrocycles have the potential to mediate such systems because the embedded peptide can mimic native protein structure and recognition elements, and the ring structures contribute to structural preorganization, lowering entropic penalties upon target binding. Compounds of this type having precise shapes and drug-like character are coveted, but are relatively difficult to synthesize. Our lab has developed methods to synthesize shape-defined macrocycles from small linear peptides. These experiments run as processes, wherein designed templates react incrementally with unprotected oligomers to form composite products. The resulting compounds retain molecular recognition elements in the oligomer, yet display that functionality as part of stable polycyclic structures. Our experimental work is based on proteinogenic amino acids and the reactivity of their nucleophilic side chains. However, using unnatural amino acids, the hypothetical scope of the chemistry becomes vast and far outpaces the capacity of our experimental format. Here, we describe the development of a computational rendering of our experimental platform, Composite Peptide Macrocycle Generator (CPMG). This open-source platform simulates our multi-step reaction chemistry using a large, tailored monomer set. We have used the algorithms to anticipate product outcomes of >2 billion processing sequences. We have further developed software to generate three- dimensional structures for each product. Every library member has feature constraints meant to increase the probability of it being bioavailable. We discuss efforts to merge our experimental and computational abilities into a single, iterative workflow to discover new macrocyclic ligands for challenging protein targets. We describe new computational tools and techniques to allow rapid, flexible docking of conformationally dynamic ligands onto multiple protein targets. We describe experiments to validate predictions by synthesizing novel arene amino acids and engaging them in macrocyclizations to generate previously unknown ring systems. We show how the interplay between calculations and synthesis can offer insight into molecular properties and applications.

Marine natural products are a rich source of small molecules with novel structures and potentially valuable biological activities. We have studied two new molecules of this type. Callyspongiolide is a sponge-derived macrolide harboring an arylated dien-yne appendage. Portimine is a macrobicyclic ketal scaffolded by a spirobicyclic imine isolated from a dinoflagellate. Both compounds strongly inhibit the growth of multiple human cancer cell lines at nanomolar concentrations, yet they appear to function very differently at the level of apoptotic signaling. The activity of callyspongiolide is caspase-independent and does not involve canonical apoptosis. In contrast, portimine is a selective potentiator of apoptosis that does not cause cellular damage or necrosis even at much higher doses. Its activity parallels the effects of pro-apoptotic biologics such as TNF alpha and TRAIL. As a steps towards understanding their biology, this thesis describes experiments to synthesize the highly functionalized backbones of each of these natural products in acyclic form, and subsequently convert those species into the natural products. It also outlines our collaborative discovery that callyspongiolide’s cellular target is the vacuolar H+ ATPase. Chapter one describes an asymmetric total synthesis of callyspongiolide, wherein the macrolactone core is built unconventionally from a saccharide and a monoterpene without the intermediacy of a seco-acid. Two main fragments were joined using Krische’s iridium-catalyzed asymmetric redox allylation. Fe(II)-promoted fragmentation of a perhemiketal and immediate Cu(II)-mediated β-hydride elimination selectively afforded a homoallylic alcohol, from which the macrolactone was synthesized via a carbonylative macrocyclization. The Z-acrylate was established by photolysis of the corresponding E-acrylate. Further homologation and Sonogashira coupling provided the natural product in an efficient manner. Chapter two discusses the discovery that callyspongiolide is a potent vacuolar ATPase inhibitor (IC50 = 10 nM) and the first V-ATPase inhibitor known to be expelled by Pdr5p. Chapter three discusses the re-assignment of the relative stereochemistry previously ascribed to product diastereomers resulting from imidazolidinone-catalyzed vinylogous Mukaiyama-Michael reaction of 2-trimethylsiloxyfuran and trans-crotonaldehyde. A modified procedure that uses a diphenylprolinol catalyst was subsequently developed to selectively provide the ‘syn’ diastereomeric product in high enantiomeric excess on decagram scales. Chapter four details the efforts towards the synthesis of portimine. Intramolecular Diels-Alder reaction was proposed for the construction of the macrocyclic carbon framework. A deoxygenated model system of portimine was designed and synthesized in 3 steps from the butenolide described in the previous chapter. It contains all the carbons present in portimine and allows the exploration of key transformations. En route to the natural product, the contiguous oxygenated stereocenters were established via aldol reaction followed by Narasaka-Prasad reduction. Substrates for Diels-Alder reaction were synthesized in 9 or 10 steps from the butenolide, with only one protecting group installed in the longest linear sequence. Diels-Alder cycloaddition of a dendralene substrate afforded an isomeric spiroimine framework.

