1,721,031 research outputs found
Mapping the Franck-Condon Active Modes of Flavin
Get PDFFlavins are a class of organic molecules which share a tricyclic isoalloxazine ring as part of their structure. Several flavins are biologically important, including riboflavin (RF, also known as vitamin B2), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). FMN and FAD, in particular, are notoriously cofactors in a large group of proteins known as flavoproteins. Enzymologists and biochemists frequently utilize spectroscopic tools such as ultraviolet-visible (UV-vis) spectroscopy to study the reactivity and kinetics of these enzymes. Therefore, it is useful to understand the origin of spectral features of flavins and how they are affected by the protein environment. The shape and broadening of the UV-vis bands are attributed to a combination of electronic and vibrational excitations, which can be understood through the Franck-Condon (FC) principle. The FC principle states that since electronic transitions occur on a much faster timescale than nuclear ones, excitation is essentially a “vertical” process that occurs without changes in the positions of the nuclei. This implies that the vertical excitation energy should match well with the wavelength at which flavin absorbs most strongly (λmax). However, this approximation does not appear to work well in flavins. In this study, we employed electronic structure calculations on lumiflavin, a smaller molecule sharing the same tricyclic isoalloxazine structure and UV-vis spectrum as RF, FMN, and FAD, to understand its electronic and vibrational energies. The inspection of the ground and excited state energies for lumiflavin along its vibration modes was used to determine which modes are FC active and to shed light on why flavins do not follow the vertical transition approximation. The calculations provide insights into the spectroscopic behavior of the broader class of flavin molecules and provides a foundation for explaining the unique photophysical properties of flavins.Master of Science (MS)Chemistr
Assessing the Accuracy of Electronic Structure Methods for the Description of UV-Visible Light Absorption Intensity by Organic Molecules in Solution
Get PDFUV-visible absorption spectroscopy probes the electronic structure of molecules in their ground and excited states. Numerous studies have examined electronic structure methods' ability to reproduce molecular light-absorption energies. Comparatively few studies have addressed the intensities (probabilities) of the predicted transitions. This is the topic of this dissertation.
We looked at the oscillator strengths (OSs or f-values) predicted by four wavefunction methods (CIS, TD-HF, EOM-CCSD, and LR-CCSD) and the time-dependent (TD) implementation of nineteen DFT functionals. These included eight pure, eight hybrid, and three long-range-corrected hybrid functionals. We also tested the Tamm--Dancoff approximation (TDA) for nine of the functionals. For all methods, transition dipole moments were obtained in the length, velocity, and mixed gauges. Different basis sets (Pople, Dunning, and Jensen's) were tested as well.
We compared the computations to 85 OSs derived from the experimental absorption spectra of 69 small organic molecules in solution. Most transitions in this comparison set come from conjugated molecules and have character. In the comparison, we account for the effect of the solvent on
i) the molecular wavefunctions, by using different polarizable continuum model (PCM) implementations, ii) on the electromagnetic field driving the absorption, by modeling the cavity field acting on the molecules, and iii) on the light's energy flux, by multiplying experimental OSs (f_{exp}) times the refractive index of the solvent n.
OSs computed (f_{comp}) using the TDA, CIS, or EOM-CCSD exhibit a strong gauge dependence, which is diminished in linear response theories (TD-DFT, TD-HF, and to a smaller degree LR-CCSD). Overall, for TDA and the wavefunction methods, the length gauge reproduces experiments better. LR-CCSD, EOM-CCSD, and TD-DFT f_{comp} values have ~10--20% MAEs relative to n f_{exp}. CIS and TD-HF present significantly larger errors. The TDA results in roughly twice the error of full TD for nine functionals tested. LR-CCSD and pure functionals' f_{comp} values present a 1:1 ratio with n f_{exp} while hybrid functionals systematically overestimate experimental OSs by a magnitude that increases with the % of HF exchange included in the functional.Ph
Quantum Chemical and QM/MM Calculations on Flavin and Flavin-Binding Fluorescent Proteins
Get PDFFlavin is the cofactor for the large and diverse family of proteins called flavoproteins. Upon absorption of blue light, the flavin cofactor may undergo one of several potential photophysical processes, depending on the surrounding environment. Possible photophysical processes include fluorescence from the first singlet excited state (S1), intersystem crossing to a long-lived triplet state (T1), or photoreduction to form a radical species (D1). Each of those processes has been exploited for applications such as biosensing, bioimaging, FRET process, optogenetics, and singlet oxygen generation. However, the dependence of flavin’s photophysics (mechanism and kinetics) on its protein environment is not well understood. As a step towards developing this understanding, we simulated UV-vis and FT-IR spectra of flavin in a different polar environment to understand the effect of hydrogen bonding interaction on absorption and fluorescence energies and the vibrational frequencies of flavin. We also generated electrostatic spectral tuning maps (ESTMs) for each of flavin’s redox and protonation states to see how electrostatics influence their energetics. We also developed an automated quantum mechanical/molecular mechanical (QM/MM) protocol for simulating flavoproteins. With the guide of ESTMs and the QM/MM calculations, we focus on studying the spectral tuning of iLOV, an engineered flavin-binding fluorescent protein. Specifically, we suggest a novel mutant iLOV-Q430E that provides a red-shifted absorption and fluorescence maximum wavelength, which has been experimentally verified.Doctor of Philosophy (PhD)Chemistr
