Chemistry Research
Festival of Science was cancelled April 2020 and April 2021 due to the COVID-19 Pandemic.
Festival of Science was held Friday, April 26, 2019
David Bain, Chemistry, Faculty Sponsors: Samantha Glazier and Catherine Jahncke
“Kinetic Characterization of Chemotherapy Drugs”
Abstract: Certain flat, planar molecules can insert between DNA base pairs (intercalate) to disrupt replication and transcription upon mitosis in cells. This makes them ideal candidates for mitigation of tumor growth, a process which relies on uncontrolled cell division. To design improved drugs, an understanding of both thermodynamic and kinetic intercalative pictures is necessary. Currently, kinetic information is technique limited. Stopped-flow fluorimetry measurements have 10 ms time resolution, unfortunately many binding reactions occur on timescales orders of magnitude faster. A homebuilt temperature jump (T-jump) apparatus could achieve up to 1 μs resolution via capacitance discharge heating coupled with relaxation kinetics. This works by converting stored electrical energy from a capacitor to thermal energy in solution where binding occurs. A faster capacitor discharge leads to quicker solution heating and increased resolution in relaxation kinetics calculations.
An interesting case study is provided by the intercalating drug proflavine. Using molecular dynamics calculations, Mukherjee et al. found that the intercalation mechanism involves both the major or minor groove of DNA. This is the first report of major groove involvement. Systematically blocking each groove before measuring kinetics via T-jump will show, experimentally, whether both grooves are involved in the intercalative binding mechanism. This technique and investigation of major groove involvement can be applied to many chemotherapy agents and will pave the way for future drug synthesis for superior anti-tumor potency.
Lisa Kozodoy, Biochemistry, Faculty Sponsors: Nadia Marano and Lorraine Olendzenski
“Isolating amyloid fibers from Haloferax volcanii”
Abstract: Amyloids are proteins that when aggregated form large insoluble fibers characterized by their cross-beta sheet structure. While these proteins are often implicated in human neurodegenerative diseases, such as Alzheimer’s, many organisms make functional amyloids. These have been studied in bacterial biofilms and are known to aid in stability and structure. Archaea also form biofilms, however, they have not been researched as extensively. Functional amyloids have been identified in the archaeon Haloferax volcanii. The aim of this study is to isolate and purify amyloid fibers from H. volcanii to elucidate more about their composition and structure. Isolation is done through differential centrifugation and sonication, following previous research by Heather Raimer ‘17. Sonicating the cells releases the amyloid fibers from the cells, which are detected using Thioflavin T (ThT) assays. Low-speed centrifugation allows for the removal of cellular debris. Ultracentrifugation then isolates the larger fibers from suspension. Lastly, tube SDS-PAGE is used to purify the amyloids from contaminating proteins because they do not enter the gel. Current research is being done to deaggregate the fibers into monomers using formic acid. The depolymerized monomers can then be resuspended and sent off to a collaborator for sequencing.
Evan Ketcham, Biochemistry, Faculty Sponsor: Matthew Skeels
“Engineering Thermodynamically Stable Mutants of Human Basic Fibroblast Growth Factor Protein”
Abstract: Fibroblast growth factor 2, also known as FGF basic, is a signaling protein that plays a variety of physiological roles in the body. Of particular interest in recent studies is its ability to promote angiogenesis following blood vessel injury. However, it is a labile protein that tends to break down quickly under the stressors of the human body. Previous research has determined, in silico, several rationally-designed mutant proteins that are predicted to be more stable than the wild type. This project seeks to create these mutants and compare their stability in vitro to FGF2. A bacterial expression system is used to create the protein, which is then purified using chromatographic methods. After purification, the linear extrapolation method (Vivian and Callis 2001) is used to experimentally determine the change in Gibbs free energy of unfolding for the protein. In this method, the unfolding of a protein is followed by monitoring intrinsic tryptophan fluorescence as it is exposed to increasing concentrations of chaotrope. These data can be mathematically transformed into an experimental ΔG value. The data have demonstrated that the trend in stability matches the computational model, with the mutants known as FGFbase (N71E, E78K, K86E, T112E) and FGFrecp (N53E, K68E, R102E, K111E, K117E) being more stable under chemical denaturing conditions than the wild type FGF2. Further research is being conducted to probe more mutants for their stabilities and assess their relative biological activities using cell cultures.
