Medicinal-Pharmaceutical Chemistry Commons^™

Fluorogenic Protein Labeling Using A Genetically Encoded Unstrained Alkene, X. Shang, X. Song, C. Faller, R. Lai, H. Li, Ronald Cerny, Wei Niu, Jiantao Guo

Department of Chemistry: Faculty Publications

We developed a new fluorogenic bioorthogonal reaction that is based on the inverse electron-demand Diels–Alder reaction between styrene (an unstrained alkene) and a simple tetrazine. The reaction forms a new fluorophore with no literature precedent. We have identified an aminoacyl-tRNA synthetase/tRNA pair for the efficient and site-specific incorporation of a styrene-containing amino acid into proteins in response to amber nonsense codon. Fluorogenic labeling of purified proteins and intact proteins in live cells were demonstrated. The fluorogenicity of the styrene–tetrazine reaction can be potentially applied to the study of protein folding and function under physiological conditions with low background fluorescence interference.

Structure And Characterization Of A Class 3b Proline Utilization A: Ligand-Induced Dimerization And Importance Of The C-Terminal Domain For Catalysis, David A. Korasick, Thameesha T. Gamage, Shelbi Christgen, Kyle M. Stiers, Lesa J. Beamer, Michael T. Henzl, Donald F. Becker, John J. Tanner

Department of Chemistry: Faculty Publications

The bifunctional flavoenzyme proline utilization A (PutA) catalyzes the two-step oxidation of proline to glutamate using separate proline dehydrogenase (PRODH) and L-glutamate-y-semialdehyde dehydrogenase active sites. Because PutAs catalyze sequential reactions, they are good systems for studying how metabolic enzymes communicate via substrate channeling. Although mechanistically similar, PutAs vary widely in domain architecture, oligomeric state, and quaternary structure, and these variations represent different structural solutions to the problem of sequestering a reactive metabolite. Here, we studied PutA from Corynebacterium freiburgense (CfPutA), which belongs to the uncharacterized 3B class of PutAs.A2.7A˚ resolution crystal structure showed the canonical arrangement of PRODH, L-glutamate-y-semialdehyde dehydrogenase, …

Amino Acid Catabolism In Staphylococcus Aureus And The Function Of Carbon Catabolite Repression, Cortney R. Halsey, Shulei Lei, Jacqueline K. Wax, Mckenzie K. Lehman, Austin S. Nuxoll, Laurey Steinke, Marat Sadykov, Robert Powers, Paul D. Fey

Department of Chemistry: Faculty Publications

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced …

Laterally Extended Atomically Precise Graphene Nanoribbons With Improved Electrical Conductivity For Efficient Gas Sensing, Mohammad Mehdi Pour, Andrey Lashkov, Adrian Radocea, Ximeng Liu, Tao Sun, Alexey Lipatov, Rafal A. Korlacki, Mikhail Shekhirev, Narayana R. Aluru, Joseph W. Lyding, Victor Sysoev, Alexander Sinitskii

Department of Chemistry: Faculty Publications

Narrow atomically precise graphene nanoribbons hold great promise for electronic and optoelectronic applications, but the previously demonstrated nanoribbon-based devices typically suffer from low currents and mobilities. In this study, we explored the idea of lateral extension of graphene nanoribbons for improving their electrical conductivity. We started with a conventional chevron graphene nanoribbon, and designed its laterally extended variant. We synthesized these new graphene nanoribbons in solution and found that the lateral extension results in decrease of their electronic bandgap and improvement in the electrical conductivity of nanoribbon-based thin films. These films were employed in gas sensors and an electronic nose …

Nanomaterials As Stationary Phases And Supports In Liquid Chromatography: A Review, Sandya Beeram, Elliott Rodriguez, Suresh Doddavenkatanna, Zhao Li, Allegra Pekarek, Darin Peev, Kathryn Goerl, Gianfranco Trovato, Tino Hofmann, David S. Hage