Separation of Short and Medium-Chain Fatty Acids Using Capillary Electrophoresis with Indirect Photometric Detection
Get PDFShort and medium-chain fatty acids (SMCFA) are known as essential metabolites found in gut microbiota that function as modulators in the development and progression of many inflammatory conditions as well as in the regulation of cell metabolism. Currently, there are few simple and low-cost analytical methods available for the determination of SMCFA. This work focuses on the identification and measurement of SMCFA in rat feces utilizing capillary electrophoresis with indirect photometric detection (CE-IPD). In chapter 2, several parameters are optimized for maximum resolution, efficiency, and signal-to-noise ratio of FAs. The developed CE-IPD method is next validated in chapter 3 to quantify the FAs in healthy rat feces. Application to a pilot study on the influences of developmental stages (adult vs. adolescent) and various drug treatments on fecal SMCFA concentration in rats demonstrates the proposed CE-IPD method is an efficient tool for investigations on biological functions of SMCFA in clinical laboratories.Master of Science (MS)Chemistr
Ultrabright Fluorine Containing Squaraine Fluorophores Used for Biological Imaging
Get PDFThis thesis outlines the relevance of fluorinated compounds in organic chemistry, more specifically in different scaffolds of fluorophores and the applications reported as well as highlighting the potential for different applications of the selected compounds in the first chapter. The second chapter describes the synthesis, characterization, optical, and physicochemical properties of a series of fluorinated squaraine dyes and highlights their potential in biological applications. The chemical structure of synthesized compounds was characterized using 1H and 19F nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Biological distribution studies and further imaging studies were conducted following the full characterization of compounds.Master of Science (MS)Chemistr
Prodrug Strategies for Drug Delivery: from Carbon Monoxide to PROTACs
No full textThis dissertation studies the applications of prodrug strategies in drug delivery. The prodrugs under study are compounds that can deliver carbon monoxide or PROTACs under physiological conditions.
Chapter 1 describes a new structural scaffold of CO prodrugs through the use of an adamantane moiety to afford stabilization of a critical precursor structure, cyclopentadienone, and to allow for optimization of water solubility. The prodrugs are tested in a cell culture model to confirm CO release from such prodrugs and demonstrate their efficacy in anti-inflammation studies.
Chapter 2 reviews the field of using click chemistry in the development of PROTACs, which are hetero-bifunctional molecules designed to mediate the disposal of a target protein via recruitment of the ubiquitination-proteasome degradation machinery. Click chemistry has unique advantages for tethering two or more molecular entities of choice under near-physiological conditions and therefore has been applied to the development of PROTACs in various ways. Chapter 2 provides a succinct summary of this field with a critical analysis of various factors that need to be considered for optimal results.
Chapter 3 describes our effort to develop a reversible assembly approach to the design of PROTACS.Doctor of Philosophy (PhD)Chemistr
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Leveraging Chemical Tools for Improving Therapeutic Delivery of Carbon Monoxide and Potency of Anthraquinone-based MDM2 Inhibitors
No full textCarbon monoxide (CO) is an endogenous signaling molecule with known pharmacological activities in a range of animal model studies, including inflammation, cancer, and organ protection. With our long-standing interest in the development of organic CO prodrugs, we developed a new class of CO prodrugs immobilized on silica microparticles which are generally recognized as safe by the US FDA. Amidation-based conjugation with silica is shown to provide 0.2 mmol/g loading density, effective prodrug activation in buffer, and stable tethering to prevent detachment. One representative silica conjugate, SICO-101, is shown to exhibit anti-inflammation activity in LPS-challenged RAW264.7 cells and to deliver CO systemically in mice through oral administration and GI CO release.
For improved therapeutic delivery of CO, we developed an enrichment-triggered release approach for the activation of CO prodrug pair for CO delivery to lysosomes. This approach exploits the concentration-dependent nature of bimolecular reactions and increased concentrations of prodrug components after enrichment for targeted delivery. For this, we tether CO prodrug pair, each with a morpholine moiety for enrichment in lysosomes for their bio-orthogonal activation and subsequent release of CO in a kinetically-controlled fashion. In this regard, our LC-MS experiments showed morpholine-facilitated enrichment of CO prodrug pair in lysosomes led to 13-fold increase in CO release compared to the non-targeted controls. Furthermore, LysoTracker Red co-localization experiments confirmed the presence of the fluorescent product after CO release in lysosomes.