Jiarui Zhang, Chemistry, Faculty Sponsor: Larry French
“Synthesis of Anthraquinone Derivatives by Editing DNA Binding Functional Groups”
Abstract: Anthraquinone is a polycyclic aromatic hydrocarbon, and its derivatives have appeared in many anticancer chemotherapeutic drugs, such as Doxorubicin, Mitomycin, and many other biologically active compounds. The anthraquinone moiety may act as a DNA intercalator and its derivatives can slide between nucleotide bases forming covalent bonds with DNA to act as cytotoxic molecules by stopping the cell cycle within various cell types. This project is to expand the variety of anthraquinone derivatives. The multistep reaction started with the Diels-Alder reaction forming the anthraquinone molecule. The Diels-Alder reaction allows the formation of a six-membered ring by creating two sigma bonds, and has been utilized in many reactions of organic synthesis. The double Diels-Alder reaction utilizing myrcene as diene and benzoquinone as dienophile will form two six-membered rings flanking quinone. This multi-step synthesis can be used to generate derivates based on the multiplicity and variability of the functional groups, be editing and expanding the variety of side functional groups may cause more interactions with DNA backbone or nucleotides for potential pharmaceutical interest. In particular, we are trying to synthesize an anthraquinone that is flanked by two piperazine moiety that we hypothesize may increase the affinity of DNA binding.
Dylan Babcock, Chemistry, Faculty Sponsor: Larry French
“Sustainable Chemistry Utilizing Terpenes Synthesis of Anthraquinone Analogs from Myrcene”
Abstract: The use of terpenes in synthesis of chemical products may bring about a more sustainable future. Currently, chemical procedures include bulk chemicals such as hydrogen, benzene, and propylene obtained from oil and gas. The movement towards a more sustainable future is promoting green alternatives in industrial synthesis. This research focuses on exploring the functionalization of myrcene towards known DNA intercalators. A myrcene derived anthraquinone analog is foundation of our work with DNA intercalating agents. The molecules synthesized in this research are useful in that they eliminate the need for some environmentally harmful precursors as well as have the potential to mimic some of the structural properties that important drugs have. Analysis of these compounds is being done through GC/MS as well as C13 and H1 NMR. Future experiments will focus on the continuation of functionalizing the myrcene arms to better interact with DNA base pairs as well as finding methodology for testing our molecules efficiency as intercalators.
Sarah Potter, Chemistry, Faculty Sponsor Matthew Skeels
“Nutritional Analysis of Honey”
Abstract: Bee populations around the globe have been decreasing for the last several decades. While many factors are contributing to this decline, several are related to nutrition. A diverse diet is critical for bee’s growth, reproduction, learning, and resistance to viruses and parasites. The nutrition available to bees can be investigated by analyzing the nutritional profile of one of their major food sources, honey. The goal of this work is to develop facile methods to analyze the nutritional profile of honey. Analytes include moisture content; minerals and metals; sugars, glycerol, and organic acids. Moisture content will be measured as a percentage of weight after honey is heated to a constant mass and through refractometric methods. Samples will be subjected to microwave digestion and minerals and metals will be determined with FAAS. An enzymatic assay will be applied for glycerol and glucose determination. Organic acids will be analyzed using solid phase extraction followed by HPLC. Techniques developed will be applied to the study of monofloral honeys to compare their respective nutritional profiles. Eventually, this information will be used to create environments with plants that provide appropriate nutrition for pollinators year-round.
Lillian Devereux, Biochemistry, Faculty Sponsor Nadia Marano
Detection of Amyloid Oligomers using ANS (1-Anilinophthalene-8-sulfonic acid), Bis-ANS (4,4’ –Dianilino- 1,1’ –binaphthyl -5,5’-disulfonic acid), and DCVJ (4-dicyanovinyl-julolidine)
Abstract: Amyloid fibers are a component of bacterial biofilms that can be isolated through various protocols for analysis. The standard assay uses Thioflavin T (ThT) to identify the presence and quantity of mature amyloid fibers. ThT is a molecular rotor meaning that it rotates around a single bond. When bound to amyloid fibers this rotation is inhibited and stabilizes the excited state, thus producing measurable fluorescence. However, ThT is not an effective measure of amyloid oligomer formation, the smaller building blocks of mature fibers. ANS, Bis-ANS, and DCVJ are three alternatives to ThT that are characterized by a multi-ring structure. DCVJ acts in a similar way to ThT in that, when bound, the molecule is locked into an excited state producing fluorescence. In contrast, ANS and Bis-ANS bind to the hydrophobic regions of proteins which are exposed during amyloid fiber formation and following binding, the molecules fluoresce. Little is currently known about the binding and consequent fluorescence of these molecules to oligomers. This research will examine the ability of these three fluorophores to bind to oligomers throughout the aggregation process, giving an indication of kinetics and method of amyloid fiber formation. Insulin will be used because it forms amyloid fibers under well-defined conditions. Preliminary research shows DCVJ and ANS follow the pattern of ThT, meaning that as mature fiber concentration increases, so does the fluorescence signal. Bis-ANS, however, displays the opposite pattern, indicating it is likely detecting varying concentrations of amyloid oligomers via binding to the exposed hydrophobic residues.
Heinrich Salzmann, Chemistry, Faculty Sponsor Adam Hill
“Hafnium Containing Heterobinuclear Light Absorber Units”
Abstract: Harnessing solar energy efficiently is one way to tackle climate change and our dependency on fossil fuels. Metal to metal charge transfer in heterobinuclear units matches the energy of visible light. Hence, heterobinuclear light absorbers are potentially key features in artificial photosynthesis systems. The excited electron state is unknown but expected to undergo a spin flip. Heterobinuclear units on silica surfaces make an effective system in which to investigate intersystem crossings in the context of electron transfer to a 2,2’-bipyridine chromophore. Hafnium is heavier than previously studies early transition metals in heterobinuclear units and will have 60 times stronger spin-orbit coupling. Hafnium-cobalt oxo-bridged on silica nanoparticles have been synthesized. Infrared (IR) and Raman spectroscopy were used to ensure successful synthesis steps. Samples have been probed with 2,2’ bipyridine and analyzed with time-correlated single photon counting (TCSPC) and time-resolved fluorescence, to investigate the lifetime of the excited state.
Alissa Stone, Chemistry, Faculty Sponsor Adam Hill
“Time-correlated single-photon counting measurements of electron transfer between heterobinuclear units and bipyridine”
Abstract: Understanding the excited metal-to-metal change transfer (MMCT) states of heterobinuclear units is vital to creating artificial photosystems. To learn more about the metal electron transfer and to model artificial photosystems, binuclear units on silica nanoparticles were synthesized using air free techniques and coupled to bipyridine chromophores. Particles were pressed into transparent pellets and were characterized using UV-visible and FT-IR spectroscopy. Time-correlated single-photo counting (TCSPC) and 2D fluorescence data were collected to measure the excitation spectrum, emission yield, and lifetime of each binuclear unit/chromophore charge transfer polyad. These data provide evidence that when the energy levels of the system are in the correct rectifying arrangement, the excited state persists to the microsecond timescale.
Abigail Enders, Chemistry, Faculty Sponsors Adam Hill and Samuel Tartakoff
“Kinetics of substituted Wagner-Jauregg reactions studied by 1H-NMR with an adaptive Python program for integration of poorly resolved spectra”
Abstract: Monitoring chemical kinetics by NMR requires reproducible and accurate integration for hundreds of 1H NMR spectra, but existing software packaged with many instruments is insufficient for the purpose. Kinetics studies of the Wagner-Jauregg reaction between para-substituted styrenes and maleic anhydride for determination of the mechanism have been previously limited by NMR instrumentation and its accompanying software. This class of reactions is an example of a challenging problem in which variability between substrates and other background signals make good integrations difficult using common techniques. Custom code, written in Python and using the lmfit library incorporates the reproducible integration of multiple peaks and is adaptable for a variety of spectral features. A graphical user interface makes the code accessible to new users. With this additional analysis, the Diels–Alder-type mechanism of Wagner-Jauregg was confirmed by measuring the effects of substitutions on rate.
Any changes need to be made please let Diane Chase know.
2017 - 2018 Senior Research
Festival of Science 2018 was held Friday, April 27, 2018.
Jack Mechler - Biochemistry, Faculty Advisors: Nadia Marano and Lorraine Olendzenski
Isolating and Characterizing Functional Amyloid Fibers from Microbacterium sp.
Functional bacterial amyloids make up an important structural component of biofilms, and little is known about their variance in structure throughout nature. This research aimed to adjust isolation procedures developed by Heather Raimer (2017) for an archaeon, to obtain amyloid fibers from Microbacterium sp. that was isolated from the soil of a pig farm by Abby Korn (2014), and shown to test positive for amyloids using the Thioflavin T (ThT) assay by Hunter Berrus (2015) and Jordan Koloski (2016). ThT assays during different bacterial growth conditions showed that the Microbacterium produce more amyloids closely associated with the cells when grown on agar plates, as opposed to liquid media. Early protein isolation trials showed that amyloids more closely associated with the cells were better candidates for isolation. Therefore, plate-grown amyloids were separated from the cells using sonication, and purified from other components using differential centrifugation and polyacrylamide gel electrophoresis. To further purify this amyloidogenic material, and to characterize the monomers that it is composed of, it was depolymerized in formic acid, further purified with differential centrifugation, dried in a speedvac, and resuspended in both water and formic acid with 1M urea. The sample in formic acid and urea was run on an SDS-page gel to determine the size of its monomers and the extent of the purification, and the sample in water examined under a scanning electron microscope, according to previously derived methods. Isolating and imaging these novel functional amyloids will be another step in our understanding of bacterial amyloids.
Funding: University Fellowship
Alexis Haley - Chemistry, Faculty Advisor: Adam Hill
Luminescence of Lanthanide/Transition Metal Heterobimetallic Phosphinoamide Complexes
Due to their combination of long luminescence lifetimes and higher absorption cross-sections, heterobimetallic complexes containing lanthanides and transition metals are becoming increasingly suitable for applications in photocatalytic processes and bioimaging techniques, as well as offering the opportunity to study energy transfers between d and f orbitals. Non-radiative quenching of the excited state competes with luminescence. The competition may be optimally studied in a family of three-fold symmetric complexes with phosphinoamide ligands, which provide stability for the transition metal-lanthanide center’s dative interaction and aid in its metal-to-metal charge transfer (MMCT). The first-row transition metals Fe, Co, and Ni are paired with lanthanides La, Eu, and Tb to investigate their metal-metal interactions via their luminescent lifetimes and quantum yields. The results are compared with monometallic complexes and free metal ions in solution.
Funding: Chemistry Department
Alissa Stone - Chemistry, Faculty Advisor: Adam Hill
Synthesis of Cobalt-Zirconium Heterobimetallic Sites on a Silica Nanoparticle Substrate
Co-Zr-SiO2 nanoparticle heterobimetallics are useful for performing artificial photosynthesis due to d-d electron transfers between metals. The metal-to-metal charge-transfer (MMCT) transition should exist between Co(II) and Zr(IV) based on previous studies. Zr-SiO2 nanoparticles and Co-Zr-SiO2 nanoparticles were made using air-free techniques. The monometallic and heterobimetallic products were analyzed using FT-IR, UV-vis, and fluorescence. The heterobimetallic sites are useful in determining and understanding the MMCT transition.