Peptidomimetic macrocycles are increasingly growing as attractive scaffolds for the development of therapeutics targeting protein-protein interactions. Compounds of this type have been traditionally underrepresented in drug discovery platforms due to their structural characteristics not conforming to typical guidelines for ‘drug-likeness’. We have developed methods to simultaneously investigate new bioactive chemotypes and their pharmacological properties in a systematic manner. Several libraries of peptidyl macrocyclic and polycyclic compounds are examined here based on previously developed template methodology and novel discoveries using a highly reactive fluorinated reagent. Both methods allow us to rapidly build complexity from machine made, protecting-group-free bioactive starting materials. In this way, we can efficiently and broadly survey the chemical space of macrocycles through a diversity-oriented strategy.

In Chapter 2, we show that unprotected peptides react with commercial octafluorocyclopentene (OFCP) to afford hexafluorinated macrocycles via successive ipso substitutions of vinyl fluoride. Sequence variants having combinations of cysteine, tyrosine, histidine, and serine residues are shown to react rapidly at room temperature to give stable, isolable products. We also introduce our initial discoveries involving polysubstitution cascades with OFCP.

Chapter 3 details a broad scope of polycyclic compounds provided directly through one-pot methods. Macrocycle and spirocycle variations are also examined. We found the vinyl fluoride position to be amenable to linker systems that provide access to polycycles incorporating previously unused amino acid residues. Non-cell-based permeability assays are performed to evaluate the properties of these structures.

In Chapter 4, using cell-based permeability assays we study the pharmacological properties of macrocycles incorporating various template generations derived from a core cinnamyl alcohol motif. Structure-permeability relationships are explored with these compounds systematically via a small library.

Nature continues to provide a wide array of structurally novel, biologically active small molecules. This thesis describes synthetic studies on two such compounds. The approach used in each project are different. The first project involves a unique molecule that is produced by Nature in only trace quantities. We attempt to develop the first laboratory synthesis of the compound such that it could become available in sufficient amounts to study its biological properties in detail. In the second project, we work with a compound that is naturally abundant, yet has poor pharmacological properties. In that instance, we use synthesis to selectively modify its complex structure in ways that alter its properties while enabling conjugation to carrier proteins.Portimine is a marine-derived cyclic imine recently discovered as a selective inducer of apoptosis in cancer cells. Moreover, it showed markedly lower in vivo toxicity relative to other macrocycles in its class. Portimine’s biological activities are reminiscent of pro-apoptotic biologics such as TRAIL (TNF-related apoptosis-inducing ligand) and TNFα (tumor necrosis factor alpha). No synthesis of portimine has been published and this work outlines the synthetic studies performed en route to the natural product as well as the exploration and development of novel methodologies along the way. Another natural product, epigallocatechin-3-gallate (EGCG), is a major catechin found in tea that has been shown to inhibit tau through disaggregation of fibrils. The synthesis of several EGCG analogs as well as the biological assays performed with the ultimate goal of developing an Alzheimer’s therapeutic are described. Chapter one describes the development of a highly diastereo- and enantioselective Mukaiyama-Michael reaction for the synthesis of a key chiral intermediate utilized in the subsequent synthetic studies towards portimine. A stereochemical reassignment of the reported isomers of the desired structure is discussed aimed at the elimination of clear inconsistencies in the published reports. Both the racemic, as well as stereoselective synthesis, of both diastereomers are shown that once and for all establish the spectroscopic assignment of the products. Model studies of portimine including both the synthesis of thiophene-based as well as the more elaborate deoxygenated Diels-Alder precursor synthesized in just 3 steps from the published intermediate are reported as well. Optimization of the homoenolate cross-coupling as well as the development of the highly convergent route allowed for the rapid assembly of the chain containing all of the carbons present in portimine. Subsequent Diels-Alder studies as well as the challenges associated with utilizing an acyclic precursor provided useful information for the asymmetric synthesis of portimine. Chapter two describes the synthetic studies toward the natural product. Highly enantio- and diastereoselective aldol reaction as well as the alcohol-directed Narasaka-Prasad reaction allowed for the installation of the key sequence of stereocenters. Both halogenated as well as highly reactive dendralene Diels-Alder precursors have been synthesized which allowed for the in-depth studies of the cycloaddition conditions. The synthesis of a novel heterocycle provided access to the previously unknown motif that will be beneficial for the future syntheses of cyclic imine natural products. Chapter three discusses the synthesis of the EGCG analogs, and the biological studies associated with their potential as tau disaggregants. The development of the efficient click reaction assisted by copper-stabilizing Sharpless polytriazole ligand allowed for the synthesis of the conjugates of varied lengths. Subsequent nanoparticle conjugation showed the conservation of the biological activity as well as confirmed a previously hypothesized EGCG’s mode of action. Diastereoselective B-ring derivatization not only allowed for the installation of the PEGylated linkers at the new position but also for structure-activity relationship studies on EGCG’s D-ring shedding light on the importance of gallate phenols as well as the possibility for the installation of fluorine without sacrificing activity. Improved disaggregating activity of synthesized analogs showcased their promise as potential Alzheimer’s therapeutics.