Department of Chemistry: Faculty Publications

The development of various nanomaterials over the last few decades has led to many applications for these materials in liquid chromatography (LC). This review will look at the types of nanomaterials that have been incorporated into LC systems and the applications that have been explored for such systems. A number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in LC, ranging from carbon nanotubes, fullerenes and nanodiamonds to metal nanoparticles and nanostructures based on silica, alumina, zirconia and titanium dioxide. Many ways have been described for incorporating these nanomaterials into LC systems. These methods have included covalent …

A Surface-Stabilized Ozonide Triggers Bromide Oxidation At The Aqueous Solution-Vapour Interface, Luca Artiglia, Jacinta Edebeli, Fabrizio Orlando, Shuzhen Chen, Ming-Tao Lee, Pablo Corral Arroyo, Anina Gilgen, Thorsten Bartels-Rausch, Armin Kleibert, Mario Vazdar, Marcelo Andrea Carignano, Joseph S. Francisco, Paul B. Shepson, Ivan Gladich, Markus Ammann

Department of Chemistry: Faculty Publications

Oxidation of bromide in aqueous environments initiates the formation of molecular halogen compounds, which is important for the global tropospheric ozone budget. In the aqueous bulk, oxidation of bromide by ozone involves a [Br•OOO⁻] complex as intermediate. Here we report liquid jet X-ray photoelectron spectroscopy measurements that provide direct experimental evidence for the ozonide and establish its propensity for the solution-vapor interface. Theoretical calculations support these findings, showing that water stabilizes the ozonide and lowers the energy of the transition state at neutral pH. Kinetic experiments confirm the dominance of the heterogeneous oxidation route established by this precursor …

Cytotoxic Polyketides With An Oxygen-Bridged Cyclooctadiene Core Skeleton From The Mangrove Endophytic Fungus Phomosis Sp. A818, Wei Zhang, Baobing Zhao, Liangcheng Du, Yuemao Shen

Department of Chemistry: Faculty Publications

Plant endophytic microorganisms represent a largely untapped resource for new bioactive natural products. Eight polyketide natural products were isolated from a mangrove endophytic fungus Phomosis sp. A818. The structural elucidation of these compounds revealed that they share a distinct feature in their chemical structures, an oxygen-bridged cyclooctadiene core skeleton. The study on their structure–activity relationship showed that the α,β-unsaturated δ-lactone moiety, as exemplified in compounds 1 and 2, was critical to the cytotoxic activity of these compounds. In addition, compound 4 might be a potential agonist of AMPK (5'-adenosine monophosphate-activated protein kinase).

Chemoselective Alteration Of Fluorophore Scaffolds As A Strategy For The Development Of Ratiometric Chemodosimeters, Xinqi Zhou, Lauren Lesiak, Rui Lai, Jon R. Beck, Jia Zhao, Christian Elowsky, Hui Li, Cliff I. Stains

Department of Chemistry: Faculty Publications

Ratiometric sensors generally couple binding events or chemical reactions at a distal site to changes in the fluorescence of a core fluorophore scaffold. However, such approaches are often hindered by spectral overlap of the product and reactant species. We provide a strategy to design ratiometric sensors that display dramatic spectral shifts by leveraging the chemoselective reactivity of novel functional groups inserted within fluorophore scaffolds. As a proof-of-principle, fluorophores containing a borinate (RF₆₂₀) or silanediol (SiOH2R) functionality at the bridging position of the xanthene ring system are developed as endogenous H₂O₂ sensors. Both …

The Future Of Nmr-Based Metabolomics, John L. Markley, Rafael Bruschweiler, Arthur S. Edison, Hamid R. Eghbalnia, Robert Powers, Daniel Raftery, David S. Wishart

Department of Chemistry: Faculty Publications

The two leading analytical approaches to metabolomics are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Although currently overshadowed by MS in terms of numbers of compounds resolved, NMR spectroscopy offers advantages both on its own and coupled with MS. NMR data are highly reproducible and quantitative over a wide dynamic range and are unmatched for determining structures of unknowns. NMR is adept at tracing metabolic pathways and fluxes using isotope labels. Moreover, NMR is non-destructive and can be utilized in vivo. NMR results have a proven track record of translating in vitro findings to in vivo clinical …

