Chemistry & Biochemistry Theses and Dissertations

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5-MODIFIED LIPOPHILIC G-QUADRUPLEXES: STRUCTURAL STUDIES AND APPLICATIONS AS SCAFFOLDS FOR [2+2] PHOTOCYCLOADDITIONS
(2019) Sutyak, Keith Brandon; Davis, Jeffery T; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Guanosine self-assembly is a powerful tool for constructing nanostructures and materials with intriguing properties. Guanosine’s nucleobase has a hydrogen bond donor and acceptor face that in the presence of an appropriate templating cation can self-assemble to form larger G-quadruplex structures. Through the addition of a nonpolar solubilizing group on the 2, 3, or 5 position of guanosine’s ribose sugar, the G-quadruplex may be formed in organic solvents. Not surprisingly, any modification made to the guanine nucleobase can have a substantial effect on the self-assembly of the G-quadruplex core. These alterations can significantly alter or destroy the ability of the guanine base to form larger H-bond structures. For this reason, a major target for the synthetic modification of the G-quadruplex is the ribose sugar. Modifications to the sugar can be used to add new functionality or groups capable of modulating the G-quadruplexes stability and structure. The 5-position is easily esterified and is an ideal target for incorporating new functionality to the G-quadruplex, not not much was known about how esterification of the 5-position of guanosine impacted self-assembly. In this thesis, a series of 5-modified aryl esters of guanosine were synthesized G 21-G 25, comprising either activating or de-activating substituents on the aromatic rings, to systematically investigate how the addition of an ester to the 5- position of guanosine affects the self-assembly G-quadruplex. The identity of the 5-aryl ester was found to have a direct impact on the molecularity (how many Gs are in each assembly) and stability of the G-quadruplexes. The information gained from these experiments was applied to rationally design a new G-quadruplex capable of templating a [2+2] photocycloaddition reaction. With an increased knowledge of how modifications to the 5-position affect the structural integrity of the G-quadruplex, we applied this information to rationally design a new G-quadruplex system capable of templating a [2+2] photocycloaddition reaction. To achieve this goal a series of 5-cinnamate esters G 26-G 29 were synthesized and studied. Each derivative G 26- G 29 when self-assembled into a G-quadruplex an photoirradiated underwent a photocycloadditon reaction ins high yields with good stereoselectivity. These same compounds when disassembled only underwent trans-cis photoisomerization. Highlighting the need for the G-quadruplex self-assembly.
AB INITIO MODELING OF THE SELECTIVITY AND REACTIVITY OF BOTH THERMAL AND LIGHT MEDIATED ORGANIC AND ORGANOMETALLIC TRANSFORMATIONS
(2022) Dykstraa, Ryan Henry; Gutierrez, Osvaldo; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The mechanism of a reaction is the collection of events that take place that lead to the products of a chemical transformation. Though there are some events in a chemical reaction that can be observed by experiment, such long-lived intermediates, many of the events are too short lived to be measured. Due to these restrictions and the advancements in the development of moderately scaling computational tools, it is becoming commonplace to use quantum mechanical software packages to model the mechanism of a reaction. Here, I used quantum mechanical calculations alongside experimental evidence provided by multiple collaborators to understand the reactivity of both heat- and light-mediated organic transformations. In chapter 2, I investigated the role of electron donor-acceptor complexes in the generation of alkyl and acyl radicals in the presence of visible light. In addition, the pathways to the experimentally observed products, alkyl and acyl thioethers, were modeled. The lowest energy pathway to product, post-radical generation, was radical addition to the radical electron donor-acceptor complex. For a photoredox-catalyzed method to cyclopropanes from a novel halomethyl radical precursor (Chapter 3), computations strongly supported a redox-neutral reductive radical/polar crossover mechanism over radical pathways, consistent with experimental trends. Investigation of the isomerization of cinnamyl chloride to cyclopropane via a commonly used photoredox catalyst (Chapter 4) revealed that the reaction was mediated via dexter energy transfer between photocatalyst and substrate over the more commonly proposed electron transfer, affording diastereoselective product formation. A dual nickel/photoredox-catalyzed coupling of sulfinate salts and aryl halides gave a mixture of aryl sulfide and aryl sulfone products (Chapter 5), suggesting that disproportionation of sulfone radical was leading to the formation of thiyl radical. Modeling the product determining steps indicated that the product distribution was controlled by radical addition of the thiyl radical to the nickel(II) species versus reductive elimination of the sulfone bound to the nickel(III) catalyst. A bicyclo[1.1.1]pentane diborylated with pinacolboryl groups, one at the arm and head position, was found to have reactivity only at the bridgehead position (Chapter 6). Calculations of a hydrozone coupling reaction performed by the Qin group found that the reactivity was due to the unique hybridization of the bridgehead position as well as increased steric interactions at the arm position. Finally, a sulfoxide synthesized from a sulfinate salt could be activated with Grignard reagent, affording coupling of the substituents originally bound to the sulfoxide. DFT calculations validated the role of the sulfurane intermediate acting as a mediator to the coupled product.