Natasha Turyasingura - Chemistry, Faculty Advisor: Emily Dixon
A Study of Gene Regulation in Yeast under Stress: The Mechanism of Binding of Rpd3 to the Promoter Region of NDP genes in Saccharomyces cerevisiae
When Saccharomyces cerevisiae yeast cells are subjected to stressful conditions, the cells respond by altering certain cellular processes to adapt and survive. One such stressful condition is nutrient deprivation. The TOR pathway has been found to be a central regulator of both cell size and proliferation. Rapamycin is a small molecule that binds to and inhibits the TOR protein eliciting a transcriptional response that is reminiscent of nutrient deprivation. This transcriptional response results in the activation and repression of specific groups of genes. The repression of select genes has been studied to occur by the action of the histone deacetylase Rpd3. Rpd3 binds to the promoters of rapamycin-repressible genes via the transcriptional repressors Dot6/Tod6 to the GATGAG sequence in the promoter region. Here, we investigate whether Rpd3 binds to the promoters of NDP genes to activate them in the same way that it binds to the promoters of genes repressed by Rpd3. To do this, we approach the question in two ways: we delete the GATGAG sequence in the promoter of the GAP1 gene to determine if Rpd3 still binds to the GAP1 promoter; and we tag Rpd3 in a Dot6/Tod6 mutant to determine whether the presence of Dot6/Tod6 is necessary for the binding of Rpd3 to the promoter of GAP1 (and other NDP genes).
Funding: Biology Department
Philip Park, Chemistry, Faculty Advisor: Matthew Skeels
Determination of Lead and Other Heavy Metal Contamination of Maple Syrup - Produced in St. Lawrence and Chittenden Counties
This study investigates the magnitude of heavy metal contamination in maple syrup. The ingestion of lead (Pb) by humans can be toxic, potentially leading to health-related issues, such as memory loss, heart disease, and reduced fertility. While the United States banned the use of leaded paints for household uses in 1978, lead contamination remains a concern from existing sources of leaded paint and water pipes, solder, and smelting. In the maple syrup industry, many pieces of equipment, including the metal taps and tin collecting buckets used in the sugaring process, have been known to contain leaded materials. To determine the extent of lead contamination in maple syrups we evaluated syrup samples with a Perkin Elmer Flame/Graphite Furnace Atomic Absorption Spectrometer. The syrup will be collected from a variety of sugar farms in St. Lawrence County (NY) and Chittenden County (VT). In addition to lPb, the concentration of other metals, including Copper (Cu), Zinc (Zn), Mercury (Hg), and Iron (Fe) will also be determined. By analyzing the concentration of lead and other metals in maple syrup, it could show the extent to which different pieces of equipment in the sugaring process are able to contaminate maple syrup.
Rachel Pineda - Chemistry, Faculty Advisor: Matthew Skeels
Comparative Assessment of Heavy Metal Concentrations in Various Vegetables via GFAAS/FAAS
Dietary exposure to heavy metals, namely lead (Pb), copper (Cu), and zinc (Zn), has been identified as a risk to human health through the consumption of vegetable crops. This study investigates the magnitude of heavy metal contamination in vegetable samples at 2 sites: Philadelphia, PA (urban) and Canton, NY (rural). The foodstuffs investigated in this study include pumpkins, potatoes, tomatoes, peas, carrots, turnips, peppers, etc. These produces were dried in a convection oven for an average of 19 days, ground into fine particles using a Wiley mill, subjected to acid digestion using nitric acid, and analyzed in independent replicates of 2 using Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS) and in replicates of 3 using Flame Atomic Absorption Spectrophotometry (FAAS). High concentrations of heavy metals in vegetables might be related to their growth in or near industrial settings. The effect of industrialization on the production of vegetables from small scale gardens will be assessed. .