Overexpression of ubiquitin ligase MDM2 causes depletion of the p53 tumor-suppressor and thus leads to cancer progression. Previously, we have developed potent anthraquinone compounds having the ability to upregulate p53 via inhibition of MDM2 in both in-vitro and in-vivo models of acute lymphocytic leukemia. Earlier work was focused on mechanistic work, pharmacological validation of the compounds in animal models and mapping out structural space that allows for further modification and optimization. Herein, we describe our work in optimizing the substituents on the two phenol hydroxyl groups. It was found that the introduction of an alkylketone moiety led to a potent series of analogs with BW-AQ-350 being the most potent compound yet which exerts cytotoxicity by inducing MDM2 degradation and p53 upregulation.Doctor of Philosophy (PhD)Chemistr
Functional Importance of Non-Catalytic Residues in the Active Site of NADH:Quinone Oxidoreductase From Pseudomonas aeruginosa PA01
Get PDFNADH:quinone oxidoreductase (NQO) from Pseudomonas aeruginosa PA01 is an FMN-dependent enzyme that utilizes NADH to catalyze the two-electron reduction of a wide variety of benzoquinones and naphthoquinones. The two-electron quinone reduction by NQO is thought to play a detoxifying role in P. aeruginosa PA01 by avoiding the formation of semiquinone radicals known to cause oxidative stress in cells. Crystal structures of NQO demonstrate the enzyme contains two domains: a TIM-barrel domain and an extended domain which are connected through two βα loops. The TIM-barrel domain is the most common enzyme fold found in nature and is often utilized as a target to engineer catalytic functions during the de novo synthesis of enzymes. NQO is a mostly unexplored enzyme at the time of this thesis, where only its mechanistic and structural properties have been elucidated. In this thesis, the functional roles of two non-catalytic residues that form the active site of NQO are investigated by employing UV-visible absorption spectroscopy, molecular dynamics, and steady-state kinetics. The thesis presented below highlights the importance of non-catalytic residues in modulating the structural, biophysical, and kinetic properties of NQO, which should be taken into account during the de novo synthesis of TIM-barrel containing enzymes.
Previously solved crystal structures of NQO demonstrated the βα loop 3 (residues 75-86) from the TIM-barrel domain samples an open conformation in the ligand-free form and a closed conformation in the ligand-bound form. The relationship between loop 3 rigidity and turnover rates in NQO was investigated by replacing the conserved P78 with a glycine. Circular dichroism, fluorescence spectroscopy, and UV-visible absorption spectroscopy were employed to demonstrate the P78G mutation minimally altered the secondary structure elements of the protein and the active site environment surrounding the flavin cofactor when compared to wild-type NQO. The gate in NQO was determined to consist of loop 3 and the extended domain, where molecular dynamics demonstrated the substrate-free form of NQO samples more open gate conformations following the P78G mutation. The steady-state kinetic parameters of the mutant and wild-type enzymes revealed the mutation led to a minimal increase in the kcat/KCoQ0 value, suggesting the mutation may promote the rate of association with the quinone substrate. The results presented in this investigation suggest the structural rigidity of loop 3 plays a role in modulating domain-domain interactions that may increase the rate of substrate association in NQO.
In the second part of this thesis, the relationship between the protonation state of Y277 and the flavin cofactor’s absorption spectrum was investigated in NQO. Y277 is mainly exposed to the solvent in the active site of NQO, with one exception being a 3.0 Å separation between the oxygen atom of Y277 and the C7 methyl group of the flavin cofactor. The UV-visible absorption spectrum of NQO was investigated as a function of pH and compared to those of a mutant enzyme NQO-Y277F, which indicated Y277 deprotonates at high pH. A combination of UV-visible absorption spectroscopy and QM/MM computations were utilized to determine the effect of Y277 deprotonation on flavin’s absorption peak intensities and wavelengths. Both the biochemical experiments and computational calculations suggest that in the absence of solution ions, deprotonating Y277 will significantly alter the absorption spectrum of flavin; however, in the presence of added Na+ and Cl- ions, no spectral changes are observed between protonated and unprotonated Y277. QM/MM simulations of NQO with Y277 in the neutral and anionic forms revealed either Na+ ions move closer to the protein surface and/or Cl- ions move away from the surface as Y277 deprotonates in the active site. The results presented in this portion of the thesis establish that the solution ions surrounding NQO can interact with the negative point charge at residue 277 through a long-distance counterion effect to prevent the flavin cofactor’s absorption spectrum from being altered.Master of Science (MS)Chemistr
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