Activation, Multiple-Bond Cleavage, and Fixation of Small Molecules Employing Group 6 Cyclopentadienyl, Amidinate (CpAm) Complexes
(2012) Yonke, Brendan Louis; Sita, Lawrence R; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Small molecules (e.g. N2, CO2, N2O) represent potential cheap and abundant chemical feedstocks. Despite the use of small molecules in various biosynthetic pathways, relatively few synthetic processes for the commercial utilization of small molecules exist. To investigate potential catalytic and stoichiometric methods for the activation, multiple-bond cleavage, and fixation of small molecules, Group 6 cyclopentadienyl, amidinate (CpAm) dinitrogen and multiple metal-ligand bonded complexes were investigated. Provided the weak activation of N2 in Group 6 dinitrogen complexes, these complexes were found to serve as M(II, d4) and M(IV, d2) synthons for the respective formation of Group 6 bis(carbonyl), bis(isocyanide), as well as mononuclear oxo and imido complexes. Moreover, a general route to Group 6 CpAm imido complexes of variable substitution was demonstrated upon the reaction of Group 6 CpAm dichloride complexes 48 and 49, respectively, with lithium amides, for steric amidinate deprotonation followed amido-imido tautomerization. Utilizing the synthesized mononuclear oxo and imido complexes various catalytic atom transfer reactions were demonstrated with their mechanistic details elucidated. These catalytic processes include the first ever oxygen atom transfer (OAT) reactions involving early transition metals including the synthesis of isocyanates from N2O and isocyanides, light-mediated degenerate OATs between CO2 and CO, and light-mediated non-degenerate OATs involving N2O and CO. Likewise, thermal-mediated nitrogen atom transfer (NAT) reactions were shown for the synthesis of isocyanates from organic azides (N3R) and CO. Key to this observed reactivity was the ability of multiply bonded metal ligands to undergo reaction with adjacent Lewis acidic ligands with this reactivity having been found to likewise facilitate interligand silyl group transfer in ð-loaded Group 6 CpAm oxo trimethylsilyl imido complexes. Despite direct utilization of the small molecules N2O and CO2 in OAT reactions, NAT reactions were found to require the use of organic azides. Therefore, in an effort to directly utilize N2 in NAT reactions, a novel photolytic N-N cleavage process was devised for the Group 6 dinitrogen complexes 52 and 53. This novel photolytic N-N cleavage represents the first time a ligand set has been shown to facility distinctly different N-N cleavage mechanisms for N2 bound to different metals.
Acyclic Congeners of Cucurbit[n]uril and a Related Mechanistic Study on the Cucurbit[n]uril Forming Reaction.
(2010) Ma, Da; Isaacs, Lyle D; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Supramolecular chemistry has been a very important research area in the past several decades. In this research field, molecular containers, such as cyclodextrin, attracts special attention due to their wide applications both in academia and industry. Cucurbit[n]uril (CB[n]), as a new generation molecular container, has selective and tight binding towards lots of cations and neutral molecules. A homologous family of CB[n] has been discovered including CB[5]-CB[8], CB[10], iCB[n], ns-CB[10] and ns-CB[6]. CB[n] analogues and derivatives have also been developed. CB[n] still has several issues, such as low solubility in water, difficulty to be functionalized, and slow association and dissociation kinetics. This thesis describes efforts to address these issues by developing new CB[n] type molecular containers and carrying out mechanistic investigations. Three chapters are included in this thesis. Chapter 1 is a literature review of molecular encapsulation and molecular container chemistry. We first introduced general concepts of molecular encapsulation and present examples of molecular container, such as cyclodextrin. This is followed by an introduction to CB[n] molecular containers and their supramolecular chemistry. Chapter 2 introduces new acyclic CB[n] congeners II-5a and II-5b. II-5a and II-5b are obtained from step-wise synthesis with reasonable yields. This step-wise synthetic route avoids difficult separation process. We measured the binding constants of II-5a towards a number of guests and found the binding affinity is usually comparable to CB[7]. The recognition property of II-5a is investigated in depth. We found that the length and functional groups of the guests greatly influence the binding affinity. Nevertheless, the charge and size of the guests do not have as a big influence on the binding constants as CB[7]. We discovered that the ionic strength of the buffer is critical for the binding constant. By comparing the recognition property of II-5a and II-6, it is discovered that the substituted o-xylyene walls are important for the tight binding compounds. II-5a and II-5b are new examples of CB[n] type molecular containers. They retain most of the good recognition property of CB[n] and have advantages compared to CB[n], including 1) aromatic walls that makes further functionalization possible; 2) acyclic structure that enables fast association and dissociation kinetics. Chapter 3 describes the mechanistic study of CB[n] forming reactions. Another possible way to synthesize CB[n] molecular container is to use aldehydes instead of paraformaldehyde. But neither previous researchers nor our work has succeeded to make the aldehydes participating CB[n] forming reactions happen. Mechanistic investigation was carried out to explain why this reaction simply does not occur. We used III-7 instead of glycoluril to avoid cyclization reactions. Several reasons are discovered: 1) side products are formed, such as III-SP1 and III-SP2; 2) S-shape intermediates are yielded, such as III-15S, III-16S, III-17S and III-18S, which are not able to continue the reaction to form macrocycles; 3) a small equilibrium constant for the chain grouth reaction. This study explains why aldehydes usually do not participate in CB[n] forming reactions. This work could also lead to the discovery of certain aldehydes that can form CB[n] type macrocycles.
