Summer 2013 Research Project: Jasper Werby '14

Jasper Werby '14 discusses his 2013 chemistry Summer Undergraduate Research Project, which he undertook under the mentorship of Professor Cynthia Selassie. He focused on the development and synthesis of a new bifunctional, antimalarial drug.

Hands-on lab research is an important part of being a chemistry major at Pomona College. Students have the opportunity to use modern instrumentation, computational facilities and sophisticated software in the classroom and on independent research projects as early as their first year. Below is a list of recent summer research projects conducted by chemistry students.


Anti-Tumor Pro-drug Development: Legumain Activated Immune Agonist

Phillip Clayman ’16; Mentor: Yongkai Li (Genomics Institute of the Novartis Research Foundation)

Funding Provided By: Novartis

Atmospheric Chemistry: Determination of rate constants for acetonylperoxy/hydroperoxy radical chemistry

Julia Dohner ’16; Mentor: Frederick Grieman

Acetone, one of the most abundant oxygenated organic compounds in the remote atmosphere, reacts with the OH radical to produce acetonylperoxy radical in the troposphere. Therefore, to understand and model tropospheric chemistry accurately, acetonylperoxy chemistry must be quantitatively determined. Our research entails the laboratory study of the acetonyl peroxy reaction with hydroperoxy, an important oxidant in the troposphere, and its self-reaction, important in laboratory studies of acetonylperoxy reactions, over the temperature range T = 298 K–340 K. Previously, the rate constant of the acetonyl peroxy self-reaction has been determined only at room temperature and the reliability of this value is uncertain. The rate constants for these reactions were determined by simultaneously measuring the disappearance of acetonylperoxy and hydroperoxy using Infrared Kinetic Spectroscopy (IRKS). Using non-linear kinetics fitting (FACSIMILE software) and integrated rate law linear fitting, both the temperature dependence of the rate constant for the self-reaction and the ultraviolet absorbance cross-section for acetonylperoxy at 300 nm were determined. Using these obtained values permitted the determination of the acetonylperoxy/hydroperoxy cross-reaction rate constant over the same temperature range. Future direction includes measuring the rate constants of both reactions at lower temperatures in the atmospherically relevant range between 220 and 300 K.
Funding Provided By: Neil Smith Hansch

Bacterial Biofilm Growth and Analysis of VOCs via SPME/GC-MS

Paige Oliver ’16; Mentor: Charles Taylor; Collaborator: Michael Etzel ‘15

Bacterial attachment to implanted medical devices and subsequent development of biofilms has been found to have profound effects on hospitalized patients. Ventilator associated pneumonia (VAP) is a common occurring nosocomial infection among patients whose immune systems are already compromised. Current methods of diagnosis are too slow (culture growth takes 2-3 days), and presumptive diagnosis leads to the overuse of antibiotics, which may increase the proliferation of antibiotic resistant strains. By analyzing the volatile organic compounds (VOCs) that are given off by bacterial biofilms grown in medically relevant conditions, new biomarker VOCs may be found and used to diagnose patients in a quicker, more accurate manor. In order to identify these biomarkers, we must grow bacterial biofilms in a system analogous to the tracheal tube of a hospital patient. Then, utilizing solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry, we were able to analyze uniform P. aerguginosa bacteria in planktonic and biofilm stages. This work has helped us identify candidate biomarker VOCs associated with P. aerguginosa including: tert-butyl alcohol, ethyl tert-butyl ether (ETBE), 4-Methylbenzaldehyde, and 2,4-Di-tert-butylphenol.
Funding Provided By: Howard Hughes Medical Institute

Biofilm Associated Protein (Bap) Stability and Structure Characterization

Maximilian Hoffman ’16; Mentor: Matthew Sazinsky

We are studying BAP, a 250kD extracellular protein shown to be involved in the intercell adherence, adhesion and biofilm matrix formation in S. aureus. Both the protein and biofilm structure are conserved among bacterial strains, and the infectious homologues are consistently resistant to immune system endocytosis and antibiotic targeting. The connection between BAP and biofilm production has yet to be teased out, however, and the link could help create antimicrobials that target biofilms in many antibiotic-resistant bacteria. Thus far, we have successfully grown and purified the B and C domains in E. coli, two of the three extracellular domains. We have also performed preliminary pH and metal stability testing and calculated binding constants for the protein-metal interactions. Shortly, we will analyze how pH and metal binding affects the melting point and secondary structure of the protein. We hope to reach similar results for the other two domains of the full-length protein and crystallize the A,B, D domains. Future work will include expressing shortened versions of the full-length protein to analyze subdomain interactions through deletion studies.
Funding Provided By: Norris

Design and Surface Analysis of Nanocomposites for Next-Generation Body Armor and Ballistic Protection

Neil Forsythe ’15; Mentor: Daniel O’Leary; Collaborator: Michael Papantonakis (Naval Research Laboratory)

We present results for carbon nanofibers modified by physisorption with a range of polymer modifiers and then dispersed in different polymeric hosts with mechanical advantage and other applications in mind. In contrast to covalent bonded paths, polymer wrapping can be achieved without damaging the physical integrity of the nanofiber. In addition to improving the dispersion properties of nanofibers, we also tailor the surface chemistry properties of the modified nanomaterials to provide improved interfacial adhesion to different host polymers of interest. This is an important consideration for mechanical applications in which one needs to ensure mechanical load is effectively transferred to and shared by the nanofibers embedded in a polymer host. We have characterized the modified nanofibers using inverse gas chromatography (IGC), XPS, TGA and ATR-FTIR. Additionally, IGC was used to measure the surface properties (surface energies, acid/base properties) of the modified carbon nanofiber materials to predict their compatibility with candidate matrix polymers. Finally, nanocomposite samples were fabricated by injection molding and tested for their mechanical properties. Applications of the polymer wrapped carbon nanofiber materials developed in this work may include high strength polymer composites for vehicular applications and high strength concrete for structural applications.
Funding Provided By: US Department of Homeland Security

Determining Diffusivity of Stratum Corneum Model using QCM-D

Devin Gladys ’17; Mentor: Malkiat Johal; Collaborator: Carlos Hernandez ‘18

The stratum corneum is the outer-layer of the epidermis, and it is the main barrier of the skin, preventing most substances from entering lower layers of the skin and, ultimately, the bloodstream. A Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) was employed to determine the diffusivity constant of a stratum corneum model when exposed to water. The stratum corneum model consisted of an equimolar mixture of five fatty acids, porcine brain ceramide, and cholesterol. The mixture was applied to the QCM-D crystal as a thin film by means of an airbrush, and a QSense Humidity Module was employed to expose the film to a specific humidity level. Nitrogen gas and water were used to achieve zero and one hundred percent humidity, respectively. Ellipsometry was then employed to determine the thickness of the film. The Sauerbrey equation allowed us to determine the total mass of water deposited on the film, and a mathematical model then enabled us to determine the diffusivity constants of the model, as well as their temperature dependence, in a temperature range from 15 °C to 35 °C. In the future, the data obtained will be manipulated in order to determine solubility and permeability constants, and the same procedure will be used to expose the stratum corneum model to methyl paraben, a common ingredient in skin products that acts as a fungicide, in order to shed light as to how this widely researched drug enters the skin.
Funding Provided By: Richter (Hernandez), Howard Hughes Medical Institute (Gladys)

Developing a BPF-responsive Riboswitch Through Dual Genetic Selection

Erick Velasquez ’16; Mentor: Jane Liu; Collaborator: Marek Zorawski ‘16

The use of biosensors has many applications within medicinal, agricultural, and environmental fields offering quick and robust solutions to complex problems. It would be beneficial to create a method by which we can readily engineer biosensors that allow for the qualification and quantification of any ligand of interest. We set out to develop such a method to engineer riboswitch-based biosensors. Riboswitches are components of messenger RNAs that regulate the expression of cis-encoded genes based on the binding of a specific ligand. For example, by fusing a gene that encodes for a reporter protein to a BPF-riboswitch, we reasoned that we could qualify the presence of BPF in the environment of a cell expressing the BPF-riboswitch. Here, we set out to develop a method to generate a large library of RNA sequences from which to select an effective riboswitch for our ligand of interest (BPF). We chose to use as our starting template an existing riboswitch that turns ON gene expression in the presence of thiamine. We replaced the thiamine aptamer domain with 40 random nucleotides (N40) using a variety of strategies, including traditional DNA ligation and Gibson Assembly methods. We were able to obtain libraries of 10^6–10^7 transformants using traditional DNA ligation methods. We were able to obtain a library size of 10^8 transformants using Gibson Assembly. We plan to apply this latter library to a dual selection process so as to identify a BPF-responsive riboswitch.
Funding Provided By: National Science Foundation #CBET-1258307: PI J. Liu