Resolving The Cofactor-Binding Site In The Proline Biosynthetic Enzyme Human Pyrroline-5-Carboxylate Reductase 1, Emily M. Christensen, Sagar M. Patel, David A. Korasick, Ashley C. Campbell, Kurt L. Krause, Donald F. Becker, John J. Tanner

Department of Chemistry: Faculty Publications

Pyrroline-5-carboxylate reductase (PYCR) is the final enzyme in proline biosynthesis, catalyzing the NAD(P)H-dependent reduction of [?]1-pyrroline-5-carboxylate (P5C) to proline. Mutations in the PYCR1 gene alter mitochondrial function and cause the connective tissue disorder cutis laxa. Furthermore, PYCR1 is overexpressed in multiple cancers, and the PYCR1 knock-out suppresses tumorigenic growth, suggesting that PYCR1 is a potential cancer target. However, inhibitor development has been stymied by limited mechanistic details for the enzyme, particularly in light of a previous crystallographic study that placed the cofactor-binding site in the C-terminal domain rather than the anticipated Rossmann fold of the N-terminal domain. To fill this …

Indole-Induced Reversion Of Intrinsic Multiantibiotic Resistance In Lysobacter Enzymogenes, Yong Han, Yan Wang, Yameng Yu, Haotong Chen, Yuemao Shen, Liangcheng Du

Department of Chemistry: Faculty Publications

Lysobacter species are a group of environmental bacteria that are emerging as a new source of antibiotics. One characteristic of Lysobacter is intrinsic resistance to multiple antibiotics, which had not been studied. To understand the resistance mechanism, we tested the effect of blocking two-component regulatory systems (TCSs) on the antibiotic resistance of Lysobacter enzymogenes, a prolific producer of antibiotics. Upon treatment with LED209, an inhibitor of the widespread TCS QseC/QseB, L. enzymogenes produced a large amount of an unknown metabolite that was barely detectable in the untreated culture. Subsequent structural elucidation by nuclear magnetic resonance (NMR) unexpectedly revealed that …

Synthesis Of Cerium Oxide Nanorods, Chin Li Cheung, Zane Charles Gernhart

Department of Chemistry: Faculty Publications

Cerium oxide nanorods having a variety of aspect ratios can be produced by providing a first mixture that includes a cerium precursor material, and using microwave to heat the first mixture to a first temperature for a period of time to produce first plurality of cerium oxide nanorods having a first range of aspect ratios. A second mixture that includes a cerium precursor material heated using microwave to a second temperature for a period of time to produce second plurality of cerium oxide nanorods having a second range of aspect ratios. The first plurality of cerium oxide nanorods and the …

Human Serine Racemase Structure/Activity Relationship Studies Provide Mechanistic Insight And Point To Position 84 As A Hot Spot For Β-Elimination Function, David L. Nelson, Greg A. Applegate, Matthew L. Beio, Danielle L. Graham, David B. Berkowitz

Department of Chemistry: Faculty Publications

There is currently great interest in human serine racemase, the enzyme responsible for producing the NMDA co-agonist D-serine. Reported correlation of D-serine levels with disorders including Alzheimer’s disease, ALS, and ischemic brain damage (elevated D-serine) and schizophrenia (reduced D-serine) has further piqued this interest. Reported here is a structure/activity relationship study of position Ser⁸⁴, the putative re-face base. In the most extreme case of functional reprogramming, the S84D mutant displays a dramatic reversal of β-elimination substrate specificity in favor of L-serine over the normally preferred L-serine-O-sulfate (~1200-fold change in k_cat/K_{m …}