ACYCLIC CUCURBIT[N]URIL CONGENERS: SYNTHESIS, BINDING PROPERTIES AND MEDICINAL APPLICATIONS
(2014) Zhang, Ben; Isaacs, Lyle D; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
An urgent problem for pharmaceutical industry is that the water solubility of an estimated 40-70% of the newly developed active pharmaceutical ingredients (API) are so poor that they cannot be formulated on their own. One interesting topic is to use molecular containers as the solubilizing agents. Supramolecular chemistry has always been an interesting research area and during the past decades, various new supramolecular host*guest systems have been developed. Cucurbit[n]urils (CB[n]) are very promising molecular containers as drug delivery vehicles due to their outstanding recognition properties. In order to discover the most suitable CB[n]-type containers as solubilizing agents, acyclic CB[n]-type containers have been synthesized and their recognition and formulation properties have been studied. In this thesis, three chapters have been included to investigate the possibility of using CB[n]-type containers as solubilizing agents for pharmaceutical agents. Chapter 1 gives an introduction to supramolecular chemistry and formulation techniques using molecular containers. A literature review on the synthesis, functionalization and applications of cucurbit[n]uril is given and the application of cyclodextrins and CB[n] containers in formulation techniques is discussed. Chapter 2 describes a series of acyclic CB[n]-type molecular containers (II-2a - II-2h) with different solubilizing groups bearing different charges for evaluation as potential drug solubilizing agents. The X-ray crystal structures of the negative, positive and neutral hosts (host II-2b, II-2f, and II-2h) are reported. For neutral (II-2h) and positively charged (II-2f) hosts, intramolecular H-bonds and ion-dipole interactions between the solubilizing arms and the ureidyl C=O portals are observed as well as intrahost π−π stacking interactions which results in a self-filling of the cavity. 1H NMR and UV/Vis spectroscopy are used to measure the Ka values of hosts II-2a, II-2h, and II-2f toward guests with different charge and significant decrease is noted in binding affinities of the neutral (II-2h) and positive (II-2f). The pKa of 7H+ alone and in the presence of differently charged hosts II-2a, II-2h, and II-2f are measured and the II-2a induces the largest pKa shift. The poor recognition properties of hosts II-2h and II-2f are reflected in their phase-solubility diagrams with insoluble drugs (tamoxifen, 17-α-ethynylestradiol, and indomethacin). In all cases, the anionic host II-2a functions more efficiently as a solubilizing agent than either neutral II-2h, or cationic host II-2f. In chapter 3, we compare the ability of III-1a - III-1e to solubilize insoluble drugs relative to HP-β-CD. Phase solubility diagrams are created for mixtures of containers III-1a - III-1e and HP- β-CD with 19 drugs. We find that the solubilizing ability of the best container (III-1a - III-1e) is superior to HP-β-CD in all cases. A notable achievement is the solubilization of the developmental anticancer agent PBS-1086. The acyclic CB[n]-type containers display an affinity for the steroid ring system, aromatic moieties of insoluble drugs, and cationic ammonium groups. Compound III-1b is generally the most potent (Ka up to and exceeding 106 M-1) container whereas both III-1a and III-1b display excellent solubility enhancement toward a broad range of insoluble drugs. The broad scope of insoluble drugs that can be formulated with III-1a and III-1b - in many cases where HP- β-CD fails completely - makes acyclic CB[n]-type containers particularly attractive alternatives to cyclodextrins as solubilizing excipients for practical applications.
ADSORPTION AND MECHANISTIC STUDIES OF DIMETHYL METHYLPHOSPHONATE FOR CWA DEFEAT
(2018) Huynh, Kim Lien; Eichhorn, Bryan; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
This thesis work systematically investigated carefully designed ASZM- TEDA deconstruction–reconstruction experiments. Our objective is to understand the role of each impregnant, the factors that influence filter performance, and filter lifetime. Chemical warfare agent simulant, dimethyl methylphosphonate (DMMP), is used to evaluate the adsorption properties and reactivity of different materials using a temperature programmed desorption (TPD)/quantitative 1H-NMR method, combined with inductively coupled plasma-atomic emission spectroscopy (ICP- AES) bulk analysis. Our investigation begins with a full analysis of the current commercial gas mask filter, ASZM-TEDA. In Chapter 2, studies with ordered mesoporous carbons (OMCs) and a disordered microporous carbon (BPL) reveal that surface area and pore volume dictate total adsorption loading. It was also discovered that an ordered pore network would lead to higher DMMP desorption energies by 30–40%, confirming stronger binding to the carbon surface in pores ≤2.4 nm. In Chapter 3, our investigation of an ASZM-TEDA deconstructed analog, carbon/CuO, further our understanding of CuO functionality within the adsorbent. Our study reveals that CuO impregnation increases adsorption capacity (up to 64%) in comparison to native carbon adsorbents. The CuO surface activity (reported as DMMP area capacity) was over 3.5 times higher in comparison to the OMCs (0.075–0.078 m2 g-1), and 17 times higher than BPL (0.016 m2 g-1). In addition, the extent of DMMP decomposition is greater for nano sized endo-pore CuO relative to larger exo-pore CuO particles. In Chapter 4, ordered mesoporous metal oxides (OMMs) are described with more reactive sites and designed to presumably have better mass transfer into the pores in comparison to impregnated carbons. DMMP chemisorption to the metal oxide reactive sites allow for greater decomposition (reported as the decomposed DMMP capacity), which is 2 to 4 times higher for the OMMs in comparison to OMC. TPD measurements for meso-Al2O3 and meso-Fe2O3 suggest that DMMP conversion to methanol is roughly one-to-one, whereas meso-TiO2 undergoes greater decomposition with the cleavage of a second methoxy group. It was also discovered that meso-Al2O3 achieved the highest DMMP total volume capacity in comparison to the other materials, making it the most efficient adsorbent studied for CWA defeat.