Engineering a BPA Riboswitch

Marek Zorawski ’16; Mentor Jane Liu;

Bisphenol A (BPA) is a known endocrine disruptor and carcinogen found in polycarbonate plastic products, food packaging, and drinking water supplies. A quick field method, such as a biosensor, that tests for the presence of BPA would greatly benefit human safety and health. Riboswitches, regions of mRNAs that control downstream gene expression, can be used as biosensors to fill this need. Starting from a known riboswitch that regulates the downstream reporter gene gfp, we used PCR to replace the aptamer domain of the switch with 40 random nucleotides, thus generating random riboswitch libraries. These libraries were expressed from a plasmid upstream of a tetA-gfp reporter gene fusion. The plasmids were harbored in E. coli such that each individual E. coli bacterium expressed a unique member of the riboswitch library. Using dual genetic selection, we manipulated bacterial growth media in order to select for those riboswitches that turned on tetA-gfp expression only in the presence of BPA. We succeeded in isolating four unique riboswitches from two starting libraries by bringing each library through three rounds of selection with varying selection conditions. Ultimately, our four hits proved largely unsuccessful in producing fluorescence when incubated with BPA for either 4-6 or 16 hours compared to no-BPA controls. Further studies will include changing the method of library construction and optimizing the dual genetic selection process.
Funding Provided By: National Science Foundation #CBET-1258307: PI J. Liu

Exponential Average Walk-Length Growth in Fractal and Euclidean Square Lattices, Denaturation of Proteins, and Energy Transfer Diffusion in Dendrimers

Spencer Satz ’18; Mentor: Roberto Garza Lopez; Collaborators: Sabari Kumar '17, Don Chen '18, Neil Chan '18, Jovani Azpeitia '19

In this study, we compared the efficiency of energy transfer and diffusion across surfaces with varying lattice structures. We analyzed lattices that contain a reaction center, or trap, that is 100% absorbing, where a particle that diffuses across the lattice will stop. We compared a family of fractal lattices to a family of Euclidean square lattices and examined the geometrical factors affecting the average walklength, of random walker, such as an electron, on these lattices. Using Maple, we ran calculations that obtained the site-specific average walk lengths <n>’s before trapping and in that manner compare the efficiencies of diffusion on surfaces of various sizes, both fractal and Euclidean. These diffusion-controlled reactions depend on the morphology of the catalytic surfaces, and apply to the efficiency of catalytic processes on surfaces with different roughness. We applied this to proteins by analyzing protein folding patterns to determine sequences of events in the folding and unfolding of proteins as they denature. Our future research includes simulating different reactions; molecular dynamics will be used under preset parameters (temperature, solvent, etc.) to determine how differing conditions play a role in protein folding. Another application which we pursued was energy transfer in dendrimer-ligand reactions, in which we modeled the probabilities of movement of a “walker,” representing energy flow, from the core lattice out to ligands on its extremities.
Funding Provided By: Pomona College SURP(Kumar), Paul K. Richter and Evalyn E. Cook Richter Memorial Fund(Satz), Fred J. Robbins Chemistry Fund(Chan), Howard Hughes Medical Institute High Achievement Program (Azpeitia), Howard Hughes Medical Institute High Achievement Program(Chen)

Humidity Analysis of PEI/PAZO Polyelectrolyte Films using QCM-D

Vanessa Machuca ’18; Mentor: Malkiat Johal; Collaborator: Carlos Hernandez ‘18

A comprehensive analysis of polyelectrolyte multilayer (PEMU) films, consisting of poly(ethylene imine) (PEI) and poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido] / -1,2-ethanediyl, sodium salt] (PAZO) was conducted in order to determine the behavior of this system when exposed to varying levels of humidity. The study consisted of an analysis of the uptake of water by the film at seven different humidity levels, the swelling and deswelling processes of the film as a function of humidity, hysteresis, and the humidity dependence of diffusion through the film. A Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and a QSense humidity module were employed in order to measure mass changes as water deposited on the films, while ellipsometry was used to determine the thickness and density values. Films responded to changes in humidity in accordance with previous studies, swelling as percent humidity increased. In order to characterize hysteresis, understood as memory how the film responds to future changes in humidity after experiencing previous ones -, films were cycled between 0% and 75% humidity ten times. Further trials cycling between 0% and 100% will be performed in the future in order to reach conclusions related to hysteresis. Diffusion was found to be independent of humidity. Future directions include cycling through all seven humidity levels for hysteresis analysis and improving the film formation procedure.
Funding Provided By: Richter (Hernandez), Stutzman (Machuca)

KlenTaq mutant SFM19, when crystallized, diffracts to 3.1 Å

Ilana Ruth Cohen ’16; Mentors: Matthew Sazinsky and Aaron Leconte (Keck); Collaborator: Catherine Chiang (SCR '16)

The T. aquaticus DNA polymerase, KlenTaq, is commonly used in research and medicine to replicate short strands of DNA in vitro by PCR. This enzyme builds a complementary DNA strand with remarkable selectivity; it adds only the appropriate nucleotide across from its binding partner on the DNA template, ignoring other nucleotides and structurally similar species abundant in solution. In vivo, cell survival requires this selectivity, but a less discriminating enzyme could produce more stable and varied oligonucleotides. SFM19, a I614E/E615G Taq mutant, adds six 2’ modified nucleotides and then stalls. To identify those structural features of the mutant that explain its unusual capacity we aim to determine the X-ray crystal structure of the wild-type and mutant KlenTaq with bound DNA substrates. Existing procedures were adapted to express and purify wild type and SFM19 KlenTaq proteins. Using a crystallization screen based on previously successful conditions for crystallizing KlenTaq complexed to double-stranded DNA and a waiting nucleotide, we obtained medium-quality crystals and subjected these samples to X-ray diffraction. The wild type and mutant proteins diffracted to 2.5 Å and 3.1 Å, respectively. We will soon solve the structures, comparing these data to confirm that a ternary complex was obtained. Crystallization conditions will be refined for SFM19 in pursuit of higher quality crystals which, diffracting to a higher resolution will enable a full structural analysis.
Funding Provided By: Pomona Unrestricted

MtlA Proteolysis in Vibrio Cholerae

Elisabeth Hansen ’16; Mentor: Jane Liu

Cholera, caused by the bacterial pathogen Vibrio cholerae, remains a world health problem. V. cholerae is highly adaptable to different environments, likely due in part to gene regulation by small regulatory RNAs. We are examining one such regulatory pathway by studying the proteolysis of MtlA, the mannitol transporter protein. In the absence of environmental mannitol, the small RNA MtlS down-regulates mtlA expression at the translational and post-translational level. MtlS induction also affects the expression of other proteins, which we hypothesize may be involved in MtlA proteolysis. We are studying the potential role of four proteins in MtlA proteolysis that are either upor down-regulated by MtlS: VC1872, VC1899, YidC and GroL2. We created strains that overexpress one of each of these proteins and analyzed the affect that these proteins have on MtlA levels over time in in mannitol and mannitol-free conditions. We are also in the process of creating and testing strains in which each of the four candidate proteins is knocked out. Currently, we have determined that GroL2 does not play a role in MtlA proteolysis.
Funding Provided By: National Institutes of Health #R15AI090606: PI J. Liu

One-pot Pd-catalyzed Synthesis of Aryl Sulfonyl Fluorides using SO2 Surrogate DABSO

Ismerai Rodriguez ‘16; Mentor: Nicholas Ball; Collaborator: Evan DeLorenzo ‘17

Sulfonyl fluorides have a variety of biological and synthetic applications. They are also great starting materials for the synthesis of other sulfonyl-derived functional groups making them useful and versatile compounds. Current routes of synthesis for sulfonyl fluorides include the use of a sulfonyl chloride intermediate. While sulfonyl chlorides are inexpensive and abundant, they are air and water sensitive and have low selectivity. Synthesis of sulfonyl fluorides without the use of a sulfonyl chloride intermediate would thus be safer and more practical. Our goal was to develop a palladium-catalyzed one-pot, multicomponent conversion of aryl iodides to sulfonyl fluorides. The reaction uses Pd(OAc)2 as a catalyst, DABSO as a sulfur source, and selectfluor as a fluorination source. We found modest to excellent yields and intend to broaden our substrate scope and to optimize our method. Future directions for our research include the use of atmospheric sulfur dioxide as a sulfur source and the incorporation of non-aromatic substrate groups.
Funding Provided By: Linnares