Assessment Of Metabolic Changes In Mycobacterium Smegmatis Wild Type And Alr Mutant Strains: Evidence For A New Pathway Of D-Alanine Biosynthesis., Darrell D. Marshall, Steven Halouska, Denise K. Zinniel, Robert J. Fenton, Katie Kenealy, Harpreet K. Chahal, Govardhan Rathnaiah, Raul G. Barletta, Robert Powers

Department of Chemistry: Faculty Publications

In mycobacteria, D-alanine is an essential precursor for peptidoglycan biosynthesis. The only confirmed enzymatic pathway to form D-alanine is through the racemization of L-alanine by alanine racemase (Alr, EC 5.1.1.1). Nevertheless, the essentiality of Alr in Mycobacterium tuberculosis and Mycobacterium smegmatis for cell survivability in the absence of D-alanine has been a point of controversy with contradictory results reported in the literature. To address this issue, we examined the effects of alr inactivation on the cellular metabolism of M. smegmatis. The M. smegmatis alr insertion mutant TAM23 exhibited essentially identical growth to wild type mc²155 in the …

Transcriptional And Antagonistic Responses Of Biocontrol Strain Lysobacter Enzymogenes Oh11 To The Plant Pathogenic Oomycete Pythium Aphanidermatum, Yangyang Zhao, Guoliang Qian, Yuan Chen, Liangcheng Du, Fengquan Liu

Department of Chemistry: Faculty Publications

Lysobacter enzymogenes is a ubiquitous, beneficial, plant-associated bacterium emerging as a novel biological control agent. It has the potential to become a new source of antimicrobial secondary metabolites such as the Heat-Stable Antifungal Factor (HSAF), which is a broad-spectrum antimycotic with a novel mode of action. However, very little information about how L. enzymogenes detects and responds to fungi or oomycetes has been reported. An in vitro confrontation bioassay between the pathogenic oomycete Pythium aphanidermatum and the biocontrol bacterial strain L. enzymogenes OH11 was used to analyze the transcriptional changes in the bacteria that were induced by the oomycetes. Analysis …

Ion Mobility-Resolved Collision-Induced Dissociation And Electron Transfer Dissociation Of N-Glycopeptides: Gathering Orthogonal Connectivity Information From A Single Mass- Selected Precursor Ion Population, Venkata Kolli, Katherine N. Schumacher, Eric Dodds

Department of Chemistry: Faculty Publications

Glycopeptide-level mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses are commonly performed to establish site-specific protein glycosylation profiles that are of central importance to gaining structure-function insights on glycoproteins. Confoundingly, the complete characterization of glycopeptide connectivity usually requires the acquisition of multiple MS/MS fragmentation spectra. Complementary ion fragmentation techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) are often applied in concert to address this need. While structurally informative, the requirement for acquisition of two MS/MS spectra per analyte places considerable limitations upon the breadth and depth of large-scale glycoproteomic inquiry. Here, a previously developed method of …

Phage Display To Identify Functional Resistance Mutations To Rigosertib, Nedim Filipovic

CMC Senior Theses

In vitro protein selection has had major impacts in the field of protein engineering. Traditional screens assay individual proteins for specific function. Selection, however, analyzes a pool of mutants and yields the best variants. Phage display, a successful selection technique, also provides a reliable link between variant phenotype and genotype. It can also be coupled with high throughput sequencing to map protein mutations; potentially highlighting vital mutations in variants. We propose to apply this technique to cancer therapy. RAF, a serine/threonine kinase, is critical for cell regulation in mammals. RAF can be activated by oncogenic RAS, found in over 30% …

Peroxiredoxin Catalysis At Atomic Resolution, Arden Perkins, Derek Parsonage, Kimberly J. Nelson, O. Maduka Ogba, Paul Ha-Yeon Cheong, Leslie B. Poole, P. Andrew Karplus

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases that guard cells against oxidative damage, are virulence factors for pathogens, and are involved in eukaryotic redox regulatory pathways. We have analyzed catalytically active crystals to capture atomic resolution snapshots of a PrxQ-subfamily enzyme (from Xanthomonas campestris) proceeding through thiolate, sulfenate, and sulfinate species. These analyses provide structures of unprecedented accuracy for seeding theoretical studies, and show novel conformational intermediates giving insight into the reaction pathway. Based on a highly non-standard geometry seen for the sulfenate intermediate, we infer that the sulfenate formation itself can strongly promote local unfolding of the active site to …