Advanced Receptor Models for Exploiting Highly Time Resolved Data Acquired in the EPA Supersite Project
(2012) Ke, Haohao; Ondov, John; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Receptor models have been widely used in air quality studies to identify pollution sources and estimate their contributions. A common problem for most current receptor models is insufficient consideration of realistic constraints such as can be obtained from emission inventories, chemical composition profiles of the sources, and the physics of plume dispersion. In addition, poor resolving of collinear sources was often found. With the high quality time-, composition-, and size-resolved measurements during the EPA Supersite project, efforts towards resolving nearby industrial sources were made by combinative use of Positive Matrix Factorization (PMF) and the Pseudo-Deterministic Receptor Model (PDRM). The PMF modeling of Baltimore data in September 2001 revealed coal-fired and oil-fired power plants (CFPP and OFPP, respectively) with significant cross contamination, as indicated by the high Se/Ni ratio in the OFPP profile. Nevertheless, the PMF results provided a good estimate of background and the PMF-constrained emission rates well seeded the trajectory-driven PDRM modeling. Using NOx as the tracer gas for χ/Q tuning, ultimately resolved emissions from individual stacks exhibited acceptable tracer ratios and the emission rates of metals generally agreed with the TRI estimates. This approach was later applied to two metal pollution episodes in St. Louis during in November 2001 and March 2002 and met a similar success. As NOx measurements were unavailable at those metal-production facilities, highly-specific tracer metals (i.e., Cd, Zn, and Cu) for the corresponding units were used to tune χ/Qs and their contributions were well resolved with the PMF-seeded PDRM. Opportunistically a PM2.5 excursion during a windless morning in November 2002 allowed the extraction of an in-situ profile of vehicular emissions in Baltimore. The profiles obtained by direct peak observation, windless model linear regression (WMA), PMF, and UNMIX were comparable and the WMA profile showed the best predictions for non-traffic tracers. Besides, an approach to evaluate vehicular emission factors was developed by receptor measurements under windless conditions. Using SVOC tracers, seasonal variations of traffic and other sources including coal burning, heating, biomass burning, and vegetation were investigated by PMF and in particular the November traffic profile was consistent with the WMA profile obtained earlier.
ADVANCES IN SILOXANE-BASED COUPLING TECHNOLOGIES: APPROACHES TOWARD PANCRATISTATIN AND STREPTONIGRIN
(2014) Nytko, III, Frederick Emil; DeShong, Philip; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The ability to form carbon-carbon bonds, arguably the most important transformation in synthetic chemistry, has been critically facilitated via the implementation of transition metal catalysts with main group element-associated carbon moieties. Specifically, organosilane coupling technology previously reported in the DeShong group provides ease of access to a wide variety of structurally important carbon-carbon bond motifs. The stability, tolerance of numerous implicit functional groups, simplicity of use, and ease of synthetic access to a multitude of organosilane coupling partners, makes the coupling technology developed in the DeShong lab markedly attractive for implementation in syntheses of complex natural product targets. Two targets of specific interest are pancratistatin and streptonigrin. Synthetic approaches toward pancratistatin via complex organosilane coupling precursors proved promising, however mechanistic studies performed in the DeShong group determined that standard 18-electron palladium(0) catalysts fail in transmetallation. Therefore, a new class of 16-electron Pd(0) catalysts have been developed and surveyed for applications in siloxane based allyl-aryl coupling protocols. The ability to "tune" these catalysts' activity by varying either the cone angle or the electronic characteristics of the alkene ligands attached to palladium has also been demonstrated. Unfortunately, attempts to prepare chiral adducts in the coupling reaction utilizing chiral bicyclooctadiene derivatives as a ligand for palladium provided no significant enantioenrichment in the coupled product. Similarly, previous work in the DeShong lab toward the synthesis of streptonigrin has been reported. Particularly, the synthesis of the structurally congested pyridyl C-ring proved difficult, requiring numerous steps at low yields. Development of new synthetic pathways toward the pyridyl C-ring was undertaken, exploiting the electronically withdrawn nature of the pyridone intermediate in order to brominate and alcohol, as well as change a methyl group to an aldehyde, via an enamine intermediate. The specific goals of this work were (1) to investigate new palladium(0) catalysts for the coupling of analogues of pancratistatin precursors, and (2) to improve upon problematic portions of our previous synthesis of streptonigrin's pyridyl C-ring.
