The search for novel materials that exhibit a quantum spin liquid (QSL) ground state is a highly interdisciplinary problem; motivated by a desire to more fully understand the basic physics of this new state of matter, it requires both synthetic ingenuity as well as extensive characterization of the material's structure and magnetic properties. The QSL can be described as entangled, frustrated spins that continuously fluctuate around a lattice down to T = 0 K without ever freezing into long range magnetic order. Due to the highly entangled nature of the spins, realizing a QSL in a usable material has the potential to revolutionize quantum computing. Few viable candidates currently exist both because many frustrated materials succumb to a structural distortion that breaks the delicate balance of competing magnetic interactions, and because a long series of experimental characterization is required to rule out all other possibilities for the ground state of a novel material. The most promising candidate is a mineral called herbertsmithite (Cu3Zn(OH)6Cl2) with a kagome lattice of S = 1/2 Cu2+ ions, although magnetic defects between the kagome layers complicate precise measurements of its QSL ground state at low temperature. The paucity of experimental systems has long motivated the search for new materials to validate the extensive theoretical work performed on various QSL models, and along the way this undertaking has uncovered many fascinating materials and expanded our understanding of magnetic frustration, related ground states, and the varied effects of structural disorders. In this dissertation, I report for the first time two distinct synthetic routes to a new mineral called barlowite (Cu4(OH)6FBr) that has a Cu2+ kagome lattice. While barlowite itself is not a viable QSL candidate because it orders magnetically, substituting Zn2+ suppresses magnetic coupling between the kagome layers and pushes the system towards a QSL ground state. The two routes yield crystals with different morphologies and low-temperature magnetism albeit the same crystal structure at room temperature. An array of X-ray and neutron diffraction measurements reveals that they have structural phase transitions to different low-temperature structures. One variant becomes orthorhombic and is a simple ordered magnet, while the second variant has only a slight symmetry lowering. In this novel compound, the kagome lattice is subtly modulated with a periodic pattern of distortions, for which numerical simulations predict a pinwheel valence bond crystal ground state instead of a QSL. Using neutron scattering, we find a pinwheel q=0 magnetic structure is induced below TN = 6 K. A clear relationship between the degree of distortion of the kagome lattice and the magnetic properties is evident in these two variants, and the hexagonal one is much closer to a QSL. I synthesize two versions of Zn-substituted barlowite (Cu3ZnxCu1-x(OH)6FBr) with no magnetic order: a polycrystalline version with nearly a full Zn2+ (x = 0.95) and the first single crystals ever reported, with approximately x = 0.56. Using an array of synchrotron X-ray and neutron scattering techniques, I demonstrate that both have an ideal kagome lattice and find no evidence that Zn2+ substitutes onto the kagome layers, which would disturb the QSL. Indeed, first-principles calculations show this antisite disorder is not energetically favorable. Instead, Zn2+ substitutes onto the interlayer; in Zn-substituted barlowite, site-specific X-ray diffraction reveals that Zn2+ and Cu2+ selectively occupy distinct sites, consistent with their different Jahn-Teller activity but in contrast to herbertsmithite, in which both metals occupy the same interlayer site. I uncover an experimental handle in Zn L-edge inelastic X-ray absorption spectroscopy correlated with the loss of inversion symmetry from pseudo-octahedral to trigonal prismatic coordination. Magnetic measurements indicate that both versions of Zn-substituted barlowite are QSL candidates. That the x = 0.56 crystals do not order magnetically even with 0.44 interlayer Cu2+s indicates a surprising robustness of the QSL against interlayer impurities. The two versions display remarkably similar magnetism to each other and to herbertsmithite, indicating universal magnetic behavior of the Cu2+ kagome lattice. In terms of suitability as an ideal QSL candidate, Zn-substituted barlowite has structural advantages over herbertsmithite: its kagome layers are highly resistant to nonmagnetic defects while the interlayers can accommodate a higher amount of Zn substitution

In chemical biology, reactive carbonyl species such as aldehydes and activated esters have been routinely utilized for modification biomolecules for various purposes such as imaging, enzyme profiling, drug delivery, and caging. This work herein presents a novel application of their chemistry to functionalize and control RNA and protein function through chemically reversible polyacylation. Also due to their reactive nature and propensity to form adducts with biomolecules and cause dysfunction, there has been continued interest in determining their concentration and composition to understand how they contribute to cancer, neurological disorders, and cardiovascular diseases. In furthering this endeavor, the second part of this work describes the development of fluorescent methods to measure and profile intracellular aldehydes. Chapter 1 describes the synthesis and RNA acylation activity of a series of minimalist azidoalkanoyl imidazole reagents, with the aim of functionalizing RNA at 2'-hydroxyl groups at stoichiometric to superstoichiometric levels. Due to their simple structure, they are prepared readily in high yields. Upon reaction with RNA, we find marked effects of small structural changes on their ability to acylate and be reductively removed. One compound in the series, a glycolic acid derivative, is shown to be highly active both in acylation of RNA and in phosphine-triggered de-acylation, which enables reversible control of hybridization and folding. We also identify reagents that are ideal for long-term acylation of RNA, remaining stable even after azide reduction; this presents a novel and simple strategy for amine functionalization of RNA. Finally, an azidoacyl adduct on RNA was shown to react with a strained alkyne-containing fluorophore in a "cloak-click" strategy, suggesting a general approach to facile fluorescent labeling of RNAs. These simple azidoalkanoyl acylimidazole reagents serve as a set of molecular tools that can be employed easily for post-synthesis labeling and control of RNA irrespective of length. Chapter 2 describes RNA 2'-OH polyacylation agents with improved reversibility based on quinone methide elimination. The rapidly reversible RNA caging method was utilized to control RNA folding and function, both in vitro and in cells. Previous uncloaking chemistry made use of azide reduction and subsequent amine cyclization, requiring 2 to 4 hours for completion. Aiming to improve reversal rates and yields, we designed novel acylating reagents that utilize quinone methide (QM) elimination for reversal. The QM uncloaking/de-acylation reactions were tested with two bioorthogonally cleavable motifs, azide and vinyl ether, and their acylation and reversal efficiencies were assessed with NMR and mass spectrometry on a model RNA substrate as well as on RNAs. Among the compounds tested, the azido-QM compound A-3 displayed excellent deacylation efficiency. To test its function in caging, A-3 was successfully applied to control EGFP mRNA translation in vitro and in cells. We envision that this compound will serve as a valuable tool for biological investigation and control of RNAs. Chapter 3 discusses the potential application of chemically reversible acylating reagents to control protein function. Proteins are involved in all facets of cellular biology and have been harnessed for a wide range of technological and therapeutic purposes. To decipher their roles in complex biological systems and for additional spatiotemporal control in vitro, various caging strategies have been developed. However, simple methods applicable to native protein remain underexplored. In preliminary studies toward this goal, we examined whether NAI-N3, a chemically reversible acylating agent, could be used to control protein activity in a convenient manner. Polyacylation with NAI-N3 led to the inhibition of various proteins including trypsin, luciferase, horse radish peroxidase, and DNA polymerases. However, phosphine treatment and subsequent deacylation poorly recovered the original activity likely due to irreversible denaturation and aggregation and harsh reductive reversal conditions. Future efforts will investigate acylating reagents with enhanced reversibility such as the quinone methide probes in chapter 2 and reagents that maintain protein surface charge and with less denaturing properties. Chapter 4 describes the application of fluorogenic probes to detect intracellular aldehydic load and progress toward the development of a method to profile intracellular aldehydes. Aldehydes are formed as metabolites in multiple cellular pathways and introduced from the environment. Due to their toxicity, their cellular levels are normally tightly regulated. Because they form adducts with DNA, aldehydes have been implicated in diseases with impaired DNA repair such as Fanconi anemia. Our lab has developed quenched hydrazone ("DarkZone") dyes that output a fluorescent response to intracellular alkyl aldehydes. To analyze the aldehydic load in hematopoietic stem cells with DarkZone dyes, spectral overlap had to be minimized with fluorescent antibodies utilized for flow cytometry. To this end, novel DarkZone probes with various esterase cleavable motifs and fluorophores, Pacific Blue and V450, were explored. With the DarkZone probes, intracellular aldehydic loads in circulating human leukocytes were measured for the first time, and changes in cellular aldehyde concentration in the physiological range in response to aldehyde or ethanol challenge were detected. Additionally, we examined whether fluorophores with α-nucleophile reactive handles can be applied to determine cellular aldehyde composition. Utilization of these tools to investigate how deactivating aldehyde dehydrogenase 2 (ALDH2) mutants affect aldehyde content and whether ALDH activating molecules could be utilized to rescue cells from the genotoxicity of aldehydes is currently underway