The Synthesis Of Chemosensors For Toxic Analytes, Johnathan Hugh Broome

Dissertations

A number of chemosensors have been designed and synthesized to target cations (Zn²⁺ions), neutral molecules (cathinones), charged molecules (aminoindanes), and anions. The Zn²⁺ ion sensor featured bistriazole designed binding unit and ferrocene signaling units. Characterization of Zn²⁺ ion binding was carried out with electrochemical techniques (CV and DPV), ¹H-NMR, mass spectrometry, and molecular modelling. It exhibited a 1:1 binding stoichiometry with Zn²⁺ and had an affinity for ZnCl₂ (Log K_1:1 = 4.1 ± 0.02) over other Zn²⁺ salts.

The cathinone probe was designed to selectively bind mephedrone over common street drugs …

Staphylococcal Nuclease And Ubiquitin Local Folding Energies And Rates Using Peps-Hdx-Esi-Ms, Julie Rhee

Chemistry & Biochemistry Undergraduate Honors Theses

In this study, Protein Equilibrium Population Snapshot Hydrogen-Deuterium Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) was applied to study the local regions of model proteins, staphylococcal nuclease and ubiquitin. The hydrogen deuterium exchange (HDX) has become a key technique for studying the structural and dynamic aspects of proteins in solution. This technique creates a rapid exchange between all of the exchangeable hydrogen ions with deuterium when the protein is exposed to a solvent. The PEPS method is an equilibrium-based method used to determine the populations of the closed native and open denatured states of a protein. By combining the applications of …

Ruthenium-Catalyzed Amination Of Secondary Alcohols Using Borrowing Hydrogen Methodology, Kostiantyn O. Marichev, James M. Takacs

Department of Chemistry: Faculty Publications

A new ruthenium complex catalyzes the amination of primary and secondary alcohols and the regioselective mono- and sequential diamination of diols via the borrowing hydrogen pathway. Several variations on new intra- and intermolecular cyclizations of aminoalcohols, diols and diamines lead to heterocyclic ring systems.

Αβ-Peptide Mimics Of Z-Domain Peptides, Samuel H. Gellman, James W. Checco

Department of Chemistry: Faculty Publications

Described are αβ-peptide mimics of Z-domain scaffold pep tides, methods of making them, and methods of using them. The αβ-peptide mimics include B-amino acid residues and, optionally, disulfide bonds to stabilize the conformation of the mimics. The compounds may be truncated as compared to conventional Z-domain scaffold peptides and are resistant to proteolytic degradation due to the presence off-amino acid residues. The mimics can be made so as to bind selectively to a desired target.

Hlarp7 C-Terminal Domain Contains An Xrrm That Binds The 3' Hairpin Of 7sk Rna, Catherine D. Eichhorn, Rahul Chug, Juli Feigon

Department of Chemistry: Faculty Publications

The 7SK small nuclear ribonucleoprotein (snRNP) sequesters and inactivates the positive transcription elongation factor b (P-TEFb), an essential eukaryotic mRNA transcription factor. The human La-related protein group 7 (hLARP7) is a constitutive component of the 7SK snRNP and localizes to the 3' terminus of the 7SK long noncoding RNA. hLARP7, and in particular its C-terminal domain (CTD), is essential for 7SK RNA stability and assembly with P-TEFb. The hLARP7 N-terminal Lamodule binds and protects the 3' end from degradation, but the structural and functional role of its CTD is unclear.We report the solution NMR structure of the hLARP7 CTD and …

Analytic Ab Initio-Based Molecular Interaction Potential For The Bro·H2O Complex, Ross D. Hoehn, Sachin D. Yeole, Sabre Kais, Joseph S. Francisco