AEROBIC OXIDATION OF CIS-2-BUTENE AND 2-BUTYNE IN WATER MEDIATED BY (DPMS)PtII (DPMS = DI(2-PYRIDYL) METHANE SULFONATE)
(2010) Khanthapura, Pratheep; Vedernikov, Andrei N; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
(dpms)PtII hydroxo ethylene complex (2.1) undergoes ligand substitution--intramolecular hydroxo platination with 2-butyne in water to give PtII η1-ketonyl complex (2.2). This neutral complex is oxidized by O₂ or H₂O₂ to give PtIV secondary alkyl complex (2.4). Oxidation by O₂ was found to be pH dependent. Also described here is the preparation of PtIV oxetane (3.2) derived from non-cyclic non-strained olefin, cis-2-butene. The starting complex here is the (dpms)PtII chloro cis-2-butene (3.1) which undergoes chloride abstraction by Ag₂O to give the hydroxo analog before getting oxidized by O_{2<1sub> to PtIV oxetane.}
Airborne Characterization of Regional Aerosol Origins and Optical Properties
(2004-08-31) Taubman, Brett; Dickerson, Russell R; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The ubiquity of air pollution in the Mid-Atlantic and northeastern U.S. is a major concern for human and ecological health as well as the climate. Between February 2001 and February 2004, 160 flights on a light aircraft outfitted for atmospheric research were conducted over the region to characterize the factors that lead to severe air pollution episodes and how the pollutants impact the radiation budget. One salient discovery was that the chemistry and physics of multi-day haze and ozone episodes over the Mid-Atlantic and northeastern U.S. may be accurately represented by a two-reservoir system composed of the planetary boundary layer (PBL) and the lower free troposphere (LFT). Primary pollutants are typically emitted in the PBL, where they are subject to greater humidification effects and surface deposition. Pollutants in the LFT are not subject to direct deposition and photochemical processes are accelerated, which appears to play a larger role in particle growth processes than relative humidity. Measurements of a smoke plume from Canadian forest fires showed that the plume was separated from the underlying layer by a morning subsidence inversion. Absorption of solar radiation within the optically thick plume nearly equaled the total amount attenuated at the surface, creating a feedback loop that sustained the vertical stability and protracted the lifetime of the plume. Satellite reflectances were used to calculate the optical depth of the smoke plume with two sets of inputs; AERONET retrieved optical properties and optical properties measured aboard the aircraft. The optical depths calculated using the AERONET optical properties were the lowest, while retrievals using the in-situ values were 22-43% larger, due to greater absorption measured in-situ. Radiative forcings calculated with the in-situ optical properties matched surface and TOA measurements more closely than those calculated with the AERONET retrievals. Measurements made downwind of power plants during the North American electrical blackout showed reductions in SO2 (>90%), O3 (~50%), and light scattered by particles (~70%). These observations indicate the central role power plants play in regional air quality. Finally, statistical analyses of all flights point to the dominance of photochemical and meteorological processes in determining aerosol optical properties.
ALKYNYL AMINOISOQUINOLINES, NOVEL FLT3 KINASE INHIBITORS WITH POTENT ACTIVITY AGAINST ACUTE MYELOID LEUKEMIA
(2018) Ma, Xiaochu; Sintim, Herman O; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Acute myeloid leukemia (AML) is a type of blood cancer. If not properly treated, this subclass of leukemia can progress quickly, and be fatal after a few months. Aberrantly expressed FMS (Feline McDonough Sarcoma)-like tyrosine kinase (FLT3) is observed in approximately 30% of AML patients, so it is a promising molecular target for AML. Midostaurin (commercial name, Rydapt) was the first approved FDA drug for AML that targets FLT3. However, Midostaurin, is not a single agent drug and is only effective when used in combination with other cytotoxic drugs. Therefore, there is a need for FLT3 inhibitors that are effective as a single agent. In Chapter 2, the synthesis of a series of amidine-acetylene-isoquinoline-3-amines is described. Amidine-acetylene-isoquinoline-3-amine compounds have the potential to serve new FLT3 inhibitors with inhibition IC50 values in the nanomolar region against wild type FLT3, FLT3-ITD (internal tandem duplications) and FLT3-D835Y (a tyrosine kinase domain mutant, commonly found in AML patients). Amidine drugs are generally not orally bioavailable, the synthesis of amide-acetylene-isoquinoline-3-amines, our second generation FLT3 inhibitors are described in Chapters 3 and 4. The amide-acetylene-isoquinolines inhibit FLT3-driven AML cell lines with single digit nanomolar IC50 values and are either comparable to or better than most FLT3 inhibitors reported to date.
Allostery and GroEL: Exploring the Tenets of Nested Cooperativity
(2004-06-24) Gresham, Jennifer Suzanne; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Despite a wealth of structural and biochemical studies on the functional cycle of the <i>E. coli</i> chaperonins GroEL and GroES, no model proposed to date accounts for all the effects seen experimentally by the various allosteric ligands: ATP, ADP, SP, GroES, and K+. The work in this dissertation explores the various allosteric transitions in the GroEL reaction cycle and offers a refined model for nested cooperativity that successfully accounts for the effects of these ligands. Initial studies take advantage of a single ring variant, termed SR1, to examine the allosteric properties of GroEL in the absence of complicating interactions arising from negative cooperativity. Initial rates of ATP hydrolysis by GroEL and SR1 as a function of ATP concentration were fit to an equation that makes no arbitrary assumptions. A novel role for K+ and SP is proposed, which suggests they help regulate the negative cooperativity and control the timing of the chaperonin cycle. The kinetics of association of GroES to the trans ring of the asymmetric complex were also studied, using stopped flow fluorescence energy transfer (FRET), revealing that conditions which accelerate dissociation of the cis ligands also accelerate association to the trans ring. This, along with previous work obtained by our lab, suggests that the allosteric signal transmitted between the rings for cis ligand release is the binding of ATP to the T state of the trans ring. A mechanism for the formation of symmetrical particles, termed "footballs," is suggested.