Chemical phenomena occur across a wide range of length and time scales. Atoms vibrate on the order of 10-15 seconds, while chemical reactions may take place anywhere between 10-12 seconds and hours (or as long as a chemist is willing to wait). Quantum mechanics governs the smallest of these scales, where the Schrödinger equation tells us how to compute observable properties of quantum systems. For such small systems, highly accurate calculations can be performed, though often with formidable computational cost. At intermediate scales with hundreds of atoms, approximate quantum mechanical methods can be used to define potential energy surfaces, which can be used in conjunction with Newtonian mechanics to simulate the trajectories that molecules move in. Kinetic models bridge to even larger, continuum scales, by utilizing a set of differential equations that relate macroscopic thermodynamic variables, such as temperature and pressure, to the concentrations of chemical species. The differential equations can be integrated to arbitrary points forward in time, providing predictions of how thermodynamic variables and concentrations change. Each of the aforementioned models yields a trade-off: high accuracy can be attained in the limit of the very small, while in the limit of the large, we are restricted to using coarser models. Connecting microscopic descriptions of molecules with models of macroscopic phenomena, as well as the reverse, can be informative for models at both scales. This dissertation aims to do precisely this, progressively working our way up from small length and time scales to macroscopic scales. First, we discuss research on improving the performance of electronic structure calculations such as Hartree-Fock by constructing approximations to the wavefunction through partitions of atoms into fragments. Then, we show how machine learning can be utilized to approximate effective Hamiltonian matrix elements. After, we discuss molecular dynamics integration, deriving multiple time step integration methods, showing how explicit integrators can be easily implemented, and how the accuracy of symplectic integrators can be analyzed in terms of the shadow Hamiltonian. Last, we make the connection of accelerated molecular dynamics simulations of nitromethane to kinetic models, which can be used to predict reaction pathways with varying thermodynamic conditions and time scales, allowing for comparison of predictions to experiments on computationally challenging time scales

Photochemistry is the chemical response of molecules to light and drives many natural processes such as photosynthesis, vision, and bioluminescence. Nonradiative decay via intersystem crossing or internal conversion are mechanisms of particular interest due to the direct conversion of energy from light into mechanical motion. This presents unique difficulties to experiment due to the rapid time-scale on which these dynamics often occur. The involvement of multiple electronic states is a commensurate challenge to theoretical methods. Nonetheless, theoretical quantum chemistry approaches have had much success towards this objective and have been used in conjunction with experiment to produce powerful results. In particular, advancements such as GPU acceleration of electronic structure energy and gradient calculations have enabled on-the-fly simulation techniques such as ab initio multiple spawning (AIMS) to simulate these processes. In this dissertation, we use AIMS to produce one-to-one comparisons with experiment through direct modelling of experimental observables. We specifically highlight dynamics involved in photoisomerization of cis-stilbene and photodissociation of ortho-nitrophenol in comparison to time-resolved photoelectron spectra and ultrafast electron diffraction respectively. Additionally, we discuss enhancements of AIMS algorithms to address existing challenges in simulating excitations to higher lying electronic states

The field of glycobiology defies the central dogma. Unlike many other post-translational modifications, glycosylation of proteins and the structures of glycan trees are not genetically templated. Consequently, many of the traditional biochemical approaches to studying structure and function are not amenable to studying the structure and function of glycans. Thus, glycobiologists have developed and employed a host of tools to study and manipulate the structure and function of glycans. Chemical biologists have been key to this endeavor, providing numerous metabolic, enzymatic, genetic, and synthetic tools specifically suited to this purpose. Herein, we first review some of these tools, providing a brief historical overview, as well as some recent additions that of which the new chemical glycobiologist should be aware. Then, we describe the applications of a new generation of synthetic glycopolypeptides towards studying receptor-glycan interactions in immunity and, in some cases, bringing these materials towards the clinic through translational research. We leverage glycans to modulate inflammation, in the context of inducing inflammation for immunoncology therapies or inhibiting inflammation in hyperinflammatory conditions

Every cell in the human body is coated with a peripheral layer of glycosylation. These polysaccharides, glycoproteins, glycolipids, and proteoglycans organize into an extramembrane compartment termed the glycocalyx. The glycocalyx is the cell's first point of contact with the extracellular environment, and is a key regulator of cell-matrix and cell-cell interactions. Investigations into the architecture and dynamics of the glycocalyx stand to benefit from methods to (i) spatially classify glycans at relevant length scales, and (ii) selectively edit glycocalyx components. After an introductory Chapter 1, Chapter 2 describes a super-resolution microscopy based approach for quantification of glycocalyx parameters. Application of this method in the context of pancreatic cancer revealed that oncogenic KRAS changes the glycocalyx in part by promoting the biosynthesis of mucins. Mucins are a class of cell surface glycoprotein that are difficult to study and drug due to their dense glycosylation and resistance to proteases. Chapter 3 describes characterization of a mucin-selective protease derived from the human gut microbiome, which enables selective cleavage and release of mucins from live cells. This tool lowers barriers to the study of native mucin biology and represents a potential strategy for therapeutic intervention in mucin-expressing cancers. Chapter 4 describes unbiased screening in search of mammalian enzymes that proteolyze mucin domains, culminating in the observation that the human lysosomal protease cathepsin D exhibits such activity. The discovery that a human protease cleaves mucin domains without prior deglycosylation has implications for our basic understanding of human mucin catabolism and the consequences when it goes awry