Department of Chemistry: Faculty Publications

Radical halogen oxide species play important roles within atmospheric processes, specifically those responsible for the removal of O3. To facilitate future investigations on this family of compounds, RCCSD(T)/aug-cc-pVQZ-level electronic structure calculations were employed to generate individual-molecule optimized geometries, as well as to determine the global minimum energy structure for the BrO·H₂O complex. This information facilitated the generation of several one-dimensional potential energy surface (PES) scans for the BrO·H₂O complex. Scans were performed for both the ground state and the first excited state; this inclusion is due to a low-lying first electronic excited-state energy. These rigid-geometry PES …

Ring Closures And The Hammond Postulate, Charles A. Kingsbury

Department of Chemistry: Faculty Publications

This work reports an investigation of ring closure processes in relation to the Hammond postulate. Calculations favor the importance of thermodynamics, not kinetics, as the basis for the Hammond postulate. A kinetically rapid, but thermodynamically unfavorable reaction is shown to resemble product. The ease of ring closure to three-membered rings, compared to four-membered rings, is thought to be associated with conformational mobility and perhaps vibrations coupled to the reaction coordinate motion in the case of four-membered rings.

Live, Attenuated Vaccines And Methods Of Making And Using, Jiantao Guo, Qingsheng Li, Wei Niu, Yue Li, Nanxi Wang

Department of Chemistry: Faculty Publications

A live, attenuated HIV vaccine is provided, and methods of making a atenuated HIV vaccine are provided.

Processes And Reagents For Making Daryliodonium Salts, Stephen Dimagno

Department of Chemistry: Faculty Publications

This invention relates to processes and reagents for making diaryliodonium salts, which are useful for the preparation of fluorinated and radiofluorinated aromatic compounds.

Mutagenesis-Based Active-Site Characterization Of The Diabetes Drug Target Mitoneet, Lisa Mickley

Williams Honors College, Honors Research Projects

This project investigates the cluster stability of an outer mitochondrial membrane protein mitoNEET under mutational stress and drug binding analysis. MitoNEET is a part of a class of proteins that coordinate an Fe-S molecule into its tertiary structure. This protein is believed to facilitate the oxidative capacity of the electron transport chain by reversible loss of its coordinated Fe-S molecule. The research of this paper uses site directed protein mutagenesis to selectively alter the three cysteine, one histidine cluster coordination into more common coordination patterns such as two cysteine, two histidine or four cysteine. The ultimate goal is to optimize …

Signature Of Coexistence Of Superconductivity And Ferromagnetism In Two-Dimensional Nbse2 Triggered By Surface Molecular Adsorption, Xiaojiao Zhu, Yuqiao Guo, Hao Cheng, Jun Dai, Xingda An, Jiyin Zhao, Kangzhen Tian, Shiqiang Wei, Xiao Cheng Zeng, Changzheng Wu, Yi Xie

Department of Chemistry: Faculty Publications

Ferromagnetism is usually deemed incompatible with superconductivity. Consequently, the coexistence of superconductivity and ferromagnetism is usually observed only in elegantly designed multi-ingredient structures in which the two competing electronic states originate from separate structural components. Here we report the use of surface molecular adsorption to induce ferromagnetism in two-dimensional superconducting NbSe₂, representing the freestanding case of the coexistence of superconductivity and ferromagnetism in one two-dimensional nanomaterial. Surface-structural modulation of the ultrathin superconducting NbSe₂ by polar reductive hydrazine molecules triggers a slight elongation of the covalent Nb–Se bond, which weakens the covalent interaction and enhances the ionicity of …

Engineering Escherichia Coli For High‑Yield Geraniol Production With Biotransformation Of Geranyl Acetate To Geraniol Under Fed‑Batch Culture, Wei Liu, Xin Xu, Rubing Zhang, Tao Cheng, Yujin Cao, Xiaoxiao Li, Jiantao Guo, Huizhou Liu, Mo Xian

Department of Chemistry: Faculty Publications

Background: Geraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown.

Results: Recombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol …