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.
Hydrophobicity and Drug Delivery: A QSAR Approach
Jacob Al-Husseini ’22; Advisor: Malkiat Johal
In this work, Dual Polarization Interferometry (DPI) and Quartz Crystal Microgravimetry with Dissipation Monitoring (QCM-D) were used to examine the binding characteristics and structure-activity relationships of 12 common drugs on a model bovine serum albumin (BSA) film. By taking advantage of the different hydration sensitivities of DPI and QCM-D we were able to quantify changes in the solvent state upon drug binding to BSA. Quantifying the changes in water mass within binding pockets and upon drug-protein binding allows for a more complete understanding of binding phenomena between drug molecules and serum proteins. For the drugs tested, a quantitative structure-activity relationship (QSAR) was used to establish a correlation between drug binding (KD) and hydrophobicity (ClogP), with the latter being related to the drug’s ability to desolvate the BSA upon binding. In summary, the QSAR analysis shows that both the strength of binding and the loss of water due to binding increase with drug hydrophobicity. It is not surprising that more hydrophobic drugs lead to greater water loss from the BSA, but the corresponding increase in binding strength is noteworthy. This study underscores the importance of hydrophobicity to drug binding kinetics and may be used to further understand and improve drug design and delivery protocols.
Isotopic Perturbation of Hydrogen Bonds
Casey Morrison ’21; Advisor: Daniel O’Leary
Hydrogen bonding is an attractive force between hydrogen and electronegative atoms such as oxygen. Hydrogen bonding is the cause of many unusual physical and chemical properties of compounds containing oxygen like water’s high boiling point. Hydrogen bonding can occur both intermolecularly between different molecules and intramolecularly within the same molecule. Isotopically labeling compounds causes changes in the hydrogen bond. The deuterium isotope effect plays an important role in understanding the orientation of hydrogen bonds. Deuterated compounds are not present when scanned by the instrument, which allows for observation of isotopic shifts in the molecule’s hydrogen spectra. Compounds labelled with nitrogen-15 will have sharper peaks and will have a split in the neighboring hydrogen’s peak. The spectrum would show two peaks instead of a single peak of the averaged signals. Through synthesizing nitrogen-15 labelled phenylurea and phthalamic acid, spectroscopic graphs indicated splitting of the hydrogen peak into two separate peaks. Spectroscopic scans of deuterium labeled 2,6–dihydroxybenzaldehyde along with unlabeled 2-hydroxy-6-methoxybenzaldehyde displays how the isotopic perturbation affects the chemical shift of the hydrogens involved in the hydroxyl-aldehyde intramolecular hydrogen bond. Through viewing this poster, a better understanding about the effects isotopes have on hydrogen bonds can be reached.
Electron Transfer in Photoactive Dendritic Molecules Via a Markovian Model
Lawrence Chen ’20; Advisor: Roberto Garza
Dendrimers are radially symmetrical molecules with applications in various fields of pharmacology and photochemistry. We study the electron-transfer behaviors in a DPm⊃Py2Fn family developed by Li et al. J. Am. Chem. 128 (2006) 10527-10532. Focusing on isomers of the structures discussed by Li et al., we consider two limiting scenarios depending on the number of bipyridine ligands surrounding the DPm base molecule. Using Markovian theory, we create Markovian models and show not only that we can use computational methods to recover the experimental trends but also find that the electron transfers on a larger dendrimer (DP48⊃Py2F3) is more efficient. The initial results for this study have been published and can be found in L. Chen et al. Chem. Phys. Lett. X 2 (2019) 100009. The Markovian model can be adapted to other systems and altered in the current system to explore varying activity levels of the donor-acceptor sites of the DPm molecule family.
Identification of Odor Blend Used by Caenorhabditis elegans for Recognition of Evolved Bacteria
Emily Rainge ’22; Advisor: Charles Taylor
Caenorhabditis elegans (C. elegans) are worms that inhabit bacteria-rich environments such as rotting fruit and plant matter. They use their sense of smell to make choices about what bacterial species they can consume without succumbing to disease (Shtonda and Avery 2006). The goal of this project is to investigate what blends of chemical cues C.elegans olfactory systems use to distinguish among different bacteria. To accomplish this, we have used Gas Chromatography-Mass Spectrometry to analyze volatile organic chemicals present in the air above samples of two strains of E.coli: OP50 and HB101 (a food source that is more nutritious to C.elegans than OP50). Our preliminary results show that the volatile chemicals emitted from OP50 and HB101 differ; OP50 headspace contained dimethyl disulfide, while HB101 headspace contained 1-Butanol and 2,4-Di-tert-butylphenol. Both OP50 and HB101 emitted isobutyric acid,2-(hydroxymethyl)-1-propylbutyl ester and butyl butanoate into their headspaces. Now, we want to test whether worms can tell the difference between the odors of OP50 and HB101 using bacterial choice assays.
Genotoxicity of 4-X-Phenols
Nicholas Oo ’21; Advisor: Cynthia Selassie
Phenolic compounds are known to be powerful antioxidants. They are easily found in our world, both in nature and man-made products. They develop phenolic radicals through a donation of their hydrogen atom from the hydroxyl group. Under certain conditions, such as concentration levels, pH, or presence of metal ions, they can act as pro-oxidants, causing an increase in reactive oxygen species within cells. This increase can lead to significant DNA damage, resulting in apoptosis and consequently in DNA fragmentation. This study focuses on investigating DNA damage in L1210 mice leukemia cells caused by 4-X-Phenols and finding a relationship between the structure of the phenols and their genotoxicity. A comet single gel cell electrophoresis (SGCE) assay was used in order to quantify their DNA damage. The assay differentiated denatured and cleaved DNA fragments from undamaged DNA based on their mobility. Damaged DNA was able to migrate out of the cell, while undamaged DNA stayed in the nucleus of the cell. The movement of the DNA fragments formed a migration pattern which resulted in a comet tail shape. The longer the tail, the more DNA damage was present. From our findings, it is clear that electronic attributes play a significant role in DNA damage.. Electron donating groups (EDG) cause more damage within the cell whereas electron withdrawing groups (EWG) cause less. Furthermore, it appears that more hydrophobic substituents also increase the compound’s genotoxicity.
Preparation of theophylline riboswitches for structure elucidation by SHAPE experiments
Noelle Mitchell ’20; Advisor: Jane Liu
The Central Dogma of Molecular Biology states that genetic information in the form of DNA is converted into proteins via RNA messengers. Traditionally, proteins were thought to be the regulators of this process. Riboswitches, a class of RNAs found chiefly in some 5’ UTRs of mRNAs, can regulate transcription and translation; thus, their discovery in 2002 was notable because it suggested that RNA, too, can control this process. Primarily found in bacteria, riboswitches are known to bind small molecule ligands, causing conformational changes that influence gene expression by turning a succeeding gene ON/OFF. Previously, the Liu Lab used FACS and dual genetic selection to discover novel riboswitches(Hits) responding to the ligand theophylline. While the lab has evidence of multiple Hits of varying specificity, the structure of these Hits in the +/- theophylline state has yet to be elucidated. Selective 2’OH acylation analyzed by primer extension (SHAPE) experiments use chemical probes, reverse transcription and data analysis programs to resolve secondary structures of varying RNA segments. By amplifying a Hit sequence via PCR, transcribing that sequence, performing folding optimization experiments, and completing SHAPE experiments, we expect to uncover the structure of riboswitch Hit 3-5 in the presence and absence of theophylline. In doing so, we may determine modes of specificity, prompting a range of potential applications including those in synthetic biology and therapeutics.
Analysis of Hydrogen Bonding Patterns in Cage Diols through NMR Techniques
Sara Ryerson ’21; Advisor: Daniel O’Leary
This research focuses on developing a method to test the strength of intramolecular hydrogen bonding in diol molecules using isotope effects in nuclear magnetic resonance (NMR) spectroscopy. NMR techniques were used to determine the hydrogen shifts in a cage diol system. Because the original diol shows an NMR shift that is the average of the two OH groups in the diol system, a Williamson ether synthesis was performed to determine the individual shifts of the intermolecular bonded, or outside hydroxyl group and the intramolecular bonded, or inside hydroxyl group. The synthesis involved the conversion of the diol to a mono-methylether. The added methyl group acts as an inhibitor of intramolecular hydrogen bonding at that specific oxygen site and subsequently forces the other hydroxyl group to form an intramolecular bond. This bound hydroxyl group can be used to provide an estimate of the shift of the inside hydrogen only in the original cage diol. The inside shift was experimentally determined using the methyl ether and compared to the shift determined using a previously synthesized benzyl ether. NMR scans of the original diol molecules was used to determine the shift that is the average of the inside and outside hydrogens. The outside shift was then extrapolated from these two data points. These limiting chemical shifts can be combined with an isotope shift measurement to provide a detailed understanding of the energetics of intramolecular hydrogen bonds.
Characterizing the Regulatory Function of Novel Riboswitches Genetically Engineered in Escherichia Coli.
Chanha Kim ’22; Advisor: Jane Liu
Riboswitches are sequences in messenger RNAs (mRNAs) that can regulate expression, either positively or negatively, by binding small molecules, and they have numerous applications, such as synthetic biology, biosensor development and virus control. We aimed to characterize the regulatory function of novel theophylline-binding riboswitches that were previously engineered in the lab. Specifically, we investigated whether riboswitch 3-5 regulates expression at the transcriptional or translational level. We used in vitro protein synthesis and western blotting to determine how riboswitch 3-5 regulates protein synthesis in response to theophylline. We also performed northern blot analyses to probe whether or not riboswitch 3-5 regulates expression transcriptionally in response to theophylline. Western blot analysis suggests that riboswitch 3-5 regulates protein synthesis in response to theophylline, and northern blot analysis suggests this to be, at least in part, occurring at the level of transcription or mRNA stability. Both analyses, however, suggest that further optimization of these experiments or transitioning to other in vitro methods are necessary to determine more precisely how riboswitch 3-5 regulates expression in response to theophylline. Doing so will contribute to the greater understanding of how novel riboswitches function and may aid future researchers in innovating riboswitch applications.
Fighting Lung Infections with a Materials Science Approach
Jacqueline Henriquez ’20; Advisor: Daniel O’Leary
Lung infections caused by Burkholderia pseudomallei (meliodosis) and Francisella tularensis (tularemia) are difficult to treat due to the residence of these pathogens within alveolar macrophages, where the microorganisms survive and evade the host immune response. In recent years, polymeric-drug conjugates have been produced to fight infections which afford for targeted delivery of antibiotics into these macrophages by means of a receptor-specific targeting approach. Controlled delivery of antibiotics provides improved bioavailability in the lung space and decreased likelihood of antibiotic toxicity. We have initiated a synthesis of Amikacin prodrug monomers with a protease-cleavable valine-citrulline dipeptide linker and a polymerizable methacrylate moiety. Amikacin is an aminoglycoside antibiotic with potent activity and whose undesirable side-effects can be decreased by this selective delivery approach. The valine-citrulline dipeptide linker has been shown to be stable under physiological conditions and provides for rapid enzymatic release within cells to provide local high concentrations of Amikacin. The monomer components were synthesized with standard techniques in organic chemistry. To date, we have synthesized the Val-Cit dipeptide in 50% yield and characterized its structure with high-performance liquid chromatography-mass spectrometry (LCMS) and 1H nuclear magnetic resonance (NMR) spectroscopy. The results of these synthetic procedures will be presented.
Determining the Chemical Kinetics of Ca2+- mediated Sulfamide Synthesis via NMR Spectroscopy
Pedro Martinez ’20; Advisor: Nicholas D. Ball
Sulfur(VI)-fluorides are a focus for medicinal and synthetic chemists alike due to their relevance as molecular building blocks for pharmaceuticals. Because of the enhanced stability of sulfur(VI) fluorides versus other sulfur(VI) halides, sulfur-fluoride exchange (SuFEx) chemistry has emerged as a new kind of click chemistry to introduce SO2 moieties into organic molecules. Despite their promise, the enhanced stability of sulfur(VI)–fluorides infer challenges in their reactivity. To this end, the Ball laboratory is focusing on ways to activate sulfur(VI) fluorides using SuFEx chemistry toward sulfur-based compounds important in medical chemistry. We have established a Ca(NTf2)2/t-amyl alcohol system that successfully activates sulfonyl fluorides (RSO2F), sulfamoyl fluoride (R2NSO2F), and fluorosulfates (ROSO2F) with various nucleophiles. Ca(NTf2)2 functions as a catalyst in many organometallic systems, however, its role in our reaction has not been established. Preliminary data suggests catalytic inhibition by the released fluoride anion (F-), which may coordinate to Ca2+ impeding its further participation in S-N bond activation. In this project, we aim to determine the mechanism of this reaction to better understand what could be preventing catalysis. Using 19F NMR spectroscopy, we found that increasing the temperature of the reaction, the reactions overall concentration, and the relative concentration of Ca(NTf2)2 all increase the rate of reaction.
Synthesis of an Anthelmintic Drug, a Derivative of Mebendazole
Christabel Egemba ’21; Advisor: Cynthia Selassie
The resistance of parasitic worms to current anthelmintic drugs is growing rapidly. For this reason, there is a strong need for a new drug to combat these resistant parasitic worms. Our research aims to design and synthesize a derivative of Mebendazole, a current anthelmintic drug. This derivative of Mebendazole involves a two-step synthesis of the parent compound that will incorporate a series of substituted derivatives which will then be analyzed via a quantitative structure activity relationship (QSAR). LCMS, NMR and IR will be utilized to determine the structures of these compounds. Once the parent molecule has been synthesized, its biological activity will be compared with Mebendazole in vitro, using c.elegans as a model for the nematode system. Then the effectiveness of the substituted derivatives will also be tested and compared to the current Mebendazole in vitro.
Designing a novel DNA aptamer for pathogen detection and medical diagnoses
Kirsten Mortimer; Advisor: Charles Taylor
Aptamers are short oligonucleotide-based receptors that have been subjected to an in vitro evolutionary selection process resulting in a sequence that selectively binds to a specific target. This selection process begins with a large, randomized single stranded DNA library, followed by a target binding step, and the unbound sequences are separated and discarded. PCR amplification of the bound sequences followed by strand separation leads to the next cycle of SELEX, where conditions can be made more stringent. Small molecules are promising targets for SELEX due to their ranging physiological relevance, and their functionality as a biomarker. One such application is the detection of volatile organic compounds emitted by the bacteria causing ventilator-associated pneumonia (VAP), a lung infection that is common in patients on ventilators. Previously, the Taylor Lab has used Raman spectroscopy optics to approach the low levels of detection needed for a valid diagnosis of VAP, identifying 2-aminoacetophenone as a potential breath biomarker of VAP. Because aptamers have been shown to bind targets with very high affinity and selectivity, we plan to develop an aptamer that targets 2-aminoacetophenone. Through SELEX, the DNA library is immobilized on magnetic beads and a structure-switching mechanism causes the release of target-bound sequences, we hope this synthetic biology-based approach will produce the sensitivity needed to detect and diagnose VAP in a clinical setting.
Chemical Analysis of Traditional Vietnamese Medicinal Herbs
Rachel Hall ’22; Advisor: Chemical Analysis of Traditional Vietnamese Medicinal Herbs
Bear Bile Farming (BBF) began as a practice in Vietnam in the 1990s in response to consumer demand for bear bile and traditional medicines that contain it. Although BBF was outlawed in Vietnam in 2005, bear owners were allowed to keep their bears after certifying that they would not be harvesting bile from them. The compound found in bear bile known to have medicinal value (ursodeoxycholic acid) is now commercially available. This work focuses on the chemical analysis of herbs used in preparing traditional medicines that have the same uses as bear bile.
Our goal was to determine if any compounds extracted from these herbs have been used to treat similar ailments. Our approach has been to 1) determine which compounds are extracted from these herbs and 2) identify which of these compounds could be responsible for particular medicinal properties. Using GCMS and LCMS we found volatile and non-volatile chemical constituents of these herbs. We were then able to gather more information related to the medicinal properties of each compound using databases such as SciFinder and PubChem. Another aspect we wanted to look at was which compounds can be found in more than one herb. We have identified six compounds that were common to three or more of the herbs, all with known medicinal properties.
Designing a novel DNA aptamer for pathogen detection and medical diagnoses
Bria VarnBuhler ’20; Advisor: Charles Taylor
Aptamers are short oligonucleotide-based receptors that have been subjected to an in vitro selection process resulting in a sequence that selectively binds to a target. This process, called SELEX, begins with a randomized oligonucleotide pool. The target is then introduced and bound by sequences that have affinity for it, while unbound sequences are separated and discarded. Target-bound sequences are PCR amplified, and then converted to ssDNA for use in the next round of SELEX, in which selection conditions may become more stringent. This cycle repeats until the pool has been sufficiently enriched and contains an aptamer of desired affinity for the target.
We plan to develop a sensitive and practical method for detection of ventilator-associated pneumonia (VAP), a bacterial lung infection that is common in intubated patients. Previously, the Taylor Lab used a Raman spectroscopy optics system to measure the spectra of compounds released by P. pseudomonas to produce a “fingerprint” of VOCs unique to VAP. While the Raman system has not yet reached the sensitivity needed for use in clinical settings, the lab has identified 2-aminoacetophenone as a breath biomarker of VAP. Thus, by employing capture-SELEX, in which the DNA pool is immobilized on magnetic beads and a structure-switching mechanism causes release of target-bound sequences, we plan to develop an aptamer targeting 2-aminoacetophenone and attain the sensitivity needed to detect and diagnose VAP in a clinical setting.
Determining the Chemical Kinetics of Ca2+- mediated Sulfamide Synthesis via NMR Spectroscopy
Sam Khasnavis ’21; Advisor: Nicholas D. Ball
Sulfur(VI)-fluorides are a focus for medicinal and synthetic chemists alike due to their relevance as molecular building blocks for pharmaceuticals. Because of the enhanced stability of sulfur(VI)fluorides versus other sulfur(VI) halides, sulfur-fluoride exchange (SuFEx) chemistry has emerged as a new kind of click chemistry to introduce SO2 moieties into organic molecules. Despite their promise, the enhanced stability of sulfur(VI)–fluorides infer challenges in their reactivity. To this end, the Ball laboratory is focusing on ways to activate sulfur(VI) fluorides using SuFEx chemistry toward sulfur-based compounds important in medical chemistry. We have established a Ca(NTf2)2/t-amyl alcohol system that successfully activates sulfonyl fluorides(RSO2F), sulfamoyl fluoride (R2NSO2F), and fluorosulfates (ROSO2F) with various nucleophiles. Ca(NTf2)2 functions as a catalyst in many organometallic systems, however, its role in our reaction has not been established. Preliminary data suggests catalytic inhibition by the released fluoride anion (F-), which may coordinate to Ca2+ impeding its further participation in S-N bond activation. In this project, we aim to determine the mechanism of this reaction to better understand what could be preventing catalysis. Using 19F NMR spectroscopy, we found that increasing the temperature of the reaction, the reactions overall concentration, and the relative concentration of Ca(NTf2)2 all increase the rate of reaction.
Examining Behavioral and Chemical Interactions Between Coevolved C. Elegans And Bacterial Pathogen S. Marcescens
Joseph Lopez ’20; Advisor: Chuck Taylor
Understanding host-pathogen interactions is essential to understanding mechanisms of pathogenic infection and the resulting effects on the host. Caenorhabditis elegans has emerged as a model organism for studying these interactions over multiple generations via experimental coevolution with the pathogen. In particular, research on coevolution of C. elegans with Serratia marcescens has established that the coevolved worms develop a decreased preference for the coevolved S. marcescens; however, the underlying mechanisms are not well understood (Penley and Morran, 2017). In our research, we have employed gas chromatography-mass spectrometry (GC-MS) to compare the volatile organic compounds (VOCs) released by the ancestral and coevolved strains of S. marcescens to explore if changes in olfactory profiles played a role in the changed preference of the coevolved C. elegans. Interestingly, our preliminary results suggest no major differences between the VOCs emitted by the ancestral and coevolved bacteria. This result suggests that the cause of the decreased preference may be a result of a change in the coevolved worms. For further experimentation, we plan to investigate differences in the attraction of the ancestral and coevolved worms to VOCs released by S. marcescens using chemotaxis assays.
Studying The Role Of Mannitol And Vps Gene Transcription On Vibrio Cholerae Biofilm Formation
Eric Tang ’21; Advisor: Jane Liu
Biofilm formation provides Vibrio cholerae a powerful defense mechanism. Through the creation of a large extracellular matrix of sugars and proteins, this bacterium is able to resist environmental threats ranging from phagocytosis to lack of nutrients to even antibiotics. This physical barrier allows it to survive and thrive in harsh aquatic environments, allowing for further infections of humans. This project seeks to understand V. cholerae’s biofilm response to different extracellular carbon sources. We focused on to what degree mannitol, the most abundant sugar alcohol in aquatic environments, affects biofilm formation relative to other carbon sources and how mannitol affects transcription of the vps genes that encode proteins responsible for biofilm formation. We observed that mannitol increases V. cholerae biofilm formation and, to a small extent, also induces vpsL transcription. Our results are relatively consistent with prior literature, although optimizations to the protocols can be made to more effectively measure changes in vps transcription and biofilm formation. Further investigation of MtlA, the mannitol transporter in V. cholerae, may provide great insight in the process of biofilm regulation and clarity with the interactions between the PTS and vps genes.
Lewis Acid-Mediated Synthesis of Sulfonamides and Sulfamides From Sulfonyl Fluorides and Sulfamoyl Fluorides
Sabrina Kwan ’20; Advisor: Nicholas D. Ball
Sulfonamide and sulfamide structures are highly stable and compatible with late-stage functionalization, resulting in their wide use in pharmaceutical and agrochemical industries. The synthesis of these sulfur(VI) compounds often involves nucleophilic addition to sulfonyl chlorides, however the instability of sulfonyl chlorides and poor selectivity for nucleophiles are undesirable. The more stable sulfonyl fluorides can be activated towards nucleophilic addition by calcium triflimide, which acts as a Lewis acid. We demonstrate the high efficiency, wide applicability and simplicity of this method with optimized conditions.
Carbon Locus Functionalization in a Proposed Bifunctional Inhibitor of HDAC and DHFR in P. falciparum
Martin Acosta Parra ’20; Advisor: Cynthia R. Selassie
Antimalarial resistance remains a burgeoning obstacle in the widespread treatment of malaria in the developing world. Plasmodium, the unicellular protozoans responsible for malaria, demonstrate resistance to even the most expensive antimalarials available. As a result, malaria has escalated into a humanitarian crisis. Efforts to develop novel chemical therapies must strive not only for effectivity and economy but to address the growing problem of antimalarial resistance. Bifunctional inhibitors have been explored as a solution to drug resistance because the likelihood of resistance to a drug significantly decreases as the number of targeted enzymes increases. This work focuses on the design and synthesis of a set of bifunctional inhibitors: triazine-based N-hydroxyacetamides. As the deadliest malaria-causing species, Plasmodium falciparum is the target species of our compounds. These N-hydroxyacetamides contain hydroxamic acid and diamino triazine moieties, known to target HDAC and DHFR in P. falciparum. Common experimental and synthetic techniques in organic chemistry were used in the synthesis of these compounds. Functionalization of the triazine carbon-2 locus was achieved via a key synthetic step. The analogues having propyl, butyl, and pentyl substituents will be synthesized and their biological activity tested against DHFR and HDAC separately. A QSAR approach will be used to assess whether increased hydrophobicity affords any added antimalarial potency.
Studying the structure of rRHA PP1Y Through X-Ray Crystallography
Brendan Terry ’20; Advisor: Matthew Sazinsky; Collaborator: Joseph Ha ’19
L-Rhamnosidases are a family of non-mammalian glycosyl hydrolases that have potential applications in industrial purification of flavonoids and enzyme-directed drug delivery systems. Using x-ray crystallography, this project aims to determine the atomic structure of rRHA PP1Y, an L-rhamnosidase that, based on previous kinetic analysis, shows promise in practical use and, based on the predicted motif of its active site (as determined by computer modeling), is unique among rhamnosidases in the protein data bank. Furthermore, it is of theoretical interest as well. Indeed, with atomic structure in hand, future research may utilize various techniques in mutagenesis and additional crystallographic work to improve the catalytic efficiency and characterize the conformational mechanism of rRHA PP1Y.
Funding Provided By: Rose Hills Foundation SURP Grant, The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research
Classifying the Geographic Origins of Coffee Beans with Trace Elemental Analysis Using Wavelength-Dispersive X-Ray Fluorescence and Inductively Coupled Plasma Mass Spectroscopy
Amy Watt ’20; Advisor: Charles Taylor; Collaborator: Katheryn Kornegay ’20
Classifying the geographic origin of coffee beans is important in detecting fraud and mislabeled single-origin coffee. Trace element analysis of coffee beans has the potential to identify the region of origin (Americas, Africa, Asia) of a given sample. Toward this end, 29 samples of coffee were prepared through roasting of green beans and grinding of roasted beans. Samples used were a variety of green, roasted, and pre-ground samples. First, approximately 3-gram samples of each coffee were pressed into pellets and analyzed via Wavelength-Dispersive X-Ray Fluorescence (XRF) in triplicate. Next, those samples were analyzed via Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Sample classification using the XRF data was performed via Principal Component Analysis (PCA). PCA indicated that Mn, Br, and Rb are important elements for differentiating between samples. Br was a key indicator for samples from South-East Asian countries such as Philippines and Vietnam while Mn and Rb varied across all regions. Since many elements were present below XRF detection limits, various ICP-MS methods were explored to both verify and supplement data collected via XRF.
Funding Provided By: The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research
Anodic Stripping Cyclic Volammetry
Michael Ko ’18; Advisor: Edward Crane; Collaborator: Huey Sun ’20
Anodic stripping cyclic voltammetry is used to identify sulfur compounds in solution. This technique was used to observe the long-term changes of culture bottles with Fe or sulfur, TYC, and a mud sample from mud volcanoes.
Funding Provided By: Rose Hills Foundation SURP Grant
The reaction between methane and UV-activated perchlorate surfaces
Sathya Chitturi ’18; Advisor: Frederick Grieman
Accurate modeling of methane, an important biological proxy, is critical to the search for extra-terrestrial life. On Mars, methane levels are far lower than expected and have unpredictable spatiotemporal variance. Perchlorate based oxidation, in the presence of UV radiation, has recently been proposed as a mechanism for methane decomposition. In this analysis, FT-IR was used to study the time-series UV photolysis of Mg(ClO4)2. Several vibrational peak positions and intensities were recorded and used to identify reaction products by direct comparison to accepted experimental and computational values; these products include surface Mg(ClO3)2 as well as gaseous Cl2O4, Cl2O6 and ClO2. Assignments were confirmed by calculating the vibrational energies of these species using the B3LYP density functional theory (DFT) method. From this analysis, we suggest the potential of UV irradiation of the Martian surface to produce reactive chlorine oxides with the ability to oxidize methane.
Funding Provided By: The Doudna and Cate Chemistry, Biology and Molecular Biology Fund, The Doudna and Cate Chemistry Fund
SPME-GC/MS Analysis of Potential Volatile Organic Compound Biomarkers in Pathogenic Bacteria
Soleil Worthy ’18; Advisor: Charles Taylor; Collaborator: Aseal Birir ’18
Bacteria emit specific mixtures of volatile organic compounds (VOCs) as byproducts of their metabolic pathways. VOCs produced by infectious bacteria can be detected in the breath of infected individuals, presenting a breath test as an exciting possible diagnostic tool for diseases such as ventilator associated pneumonia (VAP). The goal of this project was to identify characteristic VOCs produced by the VAP causing bacteria Pseudomonas aeruginosa both in a traditional bacterial growth medium, as well as in synthetic sputum to more closely mimic the environment of the bacteria in vivo. For the initial characterization of bacterial VOCs, solid phase microextraction (SPME) -gas chromatography / mass spectrometry (GC/MS) was employed to detect VOCs in the headspace of bacterial cultures and spent bacterial growth media. When grown in Luria Broth (LB), the VOCs carbon disulfide, 2-methyl furan, 3-methyl furan, a xylene isomer, and 2-heptanone were identified in the headspace of P. aeruginosa cultures after both 6 and 24 hours of growth but were absent in the headspace of the media control, suggesting that they are being produced by P. aeruginosa. When grown in synthetic sputum, the VOCs acetone, methylene chloride, trichloromethane, and 1-undecene were identified in the headspace of P. aeruginosa cultures after both 6 and 24 hours of growth but were absent in the headspace of the media control, suggesting that they are being produced by P. aeruginosa.
Funding Provided By: The Linares Fund for Student Research in Chemistry
Examining the role of chirality in TemporinSHf’s mechanism of membrane disruption — a QCM-D study
Laura Haetzel ’19; Advisor: Malkiat Johal
Antibiotic resistance is one of the biggest threats to global health. Thus, a search for alternative antibiotic agents such as antimicrobial peptides has emerged. Temporin-SHf is highly selective for prokaryotic membranes. It has been extensively studied in vitro but not yet in vivo. Chiral forms of cell-penetrating peptides are key for in vivo applications since D-enantiomers are often resistant to endogenous proteases. This study compared L- and D-Temporin-SHfs’ abilities to degrade prokaryotic membrane models. Peptide action on prokaryotic membranes was studied via the Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). The QCM-D continuously records mass changes, thus monitoring peptide action in situ. Prokaryotic membrane models were created using the solvent-assisted lipid bilayer formation method. Both achiral and chiral membrane models were studied. QCM-D measurements revealed that D-Temporin-SHf was twice as potent as L-Temporin-SHf on average. D-Temporin-SHf degraded 14% to 22% of the membrane, whereas L-Temporin-SHf degraded 3% to 11%. Although the difference was statistically significant, doubts over the membrane models’ stability remain due to unsuccessful control experiments. There was no significant difference in the peptides’ adsorption to the membrane, indicating that all degradation differences were mechanistic. Finally, achiral membranes were much less susceptible to degradation, thus suggesting that Temporin-SHf targets chiral membrane components.
Funding Provided By: Dale N. Robertson Fund for Undergraduate Research
Detecting Equilibrium Isotope Effect in Cyclodextrin
Evelyn Romero ’19; Advisor: Daniel O’Leary
Cyclodextrin, a carbohydrate, is a macromolecule composed of various glucose molecules linked together to form a ring. Cyclodextrins are conically shaped and can come in different sizes. They can be composed of six, seven and eight glucose monomers, referred to as alpha, beta and gamma cyclodextrin respectively. These macromolecules have been shown to exhibit hydrogen bonding, specifically between the hydrogen molecules in carbon two of one glucose and carbon three of the neighboring one. This hydrogen boding has been detected through SIMPLE NMR in DMSO-d. Hydrogen bonding interactions in cyclodextrin could vary depending on the solvent cyclodextrin interacts with. We aimed then to protect the primary hydroxyl group in cyclodextrin at carbon 6. This would allow cyclodextrin to become soluble in various organic solvents. Protection of cyclodextrin was attempted using tert-butyldimethyl silyl chloride, and tert-butyldiphenyl silyl chloride.
Funding Provided By: The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research
Infrared Kinetic Spectroscopy of Acetyl Peroxy and Hydrpoeroxy Radical Reactions
Sara Murphy ’18; Advisor: Frederick Grieman
An understanding of peroxy radical chemistry is essential to a complete understanding of the effects of pollutants on ozone levels and the HOx budget in the atmosphere. This project has sought to contribute to this understanding through the study of the reaction between acetyl peroxy (CH3COH) and hydroperoxy (HO2) radicals at atmospherically relevant temperatures. Experimentally, we conduct these studies using the IRKS (Infrared Kinetic Spectroscopy) setup in the Sander’s lab at JPL. In this setup, we create chlorine radicals in a temperature-controlled flow tube via photolysis of chlorine molecules using a XeF excimer laser, which then go on to react with methanol, acetaldehyde, and oxygen to create the hydroperoxy and acetyl peroxy radicals. We then monitor the concentrations of the radicals via IR laser spectroscopy. However, the study of this reaction is complicated by side chemistry under the laboratory conditions used to perform these experiments; the radical precursors and the radicals themselves undergo a complex array of reactions that must be understood for the eventual determination of the rate constants and branching ratios of the reaction of interest (that between HO2 and CH3COH). One of these side reactions is the association reaction between hydroperoxy and acetaldehyde (a precursor to acetyl peroxy radicals). We have therefore performed experiments at various atmospherically relevant temperatures to determine the equilibrium constant for this reaction.
Funding Provided By: Rose Hills Foundation SURP Grant
Barium-mediated Conversion of Aryl Sulfonyl Fluorides to Sulfonamides
Ryan Franzese ’19; Advisor: Nicholas Ball; Collaborators: Sarah Etuk ’19, Sabrina Kwan ’20
The main focus of our research involves synthesizing organic compounds called sulfonamides. Sulfonamides are important compounds due to their application in the pharmaceutical and agrochemical industries. A common starting material towards sulfonamides are sulfonyl chlorides; however, the synthesis of sulfonyl chlorides and the compound themselves present significant challenges. As an alternative to sulfonyl chlorides, our lab group along with a collaborating group at Pfizer are interested in using sulfonyl fluorides as a starting material to sulfonamides due to their mild synthesis reaction and stability. A key challenge to this approach is that currently there are no ubiquitous reaction conditions that can synthesize sulfonamides using a wide variety of sulfonyl fluoride and amine derivatives. We will discuss an optimized Ba(NTf2), t-amyl alcohol system that generates sulfonamides from sulfonyl fluorides in high yields. Optimization studies varying Lewis acid, solvent, temperature, amine equivalence, type of amine, and time will also be discussed. Lastly, future directions towards a complete substrate scope will be presented.
Funding Provided By: The John Stauffer Endowed Summer Research Program in Chemistry, The Doudna and Cate Chemistry Fund, Biology and Molecular Biology Fund
Verifying the Miniaturization of Eddy Co-variance Instruments for Airborne Measurements
Erik Garcia ’19; Advisor: Andrew Sappey; Collaborator: David Berkinsky ’19
Eddy co variance method can be used to determine the gas exchange rate between an ecosystem and the atmosphere. This method has significant advantages over other approaches and has a variety of applications for geology and planetary science. The purpose of this project was to verify that miniaturized eddy co variance instruments, specifically the anemometer and spectrometer, were capable of accurately determining the flux of a given gas in order to mount them on quad copters for airborne measurements. The Spectrometer showed results sufficient enough in determining the gas concentration for eddy flux calculations, however the anemometer did not. The vertical wind parameter was not accurate enough and therefore led us to conclude that it was not capable of performing eddy flux measurements.
Funding Provided By: The John Stauffer Endowed Summer Research Program in Chemistry, The R. Nelson Smith ’38 and Corwin H. Hansch Fund for Summer Chemistry Research
Investigating High-Altitude Disease through the Himalayan Rescue Association
Zane MacFarlane ’18; Advisor: Frederick Grieman
High-altitude related illnesses affect a large population of outdoor recreationists traveling to areas above 2500 meters. Although high-altitude illnesses “acute mountain sickness (AMS), high-altitude pulmonary edema (HAPE), and high-altitude cerebral edema (HACE) are partially preventable and completely treatable, unnecessary deaths occur every year in severe cases often well above 3500 meters. By working with the Division of Wilderness Medicine at Massachusetts General Hospital, I was able to conduct a literature search into the causes of high-altitude illnesses â€“ i.e. the pathophysiology of hypobaric hypoxia “and potential causes for HAPE susceptibility. In addition, I interpreted de-identified, handwritten patient charts from Himalayan Rescue Association (HRA) clinics in Manang (Annapurna Circuit) and Pheriche (Everest Base Camp Trek) from 2009-2017 for data entry onto the HRA Database. Statistical analyses were drawn from the de-identified HRA patient charts from 2015-2017 to determine basic information of those afflicted with high-altitude illness including age, sex, ascent rate, and independent vs. commercial group. In the future, this database will be used to investigate the prevalence of HAPE in the presence of higher ozone levels “demonstrating a link between human health and the changing climate.
Funding Provided By: The John Stauffer Endowed Summer Research Program in Chemistry
Building and optimizing a TIR-Raman spectroscopy system for volatile organic chemical analysis
Peter Rentzepis ’18; Advisor: Charles Taylor
Sensitive and accurate analysis of volatile organic compounds (VOCs) for medical and industrial purposes is currently a consuming process involving expensive mass spectrometric systems. In this study, we have developed and optimized a novel Total Internal Reflection (TIR) Raman spectroscopy system that enables VOC detection. This system uses commercially available polymer sorbents to collect gaseous VOCs then thermally desorbs them where they partition into a thin polymer film coating within the Raman system for spectral analysis via evanescent wave excitation. We perform two-dimensional correlation analysis on time series spectra to sharpen VOC spectra while reducing interfering spectral features, and proof of principle has been established through analysis of known VOC biomarkers for selected bacterial pathogens relating to diseases such as ventilator-associated pneumonia. This summer, optical alignment modifications to the excitation beam were made to increase instrument sensitivity, and comparative quartz crystal microbalance studies were undertaken to choose polymers. Additionally, integration of a position-sensitive detector measuring the position of the reflected beam exiting the TIR-Raman system has displayed changes in the refractive index of the polymer film coinciding with Raman spectra evidence of VOC absorption and desorption. We anticipate that our low-cost, robust system can be used for a variety of medical and industrial applications.
Funding Provided By: The John Stauffer Endowed Summer Research Program in Chemistry
Characterizing VOC Detection via TIR-Raman spectroscopy
Pollyanna Leung ’19; Advisor: Charles Taylor
We are optimizing a breathalyzer-type instrument using an advanced optics, Raman spectroscopy system to make infection diagnoses more efficient than current culture methods. This instrument would hopefully be low-cost, highly sensitive, and improve patient outcome by allowing for early initiation of antimicrobial therapy. The aim is to identify volatile organic compounds (VOCs) released by bacteria in patient breath tests to diagnose ventilator-associated pneumonia (VAP), a common complication in mechanically-ventilated patients in hospitals. Since VOCs are usually at such low concentrations, a polymer is painted on top of a hemisphere that is used to collect VOCs from samples and undergoes Raman scattering. This summer, I have been using an airflow system to flow diluted VOCs at various concentrations across the polymer to characterize the lower detection limits of the Raman optics system. This step tells us how concentrated a sample would need to be for its VOCs to appear in a Raman spectra and emphasizes the importance of increasing sensitivity of the instrument. I have also been working to characterize the polymer by determining its refractive index and density. There are three candidates for the polymers which are still undergoing testing for the best sensitivity and selectivity in the resulting spectra.
Funding Provided By: Rose Hills Foundation SURP Grant
Electron Transfer in Dendrimers, Fractal and Euclidean structures, and Hydrogen bonding in proteins.
Lawrence Chen ’20; Advisor: Roberto Garza-Lopez; Collaborators: Christina Eshak ’20, Brandon Tolentino ’19, Shawn Trimble ’20
Proteins vital to electron transfer processes such as amicyanin experience denaturation and unfold to a state of no return. To study this unfolding, the three-dimensional visualization programs UCSF Chimera and PyMol were used to track changes in the alpha carbon backbone of amicyanin at different staged of denaturation. Diffusion and electron transfer across was also studied through simulating random walks across several lattice formations. Euclidean and Fractal patterns were imaged, then run through a system pf programs to yield the average walk-length of a particle from any point on the surface. These methods were also implemented on the structure of dendritic porphyrin molecules with varying fullerene ligands to simulate electron transfer reactions on the molecule’s active sites. With these results, we are able to analyze trends and behaviors that different structures and surfaces possess and determine the most efficient structure given a situation.
Funding Provided By: HHMI, Chemistry Department Funding
Chemistry and Coding: Visualizing the Origins of Kinetic Isotope Effects with Python
Zichen Liu ’18; Advisor: Daniel O’Leary
Understanding the KIE elucidates key information about molecular reactivity and reaction mechanisms. Although enthalpic and entropic contributions to the overall KIE can be examined theoretically, atomic origins are difficult to isolate. LUCKIE is a Python software programmed to find and visualize the atomic origins of the KIE of a unimolecular system. This program uses vibrational frequency and displacement data to produce a user-friendly PyMOL display that color codes atoms to distinguish large atomic contributors to the KIE. Molecules with known KIE origins are used as test cases for both validation and robustness. LUCKIE is then used to examine Mislow’s doubly bridged diketone, a molecule with an unknown KIE origin. LUCKIE’s conclusion, that the origin lies on the exo-methylene bridging protons, is being validated through theoretical deuteration patterns and computational studies of model systems.
Funding Provided By: Chemistry Department Funding
Regulatory Factors that modulate small RNA, MtlS levels in Vibrio cholerae
Theodore Lang ’19; Advisor: Jane Liu
Vibrio cholerae is a gram-negative facultative pathogen that can exist in the human intestine and aquatic reservoirs such as lakes, rivers, and freshwater. Reported infections with V. cholerae are global with numbers ranging in the thousands. We hypothesize that V. cholerae can exist in the human intestine and aquatic reservoirs because it can regulate gene expression to respond and adapt to disparate environments for survival. Previous data has shown that the carbon sources available to V. cholerae regulate the levels of small RNA, MtlS. In this experiment, I set out to confirm the relationship between carbon sugars and MtlS sRNA. Through northern blotting, mannitol was found to repress MtlS sRNA levels, while glucose and fructose were found to induce MtlS sRNA levels. In addition, LacZ assays showed that the global transcriptional regulator, CRP-cAMP, is a repressor of mtlS transcription. Altogether, the data suggests that expression of mtlS in V. cholerae is influenced by available carbon sources and CRP-cAMP. We are now conducting research into the discovery of additional regulatory factors that modulate mtlS expression in V. cholerae. The data and ongoing research contributes to a better understanding of how V. cholerae persists in disparate environments.
Funding Provided By: Chemistry Department Funding
Characterizing Growth Kinetics and Crystal Morphologies of Vanadium Oxide Thin Films Prepared through Chemical Vapour Deposition
Pin-Cheng Leonard Chen ’18; Advisor: Charles Taylor
Vanadium oxide thin films have a variety of applications ranging from thermochromic window coatings to environmental gas sensors. Of the many preparation methods of these thin films, chemical vapor deposition (CVD), is examined in this project. Vanadium dioxide (VO2) and vanadium pentoxide (V2O5) are the most commonly produced forms of vanadium oxides and possess various crystal morphologies that are quite distinct. In this work, vanadium oxytriisopropoxide (VOTiP) was used to deposit vanadium oxides on oxidized silicon surfaces in the temperature range 170°C - 625 °C. Of particular interest are the effects of temperature and precursor partial pressure on the composition, growth rate and microstructure for the thin films produced. The kinetics experiments are on-going but the microstructural analysis has been performed using a field-emission scanning electron microscope (FESEM), with cross sectional work used to determine the growth rate and thickness of samples. Comparison of crystal morphologies to other sources and past experiments reveals some discrepancies that can be attributed to the use of different precursor partial pressures. Characterization of the exact composition of these films will be done in the future using energy-dispersive X-ray spectroscopy and X-ray diffraction.
Funding Provided By: Chemistry Department Funding
Hydroxyl Scavenging Activity of X-Phenolics
Lusajo Mwakibete ’19; Advisor: Cynthia Selassie
Radicals have been known to have significant impact in many biological systems. Foods which contain antioxidants and antiradicals have been suggested as means of neutralizing these radicals. Common compounds found in many foods are phenolic in nature. This study will focus on how substituted-phenols can mitigate hydroxyl radicals and examine how various substituents on the phenols impact their antioxidant and radical scavenging ability. These results will then be subjected to quantitative structure activity relationship (QSAR) analysis. A bio assay was developed and run using deoxyribose and an EDTA-complex to form hydroxyl radicals. An X-Phenol was then added to compete against the deoxyribose in neutralizing the hydroxyl radicals. The resulting interaction was measured by the color intensity of the pink chromogen formed by the degradative malonaldehyde. UV/Vis spectroscopy was utilized for the various absorbance readings. The lighter the color of the solution indicated that the more potent X-phenols were effective at neutralizing radicals. Preliminary data attained shows that phenols with electron donating groups (EDG) were most effective at neutralizing radicals compared to phenols bearing electron withdrawing groups. This is demonstrated by 4-aminophenol that has the highest Log (1/IC50) value and thus the lowest IC50 which suggests that its lowest dosage can neutralize most hydroxyl radicals compared to other X-Phenols.
Funding Provided By: HHMI
Probing the Equilibrium Isotope Effects in 1,3-Disiloxanediols
Kavoos Kolahdouzan ’18; Advisor: Daniel O’Leary
A series of structurally-varied disiloxane diols have presented intriguing hydrogen bonding properties and can function as anion-binding catalysts. To garner fundamental information about the solution hydrogen-bonding behavior in this series of compounds, a comparative 1H NMR study using an NMR isotopic perturbation method was employed to study the intramolecular hydrogen bonding of disiloxane diols and the carbon-based analogues. A comparison of the OH chemical shifts for OH/OH and OH/OD isotopologues of disiloxane diols shows that, upon deuteration, a downfield isotope shift is observed in apolar solvents such as CD2Cl2. This isotope shift indicates that the deuterium has a preference for the exterior position of the intramolecular hydrogen bond. This is in stark contrast to the upfield isotope shifts observed in the carbon-based analogues, which indicates that the deuterium has a preference for the bridging position of the intramolecular hydrogen bond. Through a joint experimental and computation investigation, the origin of this contrasting hydrogen bonding behavior of both compounds in solution will be presented. In particular, current efforts are directed towards observing isotope shifts in a variety of polar and apolar solvents as well as conducting quantum mechanical calculations to gain greater information about the intramolecular hydrogen bond characteristics in these molecules.
Funding Provided By: Chemistry Department Funding
Computational Mechanistic Investigation of SO2 Insertion into Ni Complexes
Neil Chan ’18; Advisor: Nicholas Ball
Sulfonyl-based groups sulfonamides, sulfonyl halides, etc. are molecules of interest with roles in pharmaceutics and industry. Due to their industrial importance, there is an incentive to find newer, more efficient ways to synthesize these compounds. Our molecule of interest is sulfonyl fluoride, a compound used as a medicinal precursor and biological probe. In this research project, we propose using a nickel catalyst, in lieu of more expensive palladium, to synthesize sulfonyl fluorides. As a new proposition, the mechanism of this nickel-catalyzed reaction is much less elucidated than its palladium-catalyzed counterpart. We propose to confront this problem by using computational quantum mechanics to create a reaction coordinate diagram detailing the energies of potential reactants, intermediates, products, and transition states involved in the reaction. The level of theory used in these computations is B3LYP-LanL2DZ/6-31G*, a density functional theory (DFT) method invoking a contracted Gaussian-type orbital (CGTO) basis set. Updated findings suggest that the oxygen atom of sulfur dioxide, not the sulfur, is the thermodynamically favored nucleophile in SO2 insertion. Additionally, thermodynamic data suggests that SO2 insertion into Ni-F bonds creates the most energetically favored intermediates, followed by Ni-C and Ni-N. Current efforts seek to determine activation barriers for each step in the reaction, as well as to identify the correct reactive Ni-catalyst structure.
Funding Provided By: Chemistry Department Funding
Studying the role of MtlR in Vibrio cholerae biofilm formation and regulation of the mannitol transporter MtlA
Jessica Wang ’18; Advisor: Jane Liu
Vibrio cholerae, the facultative bacterium that causes the disease cholera, transitions between aquatic reservoirs and the human small intestine during its life cycle. Crucial to its survival in these vastly different environments is the complex regulation of genes encoding carbon-source transporters. One such carbon-source is mannitol, a six-carbon sugar alcohol produced by algae. Mannitol is concomitantly phosphorylated and transported into V. cholerae by MtlA, which operates within the phosphotransferase system (PTS), a highly conserved system for carbohydrate uptake across bacteria. In addition to mtlA, the mtl locus also contains mtlR, encoding a regulator that showed repressive effects in Escherichia coli but has not been characterized in V. cholerae. In my research, I worked on furthering understanding of how MtlR regulates MtlA activity in V. cholerae grown in a variety of carbon sources, including mannitol, glucose, and non-mannitol, non-glucose sugars. We observed that mtlR overexpression inhibits biofilm formation, an activity positively affected by MtlA. Surprisingly, western blotting analysis demonstrated that MtlR protein levels are highest in cells grown in mannitol medium. Finally, MtlR levels remained appreciable in cells switched from mannitol to non-mannitol medium. These results contribute to a model in which MtlR represses mtlA in non-mannitol conditions, and might serve the purpose of fine-tuning MtlA levels during environmental shifts.
Funding Provided By: Chemistry Department Funding
Investigating the role of IGR4 in the fructose operon of Vibrio cholerae using peptide tags
Christina Beck ’20; Advisor: Jane Liu
Vibrio cholerae is a facultative pathogen and the causative agent of cholera—a diarrheal disease endemic in more than 50 countries today. V. cholerae uniquely functions in two distinct environments, the human small intestine and aquatic reservoirs. Small RNAs (sRNAs) have been shown to play an important role in modulating gene expression in these two environments, repressing or inducing specific genes when certain environmental signals are received. IGR4, a putative sRNA, lies between the genes fruB and fruR in the fructose operon. These genes play an important part in the fructose-specific phosphotransferase system (PTS), which is responsible for bringing fructose into bacterial cells. Thus, we have reason to believe IGR4 may affect expression of fruB or fruR, possibly affecting the PTS. In order to connect the expression patterns of IGR4, fruB, and fruR, we used multiple cloning techniques to insert either a HA tag or a FLAG tag at the C-terminal end of FruR or FruB, respectively. Once these epitope tags were inserted, we observed the expression of fruB in multiple sugars using anti-FLAG antibodies and western blot analysis. Our western blot revealed expression of fruB in fructose only—something we expected. The next step would be to quantify the effects of IGR4 on fruB expression. If we can discern regulatory effects of IGR4 on fruB, we can truly classify it as a sRNA and use its expression patterns to better understand how V. cholerae functions in two distinct environments.|
Funding Provided By: Chemistry Department Funding
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 up or 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 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 interferometry (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 two-pronged 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 L-stereoisomer 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