Pancreatic β-cells are solely responsible for secreting insulin in response to blood glucose. Loss of β-cell mass and function is a hallmark of diabetes, and reversal of this β-cell deficit is a towering goal in regenerative therapy for diabetes. Thus, the development of new β-cell-directed diabetes therapeutics to preserve and/or enhance β-cell mass and function (insulin secretion) has exceptional human health impact potential. Unfortunately, advancement in this arena is stymied by a dearth of β-cell-specific therapeutic targets. Translation of numerous promising, but un-targeted, diabetes therapeutics to the clinic has thus far failed because of systemic effects. Consequently, the next generation of β-cell-directed imaging and therapeutic modalities must be reimagined with β-cell selective targeting in mind. Chapter 1 begins by outlining the urgent need for selective β-cell targeting. A clinically successful β-cell targeting paradigm could dramatically transform diabetes care, most notably by allowing β-cell regeneration to be translated into humans. This chapter introduces current diabetes therapeutics and their potential for selective targeting to β-cells. Next, it surveys the literature of targeted β-cell delivery, highlighting successful β-cell imaging applications and their potential application to targeted drug delivery. Further, all exemplified β-cell targeting methods are reviewed, which include, most successfully, Zn2+-based targeting (Chapter 3). Finally, the potential of selective prodrug release, which has heretofore been largely unexplored in β-cells, is assessed. Within the area of β-cell therapeutics is the relatively recent idea of restoring healthy blood glucose homeostasis by stimulating β-cells to self-replicate. Chapter 2 conveys a completed research project whose goal was to overcome poor potency, a central limitation of all available pro-β-cell replication compounds. This project involved repurposing a toxic compound (OTS167) with promiscuous binding across all human kinases, and honed in using substantial synthetic chemistry, biochemistry, and cell biology to generate a highly potent, yet non-toxic, lead compound. This chapter is evidence of the medicinal chemistry aspects of this dissertation research. Chapter 3 exemplifies β-cell targeted drug delivery of a zinc-chelating small molecule. It describes assays that were established and the synthesis of a hybrid replication-promoting/zinc-chelating small molecule. New assays were developed or adapted to assess chelation in β-cells, accumulation in primary islets, and selective replication, using microscopy, liquid chromatography/mass spectrometry, and quantitative immunofluorescence. Finally, the central hypothesis that this molecule accumulates in β-cells and selectively biases replication toward β-cells relative to non β-cells was confirmed. This chapter embodies the power of the chemistry-biology interface to prod discovery in medicinal research. Chapter 4 clarifies the experimental steps involved in designing and performing the assays in Chapter 3 and elaborates some of the most crucial steps. Ensuring a high level of reproducibility is critical for capitalizing on the broad applicability of Zn2+-chelator-based targeted β-cell delivery. Chapter 5, while not directly related to either β-cell targeted delivery or β-cell replication, demonstrates that neuroendocrine neoplasms, which share commonalities with β-cells, show staining intensity for an enzyme expressed by neuroendocrine cells. This finding was robust and repeatable. This discovery illustrates the utility of examining neuroendocrine markers in real human tissues. Additionally, the presence of this chapter backs up the highly interdisciplinary nature of this dissertation, reaching from synthetic organic chemistry all the way to clinical research

Lithium-sulfur (Li-S) batteries charge by oxidizing solid lithium sulfide (Li2S) into sulfur (S8) through soluble lithium polysulfide intermediates (Li2Sx), enabling a high energy density of 2500 Wh kg-1, a five-fold increase compared to traditional Lithium ion Batteries (LiBs). Such exceptionally high energy density is enabled by the reversible reaction between sulfur and lithium sulfide (Li2S) via a series of lithium polysulfides intermediates (LiPSs, Li2Sn, 2≤n≤8). However, significant challenges remain in order to build practical Li-S batteries, which are primarily attributed to the dissolution of intermediate species (LiPSs) in the electrolytes as well as the insulating nature of both sulfur and Li2S. strategies to improve durability of Li-S batteries are key to their successful application in commercial batteries. In this thesis, polymer coating, electrolyte additive, and carbon materials were used to address issues of Li-S batteries. The first portion of my research describes an efficient design of hybrid electrode structures using a solution-processable isoindigo-based polymer incorporating polar substituents. It provides the following critical features: (1) the conjugated backbone provides good conductivity; (2) functional pyridine groups provide high affinity to polysulfide species; and (3) it possesses high solubility in organic solvents. These lead to effective coating on various carbonaceous substrates to provide highly stable sulfur electrodes. Importantly, the electrodes exhibit good capacity retention (80% over 300 cycles) at sulfur mass loading of 3.2 mg/cm2, which significantly surpasses the performance of previously reported polymer-enabled sulfur cathodes. However, a challenge that was not solved in the first part of my research is the insulating nature of both sulfur and Li2S. For examples, when charging a Li2S electrode, a significant portion of each particle is electrically isolated and can be oxidized at the localized interface between the electrode/electrolyte with sufficient charge transfer. Therefore, the Li2S exhibits a large overpotential and a limited reversible capacity that is substantially lower than the theoretical value. Hence, in the second portion of my research, we employ the redox chemistry of a quinone derivative to realize efficient, fast, and stable operation of Li-S batteries using Li2S microparticles. When adding a quinone derivative with tailored properties (e.g. oxidation potential, solubility, and electrochemical stability in the electrolyte) to an electrolyte as a redox mediator, initial charging of Li2S electrodes occurs below 2.5 V at a 0.5C rate, and the subsequent discharge capacity is as high as 1300 mAh g-1. Moreover, deposition of dead Li2S is effectively prevented with the addition of the redox mediator, thus avoiding the primary cause of increasing polarization and decreasing reversible capacity of Li-S batteries upon cycling. Another primary approach to solve the above issues is to infiltrate sulfur into nanostructured conductors, such as porous carbon materials, to realize sufficient conductivity and cycling rate performance. However, most carbon host materials were tested with high electrolyte to sulfur ratios (E/S) (generally > 15 μL/mg), which compromises the cell-level energy density. Hence, it is important to re-visit the carbon structures with desired properties to enable low E/S ratio. The third portion of my thesis is to design a flower-shaped porous carbon structure that has several advantages: (1) the material has superior high surface area (> 3300 m2/g) ; (2) the pore size is less than 5 nm, which is more suitable for low E/S ratio; (3) Incorporating Nickel nanoparticles onto the carbon flower gives stronger binding interaction with LiPSs and better reaction kinetics. Through the desired properties of carbon flower as host material for sulfur, we successfully demonstrated that higher capacity and higher cycle retention were enabled by using the carbon flower-sulfur (CF-S) electrode in low E/S ratio (< 5 μL/mg)