Perhydrolysis activity of lipase B from Candida antarctica: a model of biocatalytic promiscuity

Albert Kakkis ’16; Mentor: Cynthia Selassie

Lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosa (TLL) exhibit a behavior that is uncommon in their enzyme family: they can use either hydrogen peroxide or water to carry out the hydrolysis of ester substrates. The basis of perhydrolysis in the lipases was postulated using Chimera, a molecular graphics program. Chimera-based structural alignments of CALB and TLL with known peroxidases yielded RMSD values above 1.5 and a Q-score of 0, suggesting that large structural homologies cannot indicate perhydrolysis activity. Via active-site residue alignments, the main chain carbonyl groups of Thr 40 (CALB) and Ser 83 (TLL) were identified as potential hydrogen bond acceptors (HBAs). HBAs, when optimally positioned in the active site, can stabilize hydrogen peroxide and enable its nucleophilic attack of ester substrates. The basis for perhydrolysis could thus depend on a molecular rather than structural motif. The library of enzymes used in Chimera-based alignments will continue to expand in an attempt to validate and generalize this molecular mechanism.
Funding Provided By: Dale N. Robertson Fund for Undergraduate Research

Polymer-Drug Conjugates by Ring-Opening Metathesis Polymerization

Liliana Mora ’17; Mentor: Daniel O’Leary;

Antibiotic resistance has precipitated the search for innovative ways of killing bacteria. One idea that shows much promise is that of synthetic polymer-drug conjugates. Their potential comes from the ability to control their chemical properties, which depends on what monomers make up the chain. If the conditions are right, the polymer will form a nanoparticle delivery system with slow release of antibiotics such as Ciprofloxacin, making it more efficient and accessible. In past research, cis-5-Norbornene-exo-2,3-dicarboxylic anhydride has been used as starting material for monomers. However, the binding of 4-Hydroxybenzylamine and its effectiveness for the conjugated polymer will be explored, as only derivatives of it have been previously used. The project’s synthetic approach involved the isomerization of cis-5-norbornene-endo-2,3-dicarboxylic anhydride to then react with 4-hydroxybenzylamine and form a monomer. The drug, Ciprofloxacin, was protected with Boc2O in order to activate it with NHS and react with the monomer in an esterification reaction. A microwave reactor and flash chromatography were key in the production and purification of the polymer’s building blocks. Further research would encompass taking the monomers into ring-opening metathesis polymerization, otherwise known as ROMP, to build the copolymer. ROMP is a powerful method for the synthesis of the polymer chain due to the high control over the polymer’s molecular weight and molecular distribution.
Funding Provided By: Neil Smith Hansch

Probing Alzheimer’s Pathology with Diazirine tagged tri-peptide

Doug Goldstein ’16; Mentor: Daniel O’Leary and Karen Parfitt; Collaborator: Greg Copeland (City of Hope)

Alzheimer’s disease (AD) is a widespread condition characterized symptomatically by memory loss and cognitive decline and molecularly by the build-up of amyloid plaques in the brain. Surprisingly, the precursor to these harmful Aβ plaques (Amyloid precursor protein or APP) can also be cleaved to form secreted amyloid precursor protein alpha (sAPPα), which has been shown to increase synaptic performance and long term potentiation. This means that AD is not just characterized by an increase in Aβ oligomers, but also by a decrease in sAPPα. Therefore, understanding the mechanism through which sAPPα works against Aβ to promote cognitive function in a healthy brain could provide an entree into counteracting cognitive decline in an AD brain. sAPPα has a unique set of 16 amino acids, of which contain its active domain: an RER tripeptide. In order to investigate sAPPα, this RER tripeptide was synthesized and tagged with a diazirine crosslinking agent. Once this process has been optimized, the diazirine RER tripeptide will be injected into a rat model so that pull down assays can be used to determine what RER associates with. This finding will allow for a better understanding of the role sAPPα plays in AD and may even lead to a novel therapeutic co-opting sAPPα’s ability to counteract Aβ’s negative effects.
Funding Provided By: Fletcher Jones

Protein engineering of toluene/o-xylene monooxygenases (ToMO) mutants to enhance terminal hydroxylation of alkanes.

Kimberly Ona Ayala ’16; Mentor: Matthew Sazinsky; Collaborator: Nahlee Lin ‘17

Carboxylate-bridged diiron hydroxylases found in bacterial multicomponent monooxygenases (BMM) systems hydroxylate unactivated hydrocarbons. Their ability to metabolize organic compounds with high regiospecificity and stereospecificity has potential applications in laboratory synthesis and bioremediation. We aim to compare the hydroxylating capabilities and structural features of two archetypal BMMs: toluene/ortho-xylene monooxygenase (ToMO) and soluble methane monooxygenase (sMMO). Both have similar global topology and nearly identical reduced active site structures, but differ in configuration and substrate preference when activated by their respective regulatory proteins. We identified three key structural distinctions to account for the difference in substrate specificity: diiron center rigidity, catalytic intermediates, and manner of substrate access to the diiron center. We explored these differences by performing site-specific mutagenesis on the α-subunit of ToMOH to make mutants T201S and E111N. We report a decreased rate of hydroxylation by the mutant E111N (1.014 μM/min) over the wild type (3.9 μM/min). We believe the E111N mutant has catalase activity, converting H2O2 into O2 and H2O, and turns over H2O2 significantly faster in the absence of the regulatory protein, ToMOD. These preliminary results suggest hydrogen bonding in the second coordination sphere around the iron atoms plays a significant role in controlling the type of chemistry of the metal center.
Funding provided by Telzer/Hansch (Ona Ayala), Pomona College Chemistry Department (Lin)

QCM-D Study of MPS-PPV:Surfactant Bilayers

Conner Kummerlowe ’16; Mentor: Malkiat Johal

Poly p-phenylvilyene (PPV) is an optically active polymer with applications in solar cells, biosensors, and light emitting devices. PPV derivatives degrade in the presence of water, rendering them unusable. We studied bilayers of MPS-PPV and the positively charged polymer polyethylenimine (PEI) with varying levels of the surfactant dodecyl trim-ethyl ammonium bromide (DTAB) in aqueous and gas phase Quartz Crystal Microbalance (QCM-D) systems. The goal of this study is to characterize the interactions between MPS-PPV and water and to develop a means of excluding water from PPV bilayers.
Funding Provided By: Norris

QSAR on the radical scavenging properties of a series of BHT derivatives

Samantha Morrison ’16; Mentor: Cynthia Selassie

Many studies have found that phenolic compounds, present in various plants, fruits, and vegetables, seem to have antioxidant protective effects. These compounds can act in a protective way through radical scavenging of free radicals, potentially resulting in a decreased formation of reactive oxygen species (ROS) that are damaging to cells. The phenolic compound 2,6-di-tertbutyl-4-methyl phenol (BHT) is widely used in food products as an antioxidant to prevent oxidative spoilage. In this study, the radical scavenging properties of a series of BHT derivatives are determined using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. In the DPPH assay, the stable DPPH radical is scavenged by the antioxidant phenolic compound, resulting in a disappearance of violet color detectable by UV/Vis spectroscopy. To compare the radical scavenging ability of the BHT derivatives, the DPPH assay is used to quantify the IC50 values, the concentration at which 50% DPPH radical is scavenged, of each BHT derivative. The Quantitative Structure-Activity Relationships (QSAR) paradigm is then applied to the IC50 values in order to correlate the radical scavenging abilities of the BHT derivatives to the physicochemical attributes of the phenols, such as their lipophilicities, sterics, and electronics. A systematic analysis of the overall structure-activity relationships will help to elucidate the role of structural features and their physicochemical attributes in their radical scavenging abilities.
Funding Provided By: Howard Hughes Medical Institute