Tyler Guido - Chemistry and Biology, Faculty Advisors: Ning Gao and Jill Pflugheber
Human Teeth as a Bioindicator for Environmental Exposures
The human tooth is comprised of four primary layers; enamel, dentin, pulp and cementumToxic elements such as heavy metals and fluorine can accumulate in these four layers of the tooth over time. When looking at heavy metal accumulation, these four tooth layers may serve a greater purpose as bioindicators for environmental hazards that individuals have been exposed to over time. By analyzing the concentration levels of heavy metals and fluorine in human teeth, we might be able to draw correlations between gender, age, and possibly geographic location to infer both the extent and sources of exposure for particularly harmful elements. Building upon the work of previous St. Lawrence University SYE students, improving an analytical protocol when using a Scanning Electron Microscope (SEM) with an X-ray Energy Dispersive Spectrometer (EDS) will create standard curves with significantly improved accuracy and precision. This will allow for the quantification of concentrations of toxic elements in teeth. Calibration curves with improved accuracy and precision have been created using previously made standards for elements Pb, F, Cd, and Zn. New Hg standards have also been made, using EDTA as a ligand to keep Hg stable under high vacuum when analyzing via EDS. Using these standard curves with improved accuracy and precision, quantification of heavy metals and fluoride in the dentin, enamel, and root layers of the tooth is being done using human teeth. The teeth analyzed will be classified by age, gender, and by the absence or presence of amalgam fillings.
Amanda Kennedy - Chemistry, Faculty Advisor: Ning Gao
An Analysis of Denitrification Effectiveness Using UV/Vis Spectrometry
Nitrate in groundwater systems, typically sourced from fertilizer runoff, has detrimental environmental effects as it causes eutrophication at river mouths, and can potentially cause methemoglobinemia, or “blue baby disease” when found in drinking water at high concentrations. Previous research done at Ryerson Polytechnic University has shown that chemical denitrification of groundwater is possible using powdered aluminum. This reaction was tested under laboratory conditions, in which the percent decrease in nitrate was observed using UV/Vis spectrometry with solutions held under basic conditions. Based on the lack of effectiveness in this process when attempting to get comparative results to the literature, a photocatalytic method using blue LED light was attempted to boost the percent reduction by exciting the molecules during the reaction so the reaction would proceed more effectively. PH dependency for this reaction was also explored, as there is a notable difference in absorption of UV light between initial solutions and the same solution under basic conditions, even before reacting with aluminum. Results for this experiment may have been skewed by partial reduction of nitrate ions into nitrite, as both species absorb in the same ultraviolet range, and are difficult to determine separately.
Timothy O’Brien - Chemistry, Faculty Advisor: Ning Gao
An Independent Study in Food Chemistry: A Healthy Menu
A sample menu of nutritionally balanced soups, salads, appetizers, main courses, desserts was developed to offer healthy options for a balanced, multiple-course meal or series of dishes that could be eaten over the course of a day. For each dish, the tastes, flavors, and textures are listed. To better describe flavors and the process by which foods are sensed by the body, a background in flavor science, retronasal olfaction, and neurogastronomy is provided. For each food item, carbohydrate, protein, and lipid daily values were discussed and evaluated. Macronutrient composition was analyzed as well. Sources of both soluble and insoluble fiber are mentioned and their importance in the body is discussed. Vitamins and minerals and the processes in which they are taken up by the body are considered as well as their importance in bodily functions.