The remarkable properties of glassy materials arise from their complex amorphous structures and dynamics. The heterogeneity of these materials was studied on molecular length scales and picosecond time scales using a combination of polarization-selective IR pump-probe and 2D IR experiments and a vibrational chromophore that is sensitive to local chemical and electrical environments. By monitoring the restricted orientational motion of the chromophore in a polymer, the distribution of sub-nanometer pores could be explored. The same chromophore in a hydrogen-bonding system showed extreme polarization dependence in the 2D line shape, providing evidence for significant dynamical heterogeneity in the supercooled liquid phase

Natural product synthesis and methodology development have long enjoyed a symbiotic relationship with the structural complexity observed in natural products inspiring chemists to invent new reactions, as well as providing a proving ground for the utility of existing methods in complex settings. Chapter 1 of this dissertation provides a recent perspective on catalytic methodology developed to enable the synthesis of complex natural products. A focus on the last 20 years of organic chemistry demonstrates the state-of-the-art in methods development and its direct application to accessing complex natural products efficiently. Chapter 2 details our efforts to synthesize three sulfolipid natural products isolated from algae Ochromonas danica. Key features of our synthesis of danicalipin A include the application of our catalytic, enantioselective dichlorination of allylic alcohols and a diastereoselective boron allylation of the dichloroaldehyde fragment. Subsequent syntheses of two biosynthetic precursors of danicalipin A are presented. We also describe the results of an interdisciplinary collaboration enabled by the synthetic sulfolipids to investigate the biophysical properties of these exotic molecules. Chapter 3 describes our efforts towards the total synthesis of napyradiomycin natural product azamerone. Our synthesis required the development of a catalytic, enantioselective chlorocyclization reaction to access a key chlorinated benzopyran intermediate. Subsequent B-alkyl Suzuki cross-coupling between two enantioenriched fragments, followed by the late-stage installation of the pyridazine heterocycle via a tetrazine [4+2] cycloaddition yielded the natural product. This work constitutes the first enantioselective total synthesis of azamerone

The selective formation of carbon-carbon (C-C) bonds has been a longstanding area of study in organic chemistry with broad applications in fine and commodity chemical synthesis. This thesis describes several novel ways that C-C bonds can be made utilizing alkali carbonates in a solvent-free and catalyst-free system. The first chapter provides an introduction to the synthesis of chemicals from lignocellulosic feedstocks. While edible feedstocks have been used industrially to make ethanol and lactic acid via fermentation, the conversion of lignocellulosic feedstocks to value-added chemicals has been limited. One of the few chemicals produced from lignocellulose is furfural, which is made via the acid-catalyzed dehydration of pentoses in hemicellulose. Furfural has limited applications at present. Carbonate-promoted C-H carboxylation, a new C-C bond forming reaction reported by the Kanan group in 2016, is introduced and discussed as a way to convert furfural into more valuable C6 compounds such as 2,5-furandicarboxylic acid. The second chapter investigates an alternative synthesis of benzenetricarboxylates (BTCs), high-value small molecules that are used in the synthesis of metal-organic frameworks, PVC plasticizers, and heat-resistant polyimides. BTCs are currently made from trimethylbenzenes via the Amoco oxidation, an expensive process that produces carbon monoxide and ozone-depleting methyl bromide through undesired side reactions. Here, a non-oxidative route to produce BTCs from alkali phthalates and CO2 via carbonate-promoted C-H carboxylation is studied. While it was discovered that BTCs can be made from phthalates and various alkali carbonates and CO2, competing decarboxylation, over-carboxylation, and isomerization pathways make it difficult to develop a selective synthesis. The third chapter investigates the reactivity of benzylic C-H and C-OH bonds in the presence of alkali carbonates. Using cesium carbonate, the benzylic C-H of alkali m-toluate can be carboxylated; however, an equilibrium between the starting material and desired product resulted in limited yields. The oxidation of the benzylic C-OH bond in alkali 4-(hydroxymethyl)benzoate was also studied. Interestingly, when cesium carbonate was used, the major product was dimerized starting material while the minor products were reduced p-toluate and oxidized terephthalate. In order to explain the formation of the unexpected dimers, a mechanism involving an aldehyde intermediate reacting with a deprotonated p-toluate is proposed. Several follow-up experiments that support this mechanism are also discussed. The fourth chapter studies the synthesis of a tetrahydrofuran-based polyamide monomer from furfurylamine, one of the few chemicals made industrially from inedible lignocellulose. Using carbonate-promoted C-H carboxylation, furfurylamine is converted into a furan-containing amino acid that is purified via anion exchange chromatography. Afterwards, this intermediate is hydrogenated and cyclized to a tetrahydrofuran-containing bicyclic lactam. The synthesis of this bicyclic lactam avoids the use of protecting groups and multiple stoichiometric organic reagents required by previous, longer routes, and is the potential starting point for the design of a scalable process. The fifth and final chapter explores the polymerization of the bicyclic lactam monomer to poly(aminomethyl)tetrahydrofuran (PAT). It was found that PAT can be synthesized under heated neat conditions and in a solvent at room temperature. The mechanism of PAT polymerization is believed to occur via living anionic ring opening polymerization. As such, the number average molecular weight can be controlled by varying the ratio of monomer to imide activator. The values obtained experimentally are consistent with theoretical values. PAT also exhibits an unusually high glass transition temperature, illustrating an advantage over other bio-based polymers

Bryostatin 1 is a natural product that was originally isolated from the marine sponge Bugula neritina more than 50 years ago. Following its isolation, it has demonstrated unprecedented clinical potential across a number of indications, including HIV/AIDS eradication, the treatment of Alzheimer's disease and neurological disorders, and cancer immunotherapy. Despite this unique portfolio of indications, the natural supply of bryostatin 1 from its source organism is limited and variable and maintaining a consistent supply of the natural product has hindered its clinical advancement. To enable the continued clinical evaluation of bryostatin 1, we developed a scalable total synthesis of the natural product that can sustainably supply future clinical studies of this exciting clinical candidate. Despite its promising activity, bryostatin 1 is a marine natural product that is neither evolved nor optimized for the treatment of human disease. Historically, the scarcity of isolated material and the challenges associated with making modifications of the delicate and densely functionalized bryostatin skeleton have precluded efforts to optimize the biological activity of this natural product lead through derivatization and exploration of structure-activity relationships (SAR) around the macrocycle. Drawing on the synthetic platform we developed in our scalable synthesis of bryostatin 1, we accomplished the design, synthesis, and biological evaluation of the first close-in analogs of bryostatin 1. Using a function-oriented synthesis (FOS) strategy informed by a combination of computational and biological data surrounding bryostatin's interaction with its protein target, protein kinase C (PKC), we synthesized a series of bryostatin analogs designed to maintain PKC affinity while allowing for a systematic investigation of their biological function. By leveraging the modularity of our bryostatin 1 synthesis, we developed complementary late-stage diversification strategies that provide efficient synthetic access to parallel series of bryostatin analogs with modifications in the A- and B-rings. In agreement with our pharmacophore model, these new agents retain affinity for PKC but exhibit variable PKC translocation kinetics. We further demonstrate that select analogs potently induce increased cell surface expression of CD22, a promising target for the treatment of leukemias and lymphomas, in in vitro models of acute lymphoblastic leukemia (ALL) and AIDS-related lymphomas, highlighting the potential general use of bryostatin and bryostatin analogs for enhancing antigen-targeted cancer immunotherapies. Finally, bryostatin 1 has been shown to prevent progressive neurodegeneration in a mouse model of multiple sclerosis (MS). Working with Professors Paul Kim and Michael Kornberg at Johns Hopkins, we show that several bryostatin analogs replicate the anti-inflammatory effects of bryostatin 1 on innate immune cells in vitro and lead analog SUW133 attenuates neuroinflammation and prevents the development of MS-related neurological deficits in vivo. We further demonstrate that this activity is dependent on PKC. These findings identify bryostatin analogs as promising drug candidates for targeting innate immunity in neuroinflammation and create a platform for evaluation of synthetic PKC modulators for the treatment of MS and other neuroinflammatory diseases