Sorbent Polymer Selection for Raman Spectroscphy

Aseal Birir ’18; Mentor: Charles Taylor

Bacterially produced volatile organic compounds (VOCs) present in breath are useful biomarkers for pulmonary diseases such as pneumonia. Breath analysis is an attractive alternative to invasive sampling methods for detection of VAP and other pulmonary diseases. To identify biomarkers (compounds) using a breath analysis, Raman spectroscopy system is coupled with a sorbent polymer lens coating to identify gaseous compounds such as the VOCs released by bacteria. To first identify biomarker released by bacteria, we must first optimize the instrument to help it identify the VOCs that are present. To start running tests using the Raman instrument to identify compounds, we must first optimize it by figuring out which polymer works best for each compound that may be present in the VOCs. This was done by running a sorbent selection apparatus in which we allowed 9 different polymers to all absorb one of four polymers for a period of time. A GCMS was then used to identify the amount of compound absorbed in the polymer within the four trials. The four compounds used for the experiment were EtAOc, IVA, Pentanol, and 2-Butanone. The results showed Carboxen 1012 was best for absorbing EtAOc, Carboxen 1016 was best 2-Butanone, Carboxen 1012 was best for Pentanol, and Carboxen 569 was best for IVA. These are the best polymers for each compound. This data helps us optimize the Raman Instrument to work better with compounds we expect to be released. The will help us identify more biomarkers.
Funding Provided By: Norris

Synthesis and Characterization of Porous Cross-Linked Elastomers with Applications to the Dry Cleaning of Artwork

Matthe Alexander ’17; Mentor: Robert Grubbs (Stony Brook University); Collaborator: Anna Flach (Stony Brook University)

Dirt and grime removal from paintings is a prevalent issue for art conservators in situations where the paint surfaces are sensitive to solvent based cleaning methods. However, the abrasive nature of dry cleaning sponges and risk of surface contamination are of great concern to conservators. In collaboration with the Metropolitan Museum of Art, porous cross-linked elastomers (PCEs) were studied in an effort to discover a new spongy material free of the risks present in the current cleaning methods. PCEs were synthesized by using ring-opening metathesis polymerization (ROMP) of cyclooctene (CO) with dicyclopentadiene (DCPD) as the cross-linker. Optimization of the polymerization conditions was explored by varying cross-linker concentration, monomer concentration, ruthenium catalyst, and solvent composition. The PCEs were softer, more flexible, and more “sponge-like” with a 9:1 CO to DCPD mole ratio and polymerization in a 3:1 (m/m) isopropanol to toluene system. Characterization of the elasticity of the synthesized PCEs was accomplished via oscillatory shear rheology. It was found that the storage (elastic) and loss (viscous) moduli for the PCEs were generally much larger than various commercially available sponges. This indicates that the PCEs are more “solid-like” and harder than the commercial sponges. Further research requires modifying the ROMP monomers and polymerization conditions to synthesize PCEs that have physical properties comparable to the commercial sponges.
Funding Provided By: American Chemical Society Petroleum Research Fund

Synthesis of a Meropenem-based RAFT Monomer for Drug Delivery Applications

Mario Gaviria ’18; Mentor: Daniel O’Leary

The overall goal of this project was to synthesize a polymer that will act a drug delivery system for Meropenem. Meropenem is a broad-spectrum β-lactam that will slowly release off of the polymer to allow for long-term dosage. This summer we have synthesized monomers that will be polymerized at the University of Washington through reversible-addition fragmentation chain transfer (RAFT) polymerization. We have created the monomers in four reactions. Similar to peptide synthesis methods, we first used DCC to couple mono-2-(methacryloyloxy) ethyl succinate with either Tyramine or 4-hydroxy Benzylamine. The methacrylate is the functional group that allowed us to perform RAFT polymerization. The amines act as a linker, which pushes the Meropenem off the polymer scaffold, which has been shown at the University of Washington to facilitate the hydrolysis of antibiotics off the polymer scaffold. The difference in carbon chain length between the two amines will allow us to examine the varying rates of the release of Meropenem. In order to form the activated antibiotic we coupled the carboxylic acid on Boc protected Meropenem with 2-Thiazoline-2-thiol. The Thiazoline acts as a better leaving group, and allows for the selective coupling of the activated Meropenem with the primary alcohol on the phenol-monomer.
Funding Provided By: Norris

Synthesis of Anti-Maliarial Bifunctional Histone Deacetylase and Dihydrofolate Reductase Inhibitors

Nate Billet ’17; Mentors: Cynthia Selassie and Benjamin Sveinbjornsson; Collaborator: Steven Chen ‘17

Malaria is a mosquito-borne infectious disease that is the leading cause of death in many developing countries in Africa, Latin America, and Southern Asia. Plasmodium falciparum is the most common and deadly parasite that is known to cause malaria in humans, with over 75% of cases in Africa attributed to it. While there are malaria treatments on the market, the rapid evolution of P. falciparum has given it increased resistance to currently used drugs. Histone deaceytlases (HDAC) play an important role in the regulation of gene expression. HDACs remove the acetyl group on histones, increasing their ability to bind to DNA, thus hindering genetic transcription. Recent studies have shown HDAC inhibitors to be effective in P. falciparum growth in vitro due to gene deregulation. Dihydrofolate reductase (DHFR) is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, which is an essential cofactor in the metabolism of amino acids and nucleic acids. DHFR inhibitors have also been shown to exhibit anti-malarial activity. The goal of this project is to synthesize a bifunctional anti-malarial drug by combining HDAC and DHFR inhibitor moieties onto the same molecular backbone. We predict that this dual inhibitor will show greater anti-malarial properties than their individual components. Furthermore, by including both moieties on the same molecular backbone, we can negate issues of drug metabolism and absorption that would hinder non-hybrid molecules.
Funding Provided By: Pomona Unrestricted (Billett), Stauffer (Chen)

The Synthesis and Implications of a Meropenem based RAFT Antimicrobial Monomer

Julia Swanson ’16; Mentor: Daniel O’Leary

The overall goal of this project was to synthesize an antimicrobial polymer that will act as a drug delivery system for meropenem. Meropenem is a β-lactam antibiotic that is highly desirable in treatment of a broad range of diseases. As a collaborative effort with the University of Washington, we have synthesized meropenem based reversible-addition fragmentation chain transfer (RAFT) monomers. These monomers will be studied for their release profile of a highly potent clinical antibiotic, as well as, polymerized to form a polymer drug conjugate delivery system. Based on protocol from the University of Washington, we conjugated a RAFT methacrylate monomer with either Tyramine or 4-hydroxy Benzylamine. The amines act as a linker, which separates meropenem from the polymer backbone, which has been shown to increase rates of antibiotic hydrolysis in previous studies. We then conjugated carboxybenzyl-protected meropenem to this monomer. The Cbz-meropenem RAFT monomer was purified and analyzed through NMR. Future studies will look at the hydrolysis rates of meropenem off of analogous meropenem monomers and determine the viability of this approach as a drug delivery system.
Funding Provided By: Pomona College Chemistry Department

Ultrasensitive Detection of Biomarkers in Response to Chemical and Biological Weapons Exposure

Sebastian Cevallos ’18; Mentors: Matthew Sazinsky and David Walt (Tufts University); Collaborator: Payel Ghatak (Tufts University)

Shiga toxin and ricin both belong to the same family of protein toxins and are categorized as ribosome inactivating proteins. They are known for inducing massive cell death by inhibiting protein synthesis and signaling apoptotic pathways upon entry. This causes the release of stress-induced proinflammatory cytokines in the body, as part of the body’s immediate immune response. Therefore, being able to identify the small rise in proinflammatory cytokine concentration after infection would enable earlier detection of the toxin, before major damage is done to the infected individual. However, the concentration increase is too small for conventional methods to track. But using single molecule arrays (SiMoA), it is possible to develop assays that would allow us to quantify the cytokine levels of an infected individual, increasing their chances of survival.
Funding Provided By: Pomona College Research Fund

Using TIR-Raman Spectroscopy to Identify VOCs in Liquid and Gaseous Phases

Emily Chang ’16; Mentors: Charles Taylor and Angelika Neimz (KGI); Collaborator: Hsiang-Wei Lu (KGI)