Danielle Couture - Chemistry, Faculty Advisor: Samantha Glazier
Characterizing the Binding Mode of the Doxorubicin Derivative doxy to DNA
Doxorubicin is a DNA intercalator that binds with its planar carbon rings stacked between DNA base pairs and its sugar group locked into the minor groove. A doxorubicin derivative, doxy, has been synthesized by Haley Anchukaitis and Dr. Tartakoff. Doxy’s major difference is the removal of the sugar moiety from one of the carbon rings. This difference is significant because as doxorubicin binds to DNA, the first step in the intercalation mechanism is electrostatic interaction between the positively charged sugar and the negatively charged backbone of the DNA. Once bound, the sugar moiety stabilizes the drug-DNA interaction. The new derivative is expected to either intercalate like the parent compound or bind in the minor groove of DNA. Since the derivative compound is neutral, it is only somewhat soluble in aqueous solution, and standard methods for determining the binding mode are unfeasible. In this project, a DNA melting protocol was developed to determine the binding mode of the compound. DNA intercalators stabilize DNA and raise its melting temperature, while groove binders do not influence the stability of DNA and leave the melting temperature unchanged. The preliminary results obtained from melting curves using an absorbance based method indicated that the binding mode of doxy is most similar to groove binding. The data obtained by this protocol yielded indistinct sigmoidal curves, which led to the development of a fluorescence based method. This method, which uses 1000-fold less material, can be used to determine the binding mode of novel drug compounds like doxy.
Funding: Chemistry Department
David Bain - Chemistry, Faculty Advisor: Samantha Glazier
Examining the Intercalation of Nogalamycin with DNA
There exists a class of small, flat molecules with the ability to insert (intercalate) themselves between DNA base pairs to disrupt replication and transcription. This makes them ideal candidates for cancer and tumor treatment, conditions which rely on the DNA replication process for abnormal cell growth. This project seeks to better understand the kinetics and thermodynamics behind the intercalation of one such drug, nogalamycin, with DNA, particularly, by which groove (major or minor) it intercalates. By utilizing nogalamycin’s fluorescent properties, and alternately blocking each groove with known major and minor groove binders, methyl green and Hoechst 33258 respectively, the pathway can be established via stopped-flow fluorimetry; these methods are novel and results can be compared to computational studies. Thus far, we have shown that nogalamycin undergoes small amounts of photodecay, but the fluorescence of nogalamycin is not affected by the presence of the groove binders. A binding time of nogalamycin with DNA has also been established.
Kelsey Murphy and Avery Peck - Chemistry
Faculty Advisors: Dr. Samantha Glazier and Dr. Samuel Tartakoff
Synthesis and DNA Intercalation Mechanism Characterization of Novel Chemotherapy Drug Analogs
Proflavine is a synthetic acridine drug that in recent years has been studied as a DNA intercalator in cancer treatments to kill tumor cells. The biological activity of the drug is associated with its ability to bind to DNA, however cardiotoxicity is a major issue associated with such drugs. This necessitates an increased understanding of the binding mechanisms of these drugs to optimize drug structure-activity relationships in order to minimize toxicity and increase specificity and efficacy. Although not fully understood, it was assumed that proflavine enters directly through the minor groove of DNA to reach the final intercalated state, but recent molecular dynamics calculations proposed an unusual binding mechanism involving the major groove. The major groove was previously thought to be uninvolved in the binding event, prompting the investigation of major and minor groove involvement in intercalative pathways of small drug molecules. This study seeks to functionalize proflavine, a small intercalator, at its amino groups so as to vary molecular electronics, polarity, and size to determine how molecular functionality may change DNA binding mechanisms with respect to the major and minor grooves of DNA. Our investigation utilizes fluorescence spectroscopy competitive binding studies with Hoechst 33258 and methyl green, minor and major groove binding dyes, to examine how the binding constants of small intercalating molecules respond when access to each groove is inhibited. Proflavine is subjected to a variety of synthetic manipulations in order to sample the structure-activity profile of drug intercalative mechanisms.