Peptidyl macrocycles are a compound class with a rich clinical history and great potential for drugging biological targets by mediating protein-protein interactions. Methods to forge S-S disulfide bonds largely rely on the oxidation of dithiol containing substrates. We have developed and implemented a sulfur transalkylative macrocyclization induced by an appended cinnamyl carbonate-based template. This is a mechanistically new method for the construction of peptide macrocycles. The resultant macrocyclic structures contain the functionality of the linear oligomer, while scaffolding this potential pharmacophore in a more conformationally rigid manner. We aim to improve these macrocyclic structure’s biological stability and pharmacology relative to the linear oligomer.

Chapter 2 details the synthesis of mono- and disulfides via sulfur transalkylation induced by a tethered cinnamyl cation. Scope, limitations, and competition with previously reported nucleophilic residues are discussed. Methods to synthetically elaborate these structures are demonstrated and attempted. Synthesis of a potential ghrelin O-acyl transferase inhibitor is disclosed.

Chapter 3 covers the synthesis and development of two new templates designed to forge two macrocyclic bonds in one linear oligomer molecule. The use of these templates in the synthesis of bimacrocycles with sulfur and aryl linkages is divulged.

Chapter 4 centers on the synthesis and chemistry macrocyclic trisulfides. Acidolysis of template capped peptides containing tert-butylate trisulfide residues furnishes a mixture of mono-, tri- and pentasulfide linked macrocyclic product. Confirmation of this S2 exchange event is demonstrated via independent synthesis of the relevant monosulfide congener obtained in these trisulfidation reactions. Additionally, our efforts toward the total synthesis of an antimicrobial trithiocane containing natural product via the trisulfidation reaction are detailed.

Peptidomimetic macrocycles are valuable research tools and serve as lead compounds in drug discovery. We have developed two synthetic methods to access distinct isomeric macrocycles from a given peptide sequence; internal palladium(0)-catalyzed allylation, and large-ring forming Friedel-Crafts alkylation. These methods utilize a lipophilic template designed to impart favorable drug-like properties to the composite macrocycles relative to their linear peptide precursors. Structurally diverse macrocyclic peptidomimetics derived from these methods have the potential to identify small molecules effective against challenging targets involved in exposed protein surfaces.

We have charted a symmetry-based approach to complex, dimeric members of the pyrrole–imidazole family of alkaloids; palau'amine, axinellamines, massadines and ageliferin. New methods to prepare and manipulate bis-guanidine containing intermediates in this context are discussed. These efforts culminated in the synthesis of a partially halogenated variant of the complete (±)-axinellamine A ring system and the total synthesis of (±)-ageliferin from a common spirocyclopentane intermediate. A novel N-amidinyliminium ion rearrangement was examined for the requisite ring-expansion to provide the 2-aminotetrahydrobenzimidazole core of ageliferin directly. This method was applied to epimeric intermediates to obtain structurally novel tetracycles coined ‘iso’axinellamines.

We proposed access to (±)-axinellamine A via controlled oxidation and subsequent ring-contraction of (±)-ageliferin. This led us to examine oxidative methods for the transformation of 2-aminotetrahydrobenzimidazoles. Progress toward the synthesis of (−)-ageliferin via the oxidative desymmetrization of symmetric 2-aminotetrahydrobenzimidazole intermediates is discussed. Additionally, we report synthetic access to functionalized 3,3'-bipyrrolidines, coined ‘dispacamide dimer precursors’, for the synthesis of complex pyrrole−imidazole dimers. The dimeric framework was obtained by a diastereoselective, nickel-catalyzed reductive homocoupling of an unactivated secondary bromide derived from trans-4-hydroxy-L-proline. Dimeric precursors were desymmetrized by an auto-oxidative process. The method involves a cascading sequence of indiscrete oxidation and guanidine incorporation events followed by desymmetrization.