Ventilator-associated pneumonia (VAP) is a common, but often difficult to diagnose, phenomenon in intubated patients. Traditional diagnostic methods can be invasive and take long periods of time to achieve results. However, the volatile organic compounds (VOCs) produced by bacteria and fungi associated with VAP can be used in a less invasive diagnostic procedure. Thus, we have proposed using a total internal reflection (TIR) Raman spectroscopy system to identify VOCs and the microbes associated with their production. Using a Raman-based system that students have optimized previously and 2D correlation methods, we have obtained spectra for a variety of VOCs produced by Staphylococcus aureus and Pseudomonas aeruginosa, including 2,4-di-tert-butylphenol, a compound that has only recently been shown to be produced by P. aeruginosa. We have also begun testing a new liquid stage that allows us to obtain spectra for desired solutions and we hope to eventually be able to analyze the media flow-though from P. aeruginosa biofilm and planktonic growth runs. We are also interested in finding new polymer solutions to use as the sorbent layer. To date, we have tested First Contact (a proprietary lens coating polymer), as well as poly-4-vinylpyridine and poly-2-vinylpyridine.
Funding Provided By: Stauffer


Characterizing the Elemental Composition of Coffee Using Flame Atomic Absorption Spectroscopy

Emily Chittick (2017); Mentor(s): Charles Taylor

Abstract: Research has demonstrated that the elemental composition of coffee can be used to pinpoint its location of origin (an important factor in its commercial value). Previous research in our lab focused trace elements in coffee samples acquired using wavelength-dispersive X-ray fluorescence combined with principle component analysis (PCA). Important findings included that rubidium and manganese were key in describing the majority of samples but are insufficient for characterizing growth location. This investigation adds major elements (i.e. potassium, sodium, magnesium, etc.) analyzed via flame atomic absorption spectroscopy (FAAS). FAAS has the advantage of higher throughput after sample preparation. Results were analyzed via PCA to determine which of the eight major elements investigated are most important in distinguishing samples’ origins. In addition, this work confirmed the suspicion that elevated rubidium correlates with decreased sodium in the coffees studied.
Funding Provided by: Schulz Fund for Environmental Studies

First Steps Towards Reverse Engineering a Polymeric Lens Coating Material

Ryan Dodson (2015); Mentor(s): Charles Taylor

Abstract: Ventilator-associated pneumonia (VAP) is a serious health concern. Current VAP detection methods are time-consuming and costly, so it is of clinical interest to construct an instrument that cheaply and quickly detects VAP in a patient. Towards this end, we are developing an instrument for rapid diagnosis of VAP that uses Raman spectroscopy to analyze volatile organic compounds (VOC) in a patient’s breath. In order to achieve relevant detection limits for known disease biomarker VOCs, our system employs a commercially available polymeric lens coating material to concentrate VOCs on the sensing element surface. This polymer has thus far outperformed other trial polymers but the structure of the polymer is unpublished. A thorough understanding of the polymer would permit the design of more useful polymers, and thus optimization of this aspect of the VOC detection system. In this study, I attempt to elucidate the polymer’s structure using a number of analytical methods, including elemental analysis, IR spectroscopy, Raman spectroscopy, and NMR spectroscopy. Preliminary results are promising but additional work is necessary in order to confirm the proposed structure. Next steps include synthesis and analysis of the proposed polymer.
Funding Provided by: R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry

Bacterial Biofilm VOC Analysis via GC-MS/SPME

Michael Etzel (2015); Student Collaborator(s): Paige Oliver (2016), Daniel Pak (KGI); Additional Collaborator(s): Angelika Niemz (KGI), Ilya Tolstorukov (KGI); Mentor(s): Charles Taylor

Abstract: Bacterial attachment to implanted medical devices and subsequent development of biofilms has been found to have profound effects on hospitalized patients. Ventilator associated pneumonia (VAP) is a common occurring nosocomial infection among patients whose immune systems are already compromised. Current methods of diagnosis are too slow (culture growth takes 2-3 days), and presumptive diagnosis leads to the overuse of antibiotics, which may increase the proliferation of antibiotic resistant strains. By analyzing the volatile organic compounds (VOCs) that are given off by bacterial biofilms grown in medically relevant conditions, new biomarker VOCs may be found and used to diagnose patients in a quicker, more accurate manor. In order to identify these biomarkers, we must grow bacterial biofilms in a system analogous to the tracheal tube of a hospital patient. Using silicone tubing, we were able to grow a uniform E. coli biofilm, helping us determine VOCs associated with E. coli including, indole and benzothiazole.
Funding Provided by: Pomona College SURP (ME); Fred J. Robbins Chemistry Fund (PO)

Measuring Uptake of Volatile Organic Compounds into Polymer Thin Films Using QCM-D

Alison Mercer-Smith (2015); Student Collaborator(s): Ryan Dodson (2015), Kelly Park (2012); Additional Collaborator(s): Angelika Niemz (KGI); Mentor(s): Charles Taylor

Abstract: Many pathogenic bacteria emit characteristic volatile organic compounds (VOCs) as metabolic waste. These VOCs and patterns in their abundances may be used to identify the bacteria. We are developing a spectroscopic platform for early infection detection based on VOCs produced by the pathogenic bacteria. We are exploring VOC sorption behavior for several polymers of interest using quartz crystal microbalance with dissipation monitoring (QCM-D). Our goal is to use these QCM-D measurements to build a library of polymers that will enable us to coat our lens with a polymer that has good kinetics and selectivity towards analytes of interest.
Funding Provided by: Howard Hughes Medical Institute (AMS); R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry (RD); Paul K. Richter and Evalyn E. Cook Richter Memorial Fund (KP)

Synthesis of an Anti-Malarial DHFR/HDAC Hybrid Inhibitor for Treatment Against Plasmodium falciparum

Jerry Jiang (2015); Mentor(s): Cynthia Selassie

Abstract: In the 2013 World Malaria Report compiled by the World Health Organization, there were an estimated 207 million cases and 627,000 malaria-related deaths, 90% of which occurred in sub-Saharan Africa. The lethality and preventability of this disease highlight the necessity to develop effective forms of treatment. Histone deacetylases (HDAC) play a pivotal role in gene regulation. These enzymes catalyze the removal of acetyl groups from ε-N-acetyl lysine residues on histone units upon which DNA is wound. The acetylation status of histones modulates chromatin packing which affects the accessibility of transcription factors to DNA, thereby regulating gene expression. Recent studies have demonstrated HDACIs to inhibit growth of Plasmodium falciparum in vitro, purportedly through transcriptional deregulation. An additional protein system important for malarial progression is the dihydrofolate reductase (DHFR) and thymidate synthase (TS) system. In Plasmodia, DHFR and TS cooperatively drive DNA formation via nucleotides biosynthesis. As dihydrofolate reductase inhibitors have been shown to possess significant anti-malarial activity, they are a promising moiety for the further development of a novel anti-malaria drug. Here, we outline the synthesis of a novel anti-Malaria compound. By combining HDAC and DHFR inhibitor moieties onto a single molecular backbone, we create a dual HDAC/DHFR inhibitor hybrid that we hope to display exceptional anti-malarial qualities.
Funding Provided by: Corwin Hansch and Bruce Telzer Fund

Synthesis of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3’-(4”-N-hydroxyacetamidebenzyloxy)phenyl)-s-triazine as a potential dual DHFR/HDAC Inhibitor

Briton Lee (2015); Mentor(s): Cynthia Selassie

Abstract: Malaria disease is caused by several species of Plasmodium, with one of the more lethal species being P. falciparum. Though there are different ways to treat malaria, malaria become increasingly resistant to current forms of medicine, such as artemisinin. Since dihydrofolate reductase (DHFR) is essential in the synthesis of essential biological molecules, the inhibition of this enzyme has been researched for anti-malarial properties. Multiple derivatives have been previously synthesized in the Selassie lab in order to develop quantitative structure activity relationship (QSAR) models to optimize antimalarial activity while minimizing cytotoxicity to human cells. In addition to these DHFR inhibitors, there are histone deacetylase (HDAC) inhibitors that, among other properties, have promising, potent antimalarial activity. HDACs are integral in gene regulation, as they promote the coiling of DNA by deacetylating histones and exposing positively charged lysine residues. The goal of this project is to synthesize a hybrid molecule that functions as both a DHFR and HDAC inhibitor by building off the familiar backbone of DHFR while adding critical moieties characteristic of HDAC inhibitors. We predict that having the functional sites of both DHFR and HDAC inhibitors on the same molecule will show greater antimalarial activity than either inhibitor alone, and work towards creating a more effective class of antimalarial drugs.
Funding Provided by: Howard Hughes Medical Institute