Cu enzymes play important roles in a wide range of biological processes including O2 activation. Cu active sites in these enzymes have unique geometric and electronic structures that give rise to characteristic spectral features. Thus, the investigation of these spectral features provides insight into the structure--function correlation in Cu enzymes. The focus of this thesis is to utilize X-ray absorption and emission spectroscopies to probe Cu sites in metalloenzymes and model complexes. Chapter 1: This chapter provides the background of mononuclear Cu enzymes and Cu corroles, and the introduction to the methodologies of X-ray absorption and emission spectroscopies. Chapter 2: Cu(I) active sites in metalloproteins are involved in O2 activation, but their O2 reactivity is difficult to study due to the Cu(I) d10 closed shell which precludes the use of conventional spectroscopic methods. Kβ X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kβ XES in probing Cu(I) sites in model complexes and a metalloprotein. Using Cu(I)Cl, emission features from double ionization (DI) states are identified using varying incident X-ray photon energies and a reasonable method to correct the data to remove DI contributions is presented. Kβ XES spectra of Cu(I) model complexes, having biologically relevant N/S ligands and different coordination numbers, are compared and analyzed, with the aid of density functional theory (DFT) calculations, to evaluate the sensitivity of the spectral features to the ligand environment. While the low-energy Kβ2,5 emission feature reflects the ionization energy of ligand np valence orbitals, the high-energy Kβ2,5 emission feature corresponds to transitions from molecular orbitals (MOs) having mainly Cu 3d character with the intensities determined by ligand-mediated d--p mixing. A Kβ XES spectrum of the Cu(I) site in preprocessed galactose oxidase (GOpre) supports the 1Tyr/2His structural model that was determined by our previous X-ray absorption spectroscopy and DFT study. The high-energy Kβ2,5 emission feature in the Cu(I)-GOpre data has information about the MO containing mostly Cu 3dx2−y2 character that is the frontier molecular orbital (FMO) for O2 activation, which shows the potential of Kβ XES for probing the Cu(I) FMO associated with small molecule activation in metalloproteins. Chapter 3: The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)--FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation. Chapter 4: The formylglycine-generating enzyme (FGE) catalyzes the O2-dependent conversion of Cys to formylglycine. The Cu(I) active site of the FGE is coordinated by two Cys residues in a linear geometry. With substrate binding, the Cu(I) active site becomes a trigonal planar geometry with three Cys ligands, which turns on O2 activation. The Cu(I) active site structure in the FGE is unique because the similar bis-cysteine coordination is typically observed in Cu trafficking and sensing enzymes but not in Cu-dependent oxidases/oxygenases. Here, we apply Kβ X-ray emission spectroscopy to study the Cu(I)-FGE and the substrate bound Cu(I)-FGE active sites to probe its frontier molecular orbital(s) (FMO(s)) for O2 binding and activation. Small spectral differences are observed between Cu(I)-FGE and substrate bound Cu(I)-FGE that are correlated to the site structure change due to the substrate binding to the Cu(I) using DFT calculations. Based on these Kβ XES spectra and calculations, the Cu 3dx2−y2 vs 3dz2 nature of the FMO for O2 binding and activation, equatorially vs axially is discussed. Chapter 5: The question of ligand noninnocence in Cu corroles has long been a topic of discussion. Presented herein is a Cu K-edge X-ray absorption spectroscopy (XAS) study, which provides a direct probe of the metal oxidation state, of three Cu corroles, Cu[TPC], Cu[Br8TPC], and Cu[(CF3)8TPC] (TPC = meso-triphenylcorrole), and the analogous Cu(II) porphyrins, Cu[TPP], Cu[Br8TPP], and Cu[(CF3)8TPP] (TPP = meso-tetraphenylporphyrin). The Cu K rising-edges of the Cu corroles were found to be about 0--1 eV upshifted relative to the analogous porphyrins, which is substantially lower than the 1--2 eV shifts typically exhibited by authentic Cu(II)/Cu(III) model complex pairs. In an unusual twist, the Cu K pre-edge regions of both the Cu corroles and the Cu porphyrins exhibit two peaks split by 0.8--1.3 eV. Based on time-dependent density functional theory calculations, the lower- and higher-energy peaks were assigned to a Cu 1s → 3dx2−y2 transition and a Cu 1s → corrole/porphyrin π* transition, respectively. From the Cu(II) porphyrins to the corresponding Cu corroles, the energy of the Cu 1s → 3dx2−y2 transition peak was found to upshift by 0.6--0.8 eV. This shift is approximately half that observed between Cu(II) to Cu(III) states for well-defined complexes. The Cu K-edge XAS spectra thus show that although the metal sites in the Cu corroles are more oxidized relative to those in their Cu(II) porphyrin analogues, they are not oxidized to the Cu(III) level, consistent with the notion of a noninnocent corrole. The relative importance of σ-donation versus corrole π-radical character is discussed. Appendix 1: A macrocyclic ligand (L4−) comprising two pyridine(dicarboxamide) donors was used to target reactive copper species relevant to proposed intermediates in catalytic hydrocarbon oxidations by particulate methane monooxygenase and heterogeneous zeolite systems. Treatment of LH4 with base and Cu(OAc)2∙H2O yielded (Me4N)2[L2Cu4(μ4-O)] (1) or (Me4N)[LCu2(μ-OH)] (2), depending on conditions. Complex 2 was found to undergo two reversible 1-electron oxidations via cyclic voltammetry and low-temperature chemical reactions. On the basis of spectroscopy and theory, the oxidation products were identified as novel hydroxo-bridged mixed-valent Cu(II)Cu(III) and symmetric Cu(III)2 species, respectively, that provide the first precedence for such moieties as oxidation catalysis intermediates. Appendix 2: The multifunctional protein cytochrome c (cyt c) plays key roles in electron transport and apoptosis, switching function by modulating bonding between a heme iron and the sulfur in a methionine residue. This Fe--S(Met) bond is too weak to persist in the absence of protein constraints. Here, we ruptured the bond in ferrous cyt c using an optical laser pulse and monitored the bond reformation within the protein active site using ultrafast X-ray pulses from an X-ray free-electron laser, determining that the Fe--S(Met) bond enthalpy is ~4 kcal/mol stronger than in the absence of protein constraints. The 4 kcal/mol is comparable with calculations of stabilization effects in other systems, demonstrating how biological systems use an entatic state for modest yet accessible energetics to modulate chemical function. Appendix 3: Peroxynitrite (−OON=O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative (bio)chemistry. It may derive from reaction of superoxide anion (O2•−) with nitric oxide (•NO) and has been suggested to form an as-yet unobserved bound heme-iron-PN intermediate in the catalytic cycle of nitric oxide dioxygenase (NOD) enzymes, which facilitate a •NO homeostatic process, i.e., its oxidation to the nitrate anion. Here, a discrete six-coordinate low-spin porphyrinate-FeIII complex [(PIm)FeIII(−OON=O)] (3) (PIm; a porphyrin moiety with a covalently tethered imidazole axial "base" donor ligand) has been identified and characterized by various spectroscopies (UV--vis, NMR, EPR, XAS, resonance Raman) and DFT calculations, following its formation at −80 °C by addition of •NO(g) to the heme-superoxo species, [(PIm)FeIII(O2•−)] (2). DFT calculations confirm that 3 is a six-coordinate low-spin species with the PN ligand coordinated to iron via its terminal peroxidic anionic O atom with the overall geometry being in a cis-configuration. Complex 3 thermally transforms to its isomeric low-spin nitrato form [(PIm)FeIII(NO3−)] (4a). While previous (bio)chemical studies show that phenolic substrates undergo nitration in the presence of PN or PN-metal complexes, in the present system, addition of 2,4-di-tert-butylphenol (2,4DTBP) to complex 3 does not lead to nitrated phenol; the nitrate complex 4a still forms. DFT calculations reveal that the phenolic H atom approaches the terminal PN O atom (farthest from the metal center and ring core), effecting O--O cleavage, giving nitrogen dioxide (•NO2) plus a ferryl compound [(PIm)FeIV=O] (7); this rebounds to give
[(PIm)FeIII(NO3−)] (4a). The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished

In the era of precision medicine, molecular subtyping of non--small cell lung cancer (NSCLC) has revolutionized therapeutics tailored to individual actionable mutations, particularly the use of tyro- sine kinase inhibitors (TKIs) for Epidermal Growth Factor Receptor (EGFR) mutations. However, efficient therapy selection remains challenging for some patients, and it is estimated that 20% of lung cancer patients begin therapy prior to EGFR testing. Common reasons for lack of molecular testing include insufficient tissue biopsy or inability to biopsy certain patients, and long turnaround times to results. Clearly, new technologies that are rapid, cost--effective, and highly sensitive are needed for effective companion diagnostics. This thesis outlines the development and clinical translation of a blood--based circulating tumor (ct) DNA EGFR genotyping assay using giant magnetoresistive (GMR) nanosensors as an alternative for therapy selection and response monitoring. This GMR as- say achieved analytical sensitivities of 0.01% mutant allelic fraction for 3 common "hot spot" EGFR mutations, equivalent to detecting 1 mutant allele in a background of 10,000 wild--type alleles. In a clinical study of therapy selection and response monitoring of metastatic NSCLC patients, the assay achieved high sensitivity and specificity (AUC > 0.95) and was able to accurately predict therapy response after just 2 weeks. This represents a vast improvement compared to the current clinical standard of radiographic (CT) assessment after 2--3 months, which can be a harmful delay for patients who do not respond to targeted therapy and need immediate changes in therapeutic regimen. GMR sensors were also utilized for the early detection of colorectal cancer (CRC). While CRC diagnosis in Stage I is associated with a 90% 5--year survival rate, common screening methods such as colonoscopy or fecal testing are underutilized, resulting in a majority of CRC patients diagnosed at later stages. A non--invasive blood--based ctDNA test with GMR sensors could aid in screening efforts, with methylation biomarkers indicated for improved early cancer detection compared to mu- tations. This thesis also details the technology development of integrating methylation--specific PCR with high resolution melt (HRM) analysis on GMR sensors for quantifying methylation density. The technology was first optimized in a melanoma cell line model, and then extended to a multiplexed panel of 4 putative biomarkers of early CRC. This assay achieved analytical sensitivities of 0.01% methylated allelic fraction for 3 of 4 markers, and was validated in 2 late stage CRC patient samples

Developing structure-activity relationships in CO2 electroreduction catalysts is critical to rationally synthesizing electrode materials with improved performance. While many studies have drawn correlations between the activity, selectivity, and stability of a CO2 electroreduction catalyst and catalyst particle size, shape, composition, roughness, comparatively less had been established about how bulk defects influence surface reactivity. We hypothesized that bulk defects could stabilize reactive surfaces that persist through electrolytic conditions and alter the reactivity of more ordered surfaces. In this dissertation, we detail our efforts to obtain the first direct evidence that CO2 electroreduction in enhanced at the surface terminations of grain boundaries (GBs) and dislocations. We used scanning electrochemical cell microscopy (SECCM) to show that the surface terminations of GBs engender regions of enhanced CO2 electroreduction activity that can be several µms wide. The magnitude of enhancements appeared to depend on local GB structure and ranged between 10%-300% relative to the adjacent grains. In contrast, the competing H2 evolution reaction was relatively insensitive to the presence of these defects. Mapping the lattice deformation of the regions surrounding these bulk defects using electron diffraction suggested that the local density of dislocation surface terminations, instead of local lattice strain, were correlated to the observed enhancements. Beyond electroanalytical SECCM studies, we demonstrated the utility of engineering bulk defects in CO2 electroreduction catalysts by showing that mechanical treatment enhances CO2 electroreduction by introducing new GBs and dislocations, while the H2 evolution reaction remains relatively unaffected. In addition, we report the development and validation of a scanning electron microscope (SEM)-based technique to rapidly and accurately characterize the grain orientation and defect structure of polycrystalline nanoparticles, relieving a bottleneck imposed by the inaccessibility of existing grain mapping techniques. These studies establish a new structure space for electrocatalyst design and motivate the exploration of GB and dislocation engineering to develop improved catalysts for electrochemical transformations