Allostery in GroEL: Its Role in the Refolding of Protein Substrates
(2003-12-01) Grason, John Peter; Lorimer, George H; Biochemistry
The Escherichia coli chaperonin GroEL assists in the re-folding of misfolded substrate proteins (SPs). In response to the binding of ATP, GroEL undergoes large, allosteric structural transitions, resulting in an expansion of its central cavity and a capping of the cavity by the co-chaperonin GroES. Bound SP is released into the central cavity following the structural transitions. The exact mechanism by which GroEL assists in the re-folding of SPs is unknown, though there is evidence that GroEL has the ability to forcefully unfold bound SPs, giving them another chance to fold to the native state. The studies in this dissertation concentrate on relating the allosteric domain movements of GroEL to the unfolding of SPs: 1) As a means of controlling the domain movements, an intersubunit salt bridge was replaced with a pair of cysteine residues, allowing for the controlled introduction of cross-links that could tether the GroEL rings in their closed conformation. 2) The possible allosteric basis of SP's ability to stimulate the ATPase activity of GroEL was explored using standard kinetic assays. 3) The kinetics of GroES release from the GroEL/GroES complex in response to ATP binding were studied using stopped-flow fluorescence measurements, with an emphasis on determining why SP binding accelerates the rate of release. From these studies, it was concluded that the subunits within a GroEL ring move in a single concerted motion, maximizing the potential unfolding force exerted by GroEL against bound SP. It was also found that SP stimulates ATPase activity by binding to and holding a ring in the more active, closed conformation. To do this, SP must exert a force on the ring, and in order to undergo its structural changes, GroEL must in turn perform work on the SP. GroES release is stimulated for a similar reason. Since unfolded SP increases the number of reaction cycles and decreases the amount of time SP spends encapsulated in the central cavity, it is proposed that a GroEL-assisted refolding mechanism that includes an active unfolding event makes the most sense in physiological terms.
Alterations in the Primary Structures of Ribosomal Proteins in Acquired Drug Resistance
(2012) Lohnes, Karen Lynn; Fenselau, Catherine C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Acquired drug resistance is a multifactorial process that is one of the major causes for cancer treatment failure. The anticancer drug, mitoxantrone, was recently determined to inhibit ribosome biogenesis. Changes in ribosomal protein composition and efficiency with which the ribosomes incorporate 35S-methionine has been noted in a mitoxantrone resistant MCF7 cell line when compared with a drug-susceptible parental cell line. This dissertation evaluated three proteomic workflows in order to successfully characterize the changes in the primary structures of cytoplasmic ribosomal proteins isolated from a mitoxantrone resistant breast cancer cell line that could serve some functional significance to the resistance when compared with a parental drug-susceptible cell line. A combination of the data from the three workflows allowed for the identification of 76 of the 79 human ribosomal proteins with an average sequence coverage of 76%. The N-terminal ends of 52 of the ribosomal proteins were identified using bottom-up and middle-down mass spectrometric approaches. An additional 7 N-terminal fragments were identified by top-down high resolution mass spectrometric analysis. Forty of the 52 N-terminal peptides were observed to have lost their N-terminal methionine and 19 were acetylated. Identification of the N-terminal peptides was most successful using the middle-down approach. Internal acetylations (on lysine) and phosphorylations were only noted with trypsin in-gel digestion and HPLC fraction analysis. Gel arrays of the two ribosomal populations illustrated differences in the protein compositions. Comparative densitometry imaging software indicated the presence of two novel protein spots in the drug resistant cell line as well six additional spots with increased and decreased abundances. High coverage bottom-up mass spectrometric analysis allowed for these protein spots to be assigned as isoform pairs of RPS3, RPS10, RPL11 and RPL23A. Molecular masses and top-down analyses were used to define the alterations in the ribosomal proteins in conjunction with high coverage bottom up and middle-down analyses. The change in the primary structures of these four ribosomal proteins is believed to alter access to the mRNA tunnel in the ribosome. This suggests that these ribosomes may participate in differential selective translation to allow for the cell to produce the necessary proteins during cellular stress.