Effects of Phenolic Compounds on Cytotoxicity of L1210 Cells

Samantha Morrison (2016); Mentor(s): Cynthia Selassie

Abstract: Phenolic compounds have been found to have protective, antioxidant properties, and prooxidant, cytotoxic properties. Prior in vitro and in vivo studies have characterized the radical scavenging, metal chelating antioxidant properties of phenols as well as their cytotoxic effect in mammalian cell cultures. Using this data, QSAR models were derived and it was determined that the cytotoxicity of electron-withdrawing phenols is due to their overall hydrophobicity, while the cytotoxicity of electron-releasing phenols is attributed to their ability to form phenoxy radicals. In this project we cultured L1210 murine leukemia cells and ran cytotoxicity assays on the L1210 cells to determine the IC50 for four 4-X phenols and four estrogenic phenols. The cytotoxicity assays of the 4X phenols resulted in dose response curves that indicated that the 4-X phenols caused an inhibition of cell growth; however, the estrogenic phenols seemed to have no effect on the L1210 cells. Future work on this project will involve a fluorescence/luminescence assay to assess the degree of mitotoxicity induced by the X-phenols in order to further elucidate the specific mechanism of action of phenolic compounds and the site of their critical toxic reactivity.
Funding Provided by: Neal W. Cornell Fund for Student-Faculty Research

Studying the Hydration of BSA-Ligand Complexes Using the Quartz Crystal Microbalance and Dual Polarization Interferometer

Noah Stanton (2015); Mentor(s): Cynthia Selassie; Malkiat Johal

Abstract: Rational drug design requires understanding the properties of drug-like molecules, as well as the interaction between the ligand and its binding site. However, predicting interactions between drugs and binding sites is complicated by the involvement of water. Accounting for the movement of water within binding pockets can allow for a more complete understanding of binding interactions of ligands. Our methodology takes advantage of the different hydration sensitivities of two instrumental techniques — dual polarization interferometery (DPI) and quartz-crystal microbalance with dissipation monitoring (QCM-D) — in order to track the movement of water molecules upon drugs binding to immobilized bovine serum albumin (BSA), a common bloodstream protein. We employed this methodology to determine the approximate number of water molecules involved in desolvation ligand–protein complexes upon binding. We examined eight drugs with different hydrophobicities in this manner in search for a relationship between the ligands’ physicochemical properties and its desolvation behavior with BSA. We aim to use this data and quantitative structure activity relationship (QSAR) models to relate these desolvating properties to the structure of the drugs. If such a relationship exists, it would be of great importance in using rational drug design techniques to develop drugs with higher affinity for their targets.
Funding Provided by: Sherman Fairchild Foundation

Synthesis of a dual inhibitor prodrug for inhibition of enzymes vital to the life cycle of P. falciparum

Elise Wolf (2016); Mentor(s): Cynthia Selassie

Abstract: Plasmodium falciparum is the prevalent strain of malaria-inducing protozoa responsible for most malarial related deaths worldwide. High occurrence of resistance in P. falciparum combined with its prevalence in third world countries, particularly sub-Saharan Africa, make it an area of high importance in drug development. The enzymes dihydrofolate reductase (DHFR) and falcipain-2 /3 (FP 2/3) have been highlighted as crucial to various aspects of P. falciparum survival. A dual inhibiting pro-drug targeting both enzymes, connected by an amide linkage that would be cleaved in thebloodstream, would be valuable in treatment. Treatment has previously included multiple drug therapies that are hard to monitor and, when not taken to completion, often result in further drug resistance. The first of the two drugs is a pyrimidine derivative of trimethoprim, an antimalarial and DHFR inhibitor that is decreasing in effectiveness due to mutation that confers resistance. The derivative was successfully synthesized and nitrated to give one side of the amide linkage. The second drug, a proposed FP 2/3 inhibitor, is a larger molecule also containing a pyrimidine moiety as well as other optimized functional groups, and has proven more difficult to synthesize due to the reactivity of various groups within the overall molecule. Synthesis of the pro-drug will hopefully result in two drugs that independently show antimalarial activity, and together create a twopronged attack on the malaria protozoa.
Funding Provided by: Neal W. Cornell Student-Faculty Chemistry Research Fund

Synthesis of Peptidomimetic Analogues of Temporin-SHf

Nathaniel Ash (2015); Mentor(s): Daniel O'Leary

Abstract: The previously studied antimicrobial property of Temporin-SHf (FFFLSRIF), a hydrophobic octapeptide isolated from the skin of the Pelophylax saharica, was the impetus for the synthesis of various analogues of this molecule. Because naturally occurring Temporin-SHf consists of all Lstereoisomer amino acid residues, we were interested in creating peptidomemetic molecules using R-stereoisomer amino acid residues instead. The three Temporin analogues we synthesized were the D-stereoisomer, the reverse L-stereoisomer (FIRSLFFF), and the reverse D-stereoisomer (FIRSLFFF). One of the main reasons for creating these analogues was to test their antimicrobial activities relative to Temporin. These analogues of Temporin are of interest because of the potential antimicrobial activity they could share with Temporin, and also because D-peptides are generally less susceptible to proteolysis in the stomach and in cells. In the future we would like to test the antimicrobial properties of these newly synthesized analogues as well as their proteolytic stabilities.
Funding Provided by: Fred J. Robbins Chemistry Fund

Synthesis of Hydrogen Bond Surrogate Analogs of Temporin-SHf

Phillip Clayman (2016); Mentor(s): Daniel O'Leary

Abstract: All species produce antimicrobial peptides (AMPs) as a non-specific response to infection. Increased resistance to current antibiotics necessitates the development of new therapies and AMPs could serve as a novel class of antibiotics. Temporin-SHf, an AMP isolated from a frog, is of particular interest due to its small size, high ratio of hydrophobic and aromatic residues, and broad spectrum antimicrobial activity. The α-helix structure that Temporin-SHf forms in a micellar environment is thought to be an important factor in its antimicrobial activity. A hydrogen bond surrogate (HBS) which replaces a hydrogen bond with a carbon-carbon bond was used to enhance this α-helix structure. Building the entire peptide using solid phase peptide synthesis (SPPS) proved extremely difficult as coupling to an allylated residue resulted in minimal product formation. To avoid a difficult secondary coupling, an allyl dipeptide was synthesized in solution phase then used in SPPS. Although the dipeptide coupling went to completion under standard conditions, significant racemization was observed. Racemization was minimized using propylphosphonic anhydride and pyridine in ethyl acetate though the yield was not ideal. Future work requires the synthesis of more allyl dipeptide, further optimization of the dipeptide coupling, followed by full synthesis of the HBS Temporin-SHf.
Funding Provided by: Dale N. Robertson Fund

A Chemoenzymatic and Organic Synthesis of Cytidine Diphosphate Glucose for the Characterization of PERM-1 in Ascarylose Biosynthesis in C. Elegans

Christian Gomez (2015); Additional Collaborator(s): Katy Muzikar; Mentor(s): Daniel O'Leary, Sara Olson

Abstract: Ascarylose biosynthesis in the model organism C. elegans has received little attention in the past. However, ascarylose plays a crucial role in the formation of the impermeable eggshell during C. elegans development. Furthermore, knowledge about this eggshell may aid in the prevention of infection by parasitic nematodes, which have similar shell composition. The goal of this project is to characterize the newly-identified gene product PERM-1, an enzyme which may catalyze the final transformation in ascarylose biosynthesis in C. elegans The current investigation utilized both organic synthesis and chemoenzymatic approaches to synthesize cytidine diphosphate glucose (CDPglucose), the first product in the synthetic pathway towards CDP-4-keto-3,6-dideoxyhexulose, the putative substrate for PERM-1. Along with the synthesis, conditions were developed for monitoring and analyzing reaction products using a variety of analytical techinicques, including HPLC, TLC, NMR and LC-MS.
Funding Provided by: Fred J. Robbins Chemistry Fund