Enveloped viruses are a diverse group of agents that pose a threat to global health and economic stability. An enveloped virus consists of a lipid bilayer surrounding the particle, surface glycoproteins involved in entry, and a viral genome necessary for replication. To infect a host, the virus must interact with a host membrane and undergo membrane fusion in order to release its genome for replication. Because of the conserved nature of this process, understanding virus entry at the molecular level is critical. While virus infection and entry have been extensively examined over many decades, the molecular mechanism(s) of entry and critical membrane components remain poorly characterized for many enveloped viruses due in part to the convolution of binding and membrane fusion in many cellular based assays, the requirement for receptor identification and reconstitution, and the extreme heterogeneity of the glycocalyx surrounding the cell membrane. Model membrane systems provide a platform to examine these complex mechanistic questions surrounding virus entry. This dissertation develops model membrane platforms using single particle measurements to examine mechanistic processes of virus entry for several enveloped viruses. The Boxer lab pioneered the development of DNA-lipids as membrane tethers and recently demonstrated that DNA-lipids can be used to bind influenza to a target membrane without altering lipid mixing kinetics. This dissertation expands this receptor-agnostic platform towards novel enveloped viruses which do not require a host receptor for fusion and explores the tunability of receptor agnostic platforms. Controlled complexity is introduced into a model membrane platform in the effort to reconstruct essential components of the glycocalyx to understand its role in Influenza entry. This dissertation seeks to examine the mechanism of virus entry with the least number of confounding variables while still reconstructing critical host components. These single virus measurements deconvolve viral binding and membrane fusion to identify previously obfuscated components in each process

Since its discovery in jellyfish around the early 1960s, green fluorescent protein (GFP) and its derivatives have become the most widely used in vivo imaging tools for biological studies. Mutants of GFPs have been engineered to exhibit a palette of colors, wide range of fluorescence quantum yields (FQYs), and photoswitching characteristics, enabling advanced applications such as optogenetics and super-resolution microscopy. However, tailoring GFPs for specific purposes heavily relies on extensive screening and/or directed evolution as it is unclear how the protein environment modulates the photophysics of the chromophore in quantitative terms. Thanks to the semisynthetic split GFP method and the recently popularized technique for incorporating noncanonical amino acids (i.e., amber suppression), we created conventionally inaccessible GFP mutants with systematically altered electrostatic properties and/or steric bulk of the chromophore and the surrounding protein environment. Correlations between various photophysical properties of these mutants led to transparent models describing how the protein-chromophore interactions affect the chromophore's color and FQY. The models provide quantitative predictions for designing GFPs with desired phenotypes and infer physically imposed limitations. In addition, our finding of how electrostatics biases photoisomerization pathways in GFPs could shed light on the general phenomenon of bond-specific photoisomerization, a defining characteristic of the retinal chromophore within rhodopsins central to vision and important to the field of photoactive molecular devices

The key bottleneck in many ab initio computational chemistry calculations is the electronic structure, i.e. what are the electrons doing in response to the atomic nuclei and/or other external influences. This is particularly problematic when considering that many electronic structure calculations are needed in most workflows, such as geometry optimization or molecular dynamics trajectories. As a result, many large or accurate calculations rely on high-performance computing (HPC) systems, such as large distributed systems (e.g. supercomputers with many compute nodes) or specialized coprocessors [e.g. graphical processing units (GPUs)]. The current state of scientific software for quantum chemistry centers on monolithic packages designed to run on remote clusters with batch job submission; however, this strategy limits the speed of method development and creates a significant barrier for educating aspiring chemists and the general public. In line with recent trends of code abstraction and encapsulation in the community, I present my work on a new socket-based interface for our GPU-accelerated electronic structure package, and furthermore my development of a cloud-based framework for distributing electronic structure calculations (on either academic HPC systems or renting commercial cloud resources) utilizing this new interface. I will demonstrate how this platform was applied to several key quantum chemistry workflows that require high-throughput calculations, including dataset generation, excitation energy transfer in multichromophoric systems, and automated reaction network discovery. Additionally, I detail the advancements made in real-time ab initio interactive molecular dynamics (AI-IMD) simulations to use virtual reality (VR) headsets to provide a virtual playground for interacting with quantum chemistry for students and other non-expert users

Diagnostics and personalized therapeutics have seen a growing demand in recent years, driving the need for medical devices that respond to an individual's therapeutic needs. The production of such devices would have great implications, leading to early stage detection of viruses and diseases, improvement in the precision of drug dosing, and significantly reduced healthcare practitioner and patient burden. Towards this aim, biosensors have been developed that can respond to specific molecules in a complex biological environment. These biosensors detect specific analytes and produce a quantitative output (i.e. kinetic parameters, signal intensity), which provides precise information tailored to an individual and overcome limitations of other detection methods that are costly and require time-intensive sample preparation and processing. Continuous monitoring of analytes across a dynamic range of molecules and detection limits has led to significant advancements in the form of wearables and stretchable electronics, though adoption of implantable biosensors has been challenging. These limitations often stem from biofouling, which begins with non-specific protein adsorption in the body that accelerates the degradation of device performance that eventually renders them ineffective. A description and current state of biodevices and materials to combat biofouling is surveyed in Chapter 1. The choice of material for these biosensors is carefully curated to each application. Mechanical, chemical, and physical properties dictate how the body responds to these implanted devices and thus, affect device performance. Thus, there is pressing need for a deep understanding of these properties that leads to precision in the tuning of the materials. In Chapter 2, mechanical and chemical effects from the introduction of dangling chains in elastomeric systems are explored. Further, we develop a single-chain polymeric nano-carrier platform that offers highly controlled and precise properties (i.e. size, stability, cargo delivery), making it desirable as a drug delivery system. As these devices and platforms are translated to clinical applications, limitations from output quality, lifetime, and specificity have prevented widespread adoption and implementation of implantable biosensors. However, accompanying the rise of these devices is the introduction and development of materials that exhibit anti-biofouling properties. Biomaterials, specifically hydrogels, can be applied as coatings as they exhibit highly tunable properties that provide efficacy against fouling and the immune response, offering the potential to drive clinal translation of implanted devices to drive personalized, point-of-care diagnostics. In Chapter 3, we develop a library of acrylamide-based materials that can be used for their anti-blood biofouling properties and subject them to severe fouling conditions to screen their function and to select a top performing material. We explore the molecular features of the monomers underlying their performance and assess the mechanical properties of this material that allow it to serve as a soft materials interface with the body. In Chapter 4, we apply our top performing hydrogel to a selection of electrochemical biosensors to improve signal intensity in vitro and in vivo in whole blood conditions. In Chapter 5, translation to subcutaneous biocompatibility is explored. Through the use of insulin infusion pumps, we explore the translation of anti-biofouling materials to mitigate the foreign body response. Finally, towards future work of refining and expanding our material library, we develop a barcoded micro-particle system that can be delivered to the inter-peritoneal space for extensive material library screening