Amidinate Based Catalysts for the Stereospecific and Living Ziegler-Natta Polymerization of Alpha-Olefins
(2003-12-04) Keaton, Richard J; Sita, Lawrence R; Chemistry
The living Ziegler-Natta polymerization of alpha-olefins has been accomplished with a series of precatalysts based on cyclopentadienyl and pentamethylcyclopentadienyl zirconium amidinates (Cp and Cp*ZA's, respectively) upon activation by a borate cocatalyst at 10 °C in chlorobenzene. For the latter, the symmetry of the precatalyst determines the polymer microstructure: C(1)-symmetry gives isotactic polymer, while C(s)-symmetry gives nearly atactic material. The living behavior has been proven through kinetic analyses, narrow molecular weight distribution polymers, formation of telechelic polymers, and synthesis of well defined block copolymers. Aside from simple straight chain alpha-olefins, non-conjugated dienes and vinylcyclohexane have also been polymerized in a living fashion with this series of precatalysts. Characterization of several catalytically active derivatives through solution NMR studies and single crystal X-ray analyses were successful. In the solid state, the initiator appears as a Zr-Me cation that is involved in a doubly methyl bridged dimeric structure. The presence of ether is sufficient to break up the dimer affording a monomeric species. Also, substantially increasing the steric hindrance of the amidinate ligand yields a monomeric structure. The Zr-Me cations undergo rapid methyl group exchange as evidenced through a crossover experiment between C(1)- and C(s)-symmetric initiators. Similarly, the methyl cations can engage in methyl-polymer group exchange, thus providing a new method toward stereoblock copolymer production. Insertion of cyclopentene is also successful into the Zr-Me bond, though further propagation steps do not occur. The product of initiation is the previously unobserved cis-1,2-product, which upon warming quantitatively isomerizes to the cis-1,3-product. The former features a beta-hydrogen agostic interaction with a low J value of 87.7 Hz. Upon isomerization, two beta-agostic hydrogens are present, with J values of 97.5 and 107.2 Hz. Oligomeric polymers were prepared from low ratios of monomer to initiator. Extensive NMR studies showed a 9 : 1 selectivity for the enantiofacial selectivity of the initiation step and perfect stereospecificity thereafter. Quenching the polymerization after extremely long times, or performing the polymerization at higher temperatures, afforded evidence that beta-hydride elimination was a rare, yet active, path, and that chain-walking occurs along the alkyl chain of the last inserted monomer unit.
Analysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalis
(2017) Gebbie, Margo Page; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In this project, we studied the genetic regulatory mechanisms that affect utilization of ethanolamine, an abundant compound in the gastrointestinal environment. In Enterococcus faecalis, the ethanolamine utilization (eut) gene cluster encodes for a two-component regulatory system (TCS), comprised of a histidine kinase, EutW, which autophosphorylates upon sensing EA, and a cognate response regulator, EutV, which dimerizes upon receiving the phosphoryl group from EutW and binds the nascent transcript to prevent premature transcription termination. This TCS is responsible for coupling sensing of ethanolamine to production of eut transcripts. However, clues from other organisms had previously suggested that adenosylcobalamin (AdoCbl) might also be an important genetic regulatory signal for the E. faecalis eut genes. Indeed, we discovered a novel trans-acting noncoding RNA (EutX) that contained an AdoCbl-responsive riboswitch. Our data demonstrated that the riboswitch promotes a shortened form of EutX when cellular AdoCbl levels are replete. In contrast, a longer form is synthesized when AdoCbl levels are depleted. We demonstrated that structural motifs contained in the longer form of EutX act to sequester the EutV protein, preventing it from promoting transcription elongation of eut transcripts. These unexpected data revealed an important new type of regulatory mechanism for riboswitch RNAs. In support of this overall genetic regulatory model, we recapitulated the full genetic circuitry in a heterologous host. Using this system, we employed extensive site-directed mutagenesis to examine the functional importance of highly conserved EutV residues. This led to the identification of a cluster of positively charged residues, which we speculated are important determinants for RNA-binding activity. Consistent with this hypothesis, mutations of these residues resulted in loss of RNA-binding activity. Furthermore, we also explored whether the eut gene cluster was affected by additional genetic regulatory mechanisms. From these efforts, we concluded that oxygen is not a genetic regulatory feature of eut genes, in contrast to previously published speculation. However, we did find that it is likely to be repressed under conditions of high glucose. Therefore, these aggregate studies revealed new mechanisms of post-initiation genetic regulation, and showed how E. faecalis specifically controls expression of ethanolamine catabolism genes.
Analysis of Intact Proteins in Complex Mixtures
(2013) Dhabaria, Avantika; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Our goal is to develop an effective work flow for analysis of intact proteins in a complex mixture using the LC-LTQ-Orbitrap XL. Intact protein analysis makes the entire sequence available for characterization, which allows for the identification of isoforms and post translational modifications. We focus on developing a method for top-down proteomics using a high-resolution, high mass accuracy analyzer coupled with bioinformatics tools. The complex mixtures are fractionated using 1-dimensional reversed-phase chromatography and basic reversed- phase, and open tubular electrophoresis. The analysis of intact proteins requires various fragmentation methods such as collisional induced dissociation, high energy collisional dissociation, and electron transfer dissociation. This overall method enables us to analyze intact proteins, providing a better understanding of protein expression levels and post transitional modification information. We have used standard proteins to optimize HPLC conditions and to compare three methods for ion activation and dissociation. Furthermore, we have extended the method to analyze low mass proteins in MCF7 cytosol and in E. coli lysate as a model complex mixture. We have applied this strategy to identify and characterize proteins from extracellular vesicles (EVs) shed by murine myeloid-derived suppressor cells (MDSC). MDSCs suppress both innate and adaptive immune responses to tumor growth and prevent effective immunotherapy. Recently some of the intercellular immunomodulatory effects of MDSC have been shown to be propagated by EVs. Top-down analysis of intact proteins from these EVs was undertaken to identify low mass protein cargo, and to characterize post-translational modifications.