Synthesis of Malformin A1 and its ring-closed S-allyl-D-cysteine analog

David Kolin (2016); Mentor(s): Daniel O'Leary

Abstract: Malformin A1, a bicyclic pentapeptide isolated from Aspergillus niger, exhibits antibiotic properties believed to originate from its disulfide bridge. The purpose of this project is to synthesize Malformin A1 and a ring-closed S-allyl-D-cysteine analog of the peptide. Thus, comparing Malformin A1 to the allylcysteine analog will elucidate the importance of the disulfide bridge to the bioactivity of Malformin A1. Throughout the course of the summer, Fmoc-S-allyl-D-cysteine and Fmoc-Sbenzyl-D-cysteine were synthesized on a large scale in preparation for solid-phase peptide synthesis. To synthesize the desired amino acids, first Dcysteine’s sulfur group was protected using either allyl or benzyl bromide. Then, the amino ends of the residues were protected by the Fmoc group. Solidphase synthesis was used to form the remainder of the benzyl-protected peptide. The next step will be to remove the benzyl groups and oxidize the cysteine residues to form the desired Malformin A1 product. For the S-allyl-D-cysteine analog, ring closing metathesis (RCM) will be performed using the novel Grubbs’ catalyst to synthesize the bicyclic analog.
Funding Provided by: Rose Hills Foundation

Synthesis of a Ligand Precursor for Kinetic Isotope Effect Studies in a Metathesis-Relevant Ruthenium Complex

Cristina Saldaña (2015); Mentor(s): Daniel O'Leary

Abstract: The C-H activation in a metathesisrelevant ruthenium complex has garnered attention since it is unprecedented for ruthenium metal centers. To better understand it, nuclear magnetic spectroscopy (NMR) techniques have been employed. Specifically, they have been applied to study the kinetic isotope effects (KIE) of the C-H activation step. As part of the larger goal, this study focused on efficiently synthesizing the ligand precursor for the KIE studies. To be able to observe the activation step, several markers, carbon 13 and deuterons, need to be strategically placed on the ligand precursor. Thus, cost and reaction conditions were kept in mind during the synthesis. Once yield and purity are optimized, collaboration with Caltech will bring the two components of the complex together for the KIE studies. With these studies, we hope to lower the uncertainties found in a previous study.
Funding Provided by: Fred J. Robbins Chemistry Fund

Laboratory Study of Polymerization Reactions in Titan’s Lakes

Reina Buenconsejo (2015); Additional Collaborator(s): Robert Hodyss (Jet Propulsion Laboratory), Jeffrey Hein (Jet Propulsion Laboratory); Mentor(s): Frederick Grieman

Abstract: Laboratory studies of polymerization reactions in Titan's lakes were conducted. Titan is the largest Saturnian moon and the only natural satellite in our Solar System that has a dense atmosphere. This atmosphere is made mostly of molecular nitrogen and methane which react via photolysis from solar ultraviolet (UV) light to produce a variety of other species including HCN, butadiene, ethylene, acetylene, and acrylonitrile. These products eventually reach the surface and can dissolve in Titan’s hydrocarbon lakes and seas. Once dissolved, these products may react with free radicals (such as the cyanoethynyl radical formed from photolysis of dicyanoacetylene) or each other to form larger molecular weight polymers. Before studying the radical reaction involving the products, the formation of the radical must first be understood. Dicyanoacetylene is difficult to synthesize in the laboratory so azobis (isobutyronitrile) (AIBN) was used as the free radical source. We used matrix isolation infrared and ultraviolet spectroscopy to study the photochemistry of AIBN at low temperatures (12K). A variety of microwave discharge lamps were used and irradiation times were varied to determine the effect of wavelength and irradiation time on the formation or radical AIBN. FTIR spectra taken before and after irradiation show changes occur after irradiation that may indicate the formation of the 2cyanoprop-2-yl radicals radical. Matrix-isolated ethylene will be studied next after which AIBN and ethylene will be mixed in liquid ethane to simulate the reaction in Titan’s hydrocarbon lakes.
Funding Provided by: Linares Family SURP for Chemistry

Determination of the Temperature Dependence of the Rate Constants for HO2 /Acetonylperoxy Reaction and Acetonylperoxy SelfReaction

Emily Darby (2015); Student Collaborator(s): Aileen Hui (California Institute of Technology); Additional Collaborator(s): Stanley Sander (Jet Propulsion Laboratory), Mitchio Okumura (Jet Propulsion Laboratory); Mentor(s): Frederick Grieman

Abstract: Using a high sensitivity Infrared Kinetic Spectroscopy (IRKS) method, we are able to quantitatively determine the kinetics of atmospherically important reactions of the hydroperoxy radical, HO2 . Reactions of HO 2 with small alkylperoxy radicals are of interest due to their impact on the oxidation chemistry of the troposphere. These reactions have the potential to have a significant impact on the HOX inventory, and ultimately the ozone budget of the troposphere. The acetonylperoxy radical is a particularly abundant alkylperoxy in the troposphere because it is formed from the degradation of various volatile organic compounds emitted in large amounts to the atmosphere. Because previous studies of the HO2 /acetonylperoxy reaction have resulted in conflicting values and have only been conducted at room temperature, we have studied the kinetics of the HO2 /acetonylperoxy reaction and the acetonylperoxy self-reaction. In the IRKS method, the reactions are studied using a temperaturecontrolled slow-flow tube apparatus where flash photolysis of Cl2 is used to rapidly produce the HO2 radicals from methanol. Direct observation of the rate of reaction is accomplished by simultaneously measuring the HO2 disappearance using near-IR diode laser wavelength modulation spectroscopy and the acetonylperoxy disappearance in the ultraviolet at 300 nm. By directly measuring the HO2 and acetonylperoxy disappearance over an atmospherically relevant temperature range, we accurately determined the temperature dependence of these two rate constants.
Funding Provided by: Sherman Fairchild Foundation (ED)

Infrared Kinetics Spectroscopy Studies on HO2 Radicals from Reactions of the Criegee Intermediate CH2OO

Erin Delaria (2015); Additional Collaborator(s): Linhan Shen (Caltech/JPL), Sander Stanley (Jet Propulsion Laboratory); Mentor(s): Frederick Grieman

Abstract: Ozonolysis, a major removal mechanism for unsaturated hydrocarbons in the atmosphere, proceeds through a highly reactive carbonyl oxide intermediate, known as the Criegee intermediate (CI). CIs are very reactive species with substantial internal energies. The rapid isomerization and decomposition of CIs results in the formation of OH radicals, which represents a significant nighttime source of HOX. The simplest Criegee intermediate, CH2OO, has previously been shown to undergo unimolecular dissociation into OH and HCO radicals. The subsequent reaction of HCO with O2 also results in the formation of HO2 To study the kinetics of CI decay, we performed laboratory measurements of HO2 formed from CH2OO dissociation. In our laboratory studies, CH2OO was created through pulsed laser photolysis of a CH2I2/O2/N2 gas mixture using a 351nm XeF excimer laser. The formation and decay of HO2 was monitored in the near IR using Infrared Kinetics Spectroscopy (IRKS) with a frequency-modulated signal at 6628.6 cm-1. IO, a significant sink of HO 2 radicals and a major secondary product in this photochemical system, was simultaneously monitored in the UV at 427nm. The estimated overall unimolecular decay rate of CH2OO was 3000 s-1, equivalent to a lifetime of ~300 µs. The formation rate of HO2 was estimated to be 719 s-1 at 30 torr. The rate of CH2OO decay and HO2 formation shows significant pressure dependence in the range 10-60 torr.
Funding Provided by: R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry

CRP Is a Regulator of the sRNA MtlS in Vibrio cholerae

Tina Solvik (2015); Mentor(s): Jane Liu

Abstract: In Vibrio cholerae, the small noncoding RNA MtlS represses expression of mtlA, encoding the mannitol-specific transporter. In contrast, cAMP receptor protein (CRP) is an activator of mtlA expression. Here, I investigated whether CRP has any affect on mtlS expression. I hypothesized that CRP would act as a repressor of mtlS, which would be consistent with CRP’s role in activating mtlA. To determine the effects of CRP on MtlS levels, I used northern blot analysis with wild-type and crp mutant strains. I consistently observed lower MtlS levels when CRP was absent, suggesting that CRP may be an activator of mtlS. I also studied the effects of excess CRP on MtlS levels through northern blot analysis. No significant difference in MtlS was observed between a strain that overexpressed crp and a control. To locate regulator-binding site(s) for mtlS, I used lacZreporter fusions with varying lengths of the mtlS promoter region fused to lacZ. I found increased LacZ activity in the 250+ bp strains, suggesting that a regulator of mtlS transcription may bind between the -100 and -250 region of the mtlS promoter. LacZ activity was also assayed in a lacZ-reporter strain lacking crp, and then compared to the wild-type. I found that the absence of CRP increased activity, suggesting that CRP is a repressor of mtlS. These results suggest that CRP is a regulator of mtlS, and that it may have multiple binding sites, allowing it to act as a repressor and as an activator.
Funding Provided by: National Science Foundation #CBET-1258307: PI J. Liu; American Society of Microbiology