Iron enzymes are critical to the biosynthesis of neurotransmitters, natural products and antibiotics, the regulation of cellular oxygen levels and bioremediation. The α-ketoglutarate- (αKG)- and pterin-dependent enzymes are two classes of mononuclear nonheme iron enzymes that utilize a redox active 2-electron cofactor and an Fe(II) metal center to reduce molecular oxygen to form highly reactive Fe(IV)=O intermediates that catalyze their respective chemistries. Both of these enzyme classes have a 2-His/1-carboxylate facial triad motif, which leaves three cis sites available for cofactor, substrate and oxygen binding. These enzymes use a general mechanistic strategy, which ensures coordination saturation until both cofactor and substrate are bound in the active site before reacting with oxygen. This strategy is crucial as it prevents autooxidation and ensures that the oxidizing equivalents are used for substrate oxidation (i.e. coupled turnover). Additionally, this strategy is particularly important when only cofactor is bound to the active site, as all the reducing equivalents to produce the Fe(IV)=O are available. After these enzymes activate oxygen, identifying and characterizing reaction intermediates in these enzymes is critical to the elucidation of structure/function correlations that govern their reactivity. The focus of this thesis is to define the oxygen activation mechanisms of the αKG- and pterin-dependent enzymes. By leveraging site-selective spectroscopies, the structures of the different active sites and their intermediates have been correlated with their catalytic function. 1) Role of the Facial Triad Carboxylate in the αKG-dependent Enzymes FIH (Factor Inhibiting HIF [Hypoxia Inducible Factor]) is an αKG-dependent non-heme iron enzyme that catalyzes the hydroxylation of the C-terminal transactivation domain (CAD) asparagine residue in HIF-1α to regulate cellular oxygen levels. The role of the facial triad carboxylate ligand in oxygen activation and catalysis was evaluated by replacing the aspartate residue with glycine (D201G), alanine (D201A) and glutamate (D201E). Magnetic circular dichroism (MCD) spectroscopy showed that the (Fe(II))FIH variants were all 6-coordinate (6C) and the αKG plus CAD bound FIH variants were all 5-coordinate (5C), mirroring the behavior of the wild-type (wt) enzyme. When only αKG is bound, all FIH variants exhibited weaker Fe(II)-water bonds for the sixth ligand compared to wt, and αKG bound D201E was found to be 5C, demonstrating that the facial triad aspartic acid residue plays an important role in the wt enzyme in ensuring that the (Fe(II)/αKG)FIH site remains 6C. Variable temperature, variable field (VTVH) MCD spectroscopy showed that all the αKG and CAD bound FIH variants, though 5C, have different ground state geometric and electronic structures than the wt enzyme, which impact their oxygen activation rates. Comparison of oxygen consumption to substrate hydroxylation kinetics revealed uncoupling between the two half reactions in the variants. Thus, the Asp201 residue also ensures fidelity between CAD substrate binding and oxygen activation, enabling tightly coupled turnover. 2) Evaluation of a Concerted vs Sequential Mechanism for Productive Chemistry Determining the requirements for efficient oxygen activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental, reactivity. For the αKG-dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with oxygen) and a sequential mechanism (cofactor binding and reaction with oxygen precede substrate binding) have been previously proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. MCD spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are 6-coordinate when only αKG is bound to the Fe(II)), αKG binding to Fe(II)-DAOCS results in ~45% 5-coordinate sites that, from Mössbauer spectroscopy, selectively react with oxygen relative to the remaining 6-coordinate sites. However, this reaction produces an Fe(III) species that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to Fe(II)-DAOCS produces 5-coordinate sites that rapidly react with O2 to form an Fe(IV)=O intermediate that then reacts with substrate and produces cephalosporin product through an Fe(III)-OH and substrate radical species. Thus, these results demonstrate that the concerted mechanism is operative in DAOCS and, by extension, other nonheme iron enzymes. 3) Characterization of the Facial Triad Fe(IV)=O in the αKG-dependent enzymes and their Hydrogen Atom Abstraction Reactivity The αKG-dependent enzymes catalyze a diverse range of chemical reactions using a high-spin Fe(IV)=O intermediate, which performs a hydrogen atom abstraction (HAA) reaction with the substrate. Previously, the Fe(IV)=O intermediate in the halogenase SyrB2 has been structurally characterized and has a trigonal bipyramidal geometry with its substrate in a perpendicular orientation relative to the Fe-O bond. Taurine dioxygenase (TauD) is an αKG-dependent enzyme where an Fe(IV)=O intermediate was first characterized and has the taurine substrate positioned more along the Fe-O bond. By utilizing nuclear resonance vibrational spectroscopy (NRVS) with density functional theory (DFT) calculations, this study has defined the Fe(IV)=O intermediate in TauD as also having a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide in SyrB2. Using DFT generated square pyramidal, trigonal bipyramidal and six-coordinate Fe(IV)=O structures, we have evaluated the HAA reactivities of these geometries with two different substrate orientations (one orientation more along [σ-channel] and the other more perpendicular [π-channel] to the Fe-O bond). While Fe(IV)=O HAA reactivity along the σ-channel involves abstraction of a substrate α electron directly into dz2, reaction through the π-channel requires the promotion of an α electron from dπ* to dz2 before abstraction of the substrate α electron into dπ*, thus requiring additional energy to enable reactivity. Computational reaction coordinates for the three geometries and the two substrate orientations for the (TauD)Fe(IV)=O showed similar barriers for reactivity, i.e. all competent in performing HAA. The equivalence in reactivity between the two substrate orientations is due to the compensation of the promotion energy required to access the π channel by the increased polarization of the oxo-Fe π bond leading to an earlier transition state along the C-H coordinate. 4) Direct Coordination of Pterin to Iron Enables Oxygen Activation by the Pterin-dependent Hydroxylases The pterin-dependent nonheme iron enzymes hydroxylate aromatic amino acids to perform the biosynthesis of neurotransmitters to maintain proper brain function. These enzymes perform their chemistry by activating oxygen using a cofactor and substrate bound Fe(II) site to form highly reactive Fe(IV)=O species that initiate substrate oxidation. The consensus mechanism for reactivity in the pterin-dependent enzymes does not include the pterin cofactor directly interacting with the iron center, with much speculation over the undefined oxygen activation pathway. In this study, using tryptophan hydroxylase (TPH), we have generated a pre-Fe(IV)=O intermediate and characterized its structure as a Fe(II)-peroxy-pterin species using Mössbauer, resonance Raman and NRVS spectroscopies. Additionally, we have demonstrated that the pterin carbonyl is directly bound to the Fe(II) site, which is also the case in the pterin and tryptophan bound Fe(II)-TPH site before O2 reactivity. From reaction coordinate calculations, there is a 14 kcal/mol reduction in the oxygen activation barrier when the pterin carbonyl directly binds the Fe(II) site, as this interaction provides an orbital pathway for efficient electron transfer from the pterin cofactor to the iron center. This direct coordination of the pterin cofactor enables the biological function of the pterin-dependent hydroxylases and demonstrates a unified mechanism for oxygen activation by the cofactor-dependent nonheme iron enzymes