An Analysis of the Stability, Aggregation Propensity, and Negative Cooperativity of the Escherichia coli Chaperonin GroEL
(2013) Wehri, Sarah; Lorimer, George H; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Since the discovery of chaperonin GroEL and co-chaperonin GroES, there has been a deluge of literature investigating many aspects of the system. Substrate proteins are protected from aggregation through a cycle of capture, encapsulation, and release made possible through rigid body motions of the GroE system driven by a combination of allosteric controls influenced by nucleotide, potassium and denatured protein termed substrate protein (SP). This dissertation first explores the sequential transition of GroEL that maintains the rings operating in an alternating fashion. To do this, an intra-subunit, inter-domain mutant, GroEL_{D83AT state. Steady state ATPase assays, stopped-flow fluorescence, and gel filtration chromatography were all used to demonstrate that the trans ring must access the T state before ligands can be discharged from the cis ring.

The dual-heptameric ring structure of GroEL and the post-translational assembly of the protein make creating mutants with a mutation within a single subunit of a ring almost impossible, however the ability to do so opens the opportunity for a myriad of experiments that explore the allosteric transitions of GroEL. Two potential recombination methods, acetone treatment and heat treatment, were investigated. Förster resonance energy transfer (FRET) and electrospray ionization mass spectrometry (ESI-MS) were used to study recombination facilitated by such treatments. Recombination using the acetone method resulted in a one-in-one-out subunit exchange, however aggregation complicated the exchange. Heat treatment resulted in exchange of rings.

Finally, dynamic light scattering (DLS) was used to investigate stability and aggregation on the chaperonin. It was observed that the chaperonin is stable for over 30 days while incubated continuously at 37°C in sterile buffered solution, however interesting aggregation kinetics are observed upon addition of acetone, the solvent used to strip SP from GroEL during the standard lab purification procedure. GroEL partitions into 10nm and 100nm species that are extremely stable before the appearance of macromolecular aggregates and precipitation is observed.}
Analyzing Dynamical Processes with Local Molecular Field Theory
(2023) Zhao, Renjie; Weeks, John D; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Local molecular field (LMF) theory provides a framework for describing the collective response of a system to long-range interactions in nonuniform liquids. Based on this theory, different roles played by the short and long-range components of the intermolecular interactions can be disentangled in determining relevant structural and thermodynamic properties in equilibrium. Furthermore, in dynamical processes, nonlocal long-range interactions are often associated with long relaxation times, and can contribute significantly to the stability of the system in different phases. In this thesis, LMF theory is utilized to quantify and analyze the dynamical effects arising from long-range Coulomb interactions in aqueous solutions, while elucidating how they are connected to strong local forces and fluctuations. The first half of the work concerns ionic and dipolar solvation dynamics, which plays an essential role in many solution phase chemical reactions. The physical models of Gaussian-smoothed charge and dipole distributions are conceptualized from LMF theory to investigate the molecular origins of linear and nonlinear effects in solvation dynamics. The long-range component of the solute-solvent electrostatic interaction is shown to underlie the linear response behavior of the system, while the short-range interactions introduce additional nonlinear effects. The LMF-based solvation models further demonstrate their functionality in probing the intrinsic dielectric dispersion of solvent water. The second half of the work is focused on the nucleation processes in the aqueous environment. Simulating crystal nucleation from solutions requires efficient treatments for intermolecular interactions to drive the transitions on time scales affordable to molecular dynamics simulations. For this purpose, a LMF-based molecular model is employed to capture the renormalized long-range interactions, and well-tempered metadynamics is adopted to enhance the fluctuations arising from short-range interactions. By comparing to a short-range reference model, the necessity of long-range interactions in explaining metastability is revealed. Temporal fluctuations and direct evidence for the two-step nucleation mechanism are observed through the analysis using a deep learning-based approach. The results about these two types of dynamical processes contribute to a deeper understanding of the roles of short and long-ranges interactions in the aqueous systems.
AN APPARATUS FOR LIGHT-LESS ARTIFICIAL GAUGE FIELDS AND NEW IMAGING TECHNIQUES
(2015) Perry, Abigail Reiko; Spielman, Ian B; Rolston, Steve; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The thesis presented has three components: experiments with artificial vector potentials, a new atom-chip apparatus designed and built for light-less fictitious gauge fields, and an imaging experiment. First, we introduce experiments with light-induced vector potentials using two-photon Raman coupling to simulate charged particles using charge neutral Bose-Einstein condensates (BECs). Depending on the spatial and temporal properties of the engineered vector potential, it is possible for ultracold atoms to experience different variants of an effective Lorenz force such as; magnetic fields, electric fields, and spin-orbit coupling, via coupling between an atom's internal spin and its linear momentum. In this context, we discuss the main focus of this thesis, the design and construction of an atom-chip apparatus for $^{87}$Rb BECs for experiments with light-less artificial gauge fields. Eliminating the source of heating due to spontaneous emission will open new paths to explore artificial gauge fields in alkali fermions and will be a step towards the realization of simulated topological insulators using ultracold atoms. Finally, we will describe in detail an imaging experiment performed on this new apparatus, the reconstruction of the two-dimensional column density of a BEC using multiple defocused images taken simultaneously.

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