Developing a BPA-Responsive Riboswitch Through Dual Genetic Selection

Erick Velasquez (2016); Mentor(s): Jane Liu

Abstract: Persistent organic pollutants (POPs) are non-biodegradable chemicals that can be toxic to living organisms in high concentrations. Bisphenol A (BPA) is a carbon based synthetic POP found in polycarbonated plastics, used in the production of plastic bottles. Small amounts may migrate into food resulting in health issues to both animals and humans. We set out to develop a riboswitch-based biosensor for BPA. Riboswitches are components of messenger RNAs that regulate the expression of genes based on the binding of specific small molecules. By fusing a gene that encodes for a reporter protein such as green fluorescent protein to the expression platform of a BPA-riboswitch we reasoned that we could qualify the presence of BPA in the environment of a cell harboring the BPAriboswitch. Starting with a thiamine riboswitch, we generated a library of thirty thousand variants that were then subjected to dual genetic selection. After one round each of positive and negative selection, the genetic diversity of our library decreased, indicative that we may have successfully selected for BPA-responsive riboswitches.
Funding Provided by: National Science Foundation #CBET-1258307: PI J. Liu

Effects of Aluminum Ion on Membranes of Varying Phospholipid and Cholesterol Composition: A QCM-D Study

Fiker Bekele (2016); Student Collaborator(s): Hannah Wayment-Steele (2015); Mentor(s): Malkiat Johal

Abstract: Aluminum is the most abundant metal on earth; however, its ion Al3+ is a known neurotoxin. Even though the exact role of the aluminum ion in pathogenesis is not known, its disruption of normal cell function has been widely demonstrated. The detrimental effects of Al3+ have been studied in vivo, but less is known about the physical changes it induces in membranes. We use Quartz Crystal Microbalance with Dissipation (QCM-D) to analyze changes in adsorbed mass and viscoelastic properties of supported lipid bilayers. This work will present the effect of Al3+ on lipid bilayers of varying cholesterol content. Cholesterol is a key component of membranes that aids in membrane integrity and rigidity, and hence its presence is likely to influence the degree of liposome rupture observed when Al3+ is introduced. Understanding Al3+ interactions with the lipid components of cell membranes is important for a better understanding of Al3+ as a neurotoxin in biological systems.
Funding Provided by: Howard Hughes Medical Institute(FB); Arnold and Mabel Beckman Foundation (HWS)

Characterization of the Novel Nucleic Acid Dye DANPY 1

Conner Kummerlowe (2016); Student Collaborator(s): Marek Zorawski (2016); Additional Collaborator(s): Lewis Johnson (University of Washington); Mentor(s): Malkiat Johal

Abstract: DANPY-1 is a fluorescent dye with high affinity for DNA that has potential applications as an alternative to commonly used carcinogenic dyes, such as ethidium bromide. Using a Quartz Crystal Microbalance (QCM) and a Dual Polarized Interferometer (DPI) we characterized the binding of DANPY and ethidium bromide to a DNA surface supported by polyelectrolyte multilayers. We confirmed an intercalating binding mode between ethidium bromide and DNA, and found a surface binding mode between DANPY and DNA. Furthermore, we found that ethidium bromide binding to DNA causes a conformational change that leads to hydration of the DNA layer. On the other hand, the binding of DANPY to DNA causes a slight dehydration of the DNA surface. Lastly, we analyzed the binding kinetics of these two dyes to DNA, finding that both dyes displayed a similar magnitude of affinity to DNA and are both strong binders.
Funding Provided by: Sherman Fairchild Foundation (CK); Fred J. Robbins Chemistry Fund (MZ)

Investigating Effects of Al3+ on Lipid Membranes: FRAP and Molecular Dynamics

Hannah Wayment-Steele (2015); Additional Collaborator(s): Angelika Kunze (University of Goettingen), Sofia Svedhem (Chalmers University of Technology); Mentor(s): Malkiat Johal

Abstract: Aluminum is the most common metal in the earth's crust, yet the ion Al3+ is a well-known neurotoxin. It derives toxicity from the structural changes it induces in lipid membranes upon binding, including increasing bilayer rigidity, facilitating vesicle fusion, and inducing vesicle rupture. However, the mechanism for these processes is not well understood. We implement Fluorescence Recovery After Photobleaching (FRAP) and Molecular Dynamics (MD) simulations to better understand the mechanism of the system. We provide a quantitative analysis of the effect of increasing Al3+ concentration on membrane diffusion in both zwitterionic and anionic membranes, as measured by both FRAP and MD. We also investigate the coordination structure of Al3+ with phosphatidylcholine lipid head groups, and report Al3+ coordination to the phosphate groups and acyl chain carbonyl groups. Our results indicate that addition of Al3+ to membranes causes an overall increase in rigidity and decrease in diffusion for zwitterionic membranes, and possibly a transition to gel phase in anionic membranes. Understanding Al3+-membrane interactions is important for a better understanding of the mechanism of aluminum toxicity in biological systems.
Funding Provided by: Arnold and Mabel Beckman Foundation

Random Walks on Fractal Structures

Johnny Wang (2017); Student Collaborator(s): Aaron Tsai (2017), Devin Gladys (2017), Isaac Medina (2016); Mentor(s): Roberto Garza Lopez

Abstract: Computational random walk calculations using Markovian chains can be used to predict chemical properties such as kinetics, diffusion, reaction rates, and overall dynamics of a given chemical system. We studied the efficiency of diffusion-controlled reactions on different families of 2-dimensional and 3-dimensional lattices with reaction centers located at unique sites. We calculated numerically-exact values for the absorption time (or mean walk length) of a particle performing a nearest -neighbor random walk on finite, nth generation 2D and 3D Sierpinski lattices, binary fractals and cubic structures (Garza-López, et al., J Phys. Chem. B 1999). We obtained results demonstrating that the overall efficiency of different structures in terms of walk lengths is dependent on the size, growth of the system (N), valency of the active sites (v), dimensionality of the lattice (d), and boundary conditions.
Funding Provided by: R. Nelson Smith ’38 & Corwin H. Hansch Fund for Summer Undergraduate Research in Chemistry & Biochemistry (JW); Howard Hughes Medical Institute (IM); Fletcher Jones Fdtn. (DG); Fred J. Robbins Chemistry Fund (AT)

Synthesis and Electrospinning of Imidazolium Containing Polyesters with Applications in Drug Delivery

Neil Forsythe (2016)

Abstract: Synthesis of novel imidazolium containing monomers were investigated due the potential to create electrospun scaffolds for biomedical applications. Butyl derivatives of imidazolium polyesters have the potential to lyse endosomal membranes and provide a route for endosomal escape and DNA transfection. Both methyl and butyl derivatives of imidazolium containing diol monomers were synthesized via a 3 step organic synthesis. Poly (3-(3-hydroxy-2-(hydroxymethyl)-2methylpropyl)-2-methyl-1Himidazol adipate) (PHI) was synthesized from the methyl diol via step growth polymerization at 130° – 220°C. Gel binding studies indicated that the PHI bound DNA, which offers promise in DNA transfection and drug delivery. The butyl derivative was unable to be synthesized due to the diol’s low thermal stability. Thermogravimetric analysis (TGA) in N2 indicated a Td of 137 °C while isothermal TGA above 130 °C showed degradation within 30 minutes. Poly(neopentyl adipate) (PNPA), an uncharged analog of PHI, was synthesized and electrospinability was compared to the charged, imidazolium containing polyester. Electrospinning of PNPA did not result in fiber formation due to its low molecular weight. Solution rheology confirmed the lack of an entanglement concentration (C e ). Initial electrospinning of methyl imidazolium PHI resulted in droplets forming on the collector plate instead of fibers. In order to improve electrospinability, PHI was blended at various ratios with poly(ethylene oxide) (PEO) (200,000 g/mol). An optimal blend ratio of 80:20 PHI and PEO (respectively) resulted in uniform fibers.
Funding Provided by: National Science Foundation