Funding: SYE
Abigail Enders - Chemistry, Faculty Advisor: Samuel Tartakoff
Determining the Mechanism of the Wagner-Jauregg Reaction by Analysis of Reaction Rates: A Kinetics Study
Synthetic organic chemistry focuses on converting simple starting materials into complex molecules, which are then used to make plastics, pharmaceuticals, synthetic fibers, and other valuable commercial products. The Wagner-Jauregg reaction (a reaction that forms complex, polycyclic molecules) has long been viewed as a double Diels–Alder reaction but research on the mechanism has not been conducted. A correct understanding of the mechanism should allow us to determine if there is a reactive intermediate, which could be intercepted to form new, interesting products. Our research examined competing mechanisms for the Wagner-Jauregg reaction by comparing relative reaction rates using Nuclear Magnetic Resonance spectroscopy (NMR), then conducting a Hammett analysis. Various substituted styrenyl substrates with electron withdrawing groups (EWG) and electron donating groups (EDG) were reacted with maleic anhydride and analyzed. The preliminary results show that the reaction pathway is likely analogous to the well-understood Diels–Alder reaction for many, but not all, substrates.
Funding: Ronald E. McNair Post-Baccalaureate Achievement Program
Anne Buck - Chemistry, Faculty Advisors: Samuel Tartakoff
Progress Towards Efficient Synthesis of Morphine Analogues Using the Wagner-Jauregg Reaction
A Diels-Alder cycloaddition allows for the creation of two new sigma bonds along with a new six membered ring. This reaction occurs as a result of the pi bonds found in the diene and dienophile being higher in energy and therefore less stable, interacting to produce more stable sigma bonds. The Diels Alder reaction allows for different conformations to be produced depending on how the diene interact with the dienophile resulting in new steriocenters. The Wagner-Jauregg reaction, is a specialized Diels Alder reaction in which the diene is an aromatic ring. Aromatic compounds are exceptionally sable and unreactive due to an extended conjugated network. It normally requires very harsh conditions to cause an aromatic ring to react, which is the main challenge of the Wagner-Jauregg. This method can be used in the production of opioid- like compounds. Opioid molecules are composed of 5 rings, with the proper one ring starting material, through a Wagner-Jauregg reaction it is possible to create a three-ring structure in one step. Two starting compounds were produced through Wittig reactions followed by purification via column chromatography and characterization via proton nuclear magnetic resonance. These compounds then underwent a series of variable reaction conditions, such as high temperatures, to try and promote a Wagner-Jauregg intermolecular cycloaddition with maleic anhydride. Up to the present date, we have produced a compound with highly electron withdrawing functional groups, which we are trying to currently characterize.
Funding: University Fellowship, Stradling Scholarship Fund
Haley Anchukaitis - Chemistry
Faculty Advisors: Samuel Tartakoff and Samantha Glazier
Synthesis of Doxorubicin Analogs for DNA Binding Studies
Anthracyclines are used in the medical field for their antitumor activities. One of the most common drugs of this class, doxorubicin (DOX), has been used to treat cancers in the breasts, ovaries, and lungs. The mode of action for DOX is intercalation between the base pairs of DNA, which disrupts translation and replication. The planar structure of DOX allows the rings to slide in between the base pairs, while the sugar moiety interacts with the minor groove. The sugar connects to the minor groove via hydrogen bonding between the amine of the sugar and the sugar-phosphate backbone of DNA. To determine how the absence of those hydrogen bonding interactions affect the binding mode, multiple DOX analogs were synthesized. The DOX analog 7,10-dihydro-6,11-dihydroxy-naphthacenedione required a four-step synthesis and was recovered in fairly low purity. The main difficulty with this synthesis was optimizing the double Claisen rearrangement reaction conditions. Further attempts have also been made to introduce an amine group onto this tetracyclic structure to mimic the DOX amino-sugar, making interaction with the minor grove of the DNA backbone possible. DOX-aglycone was synthesized by hydrolyzing the sugar off of the fourth ring. The analogs are currently undergoing kinetic studies to test how well and with what mechanism they bind to DNA.
Funding: University Fellowship: Robert L. Stiles P’71, University Fellows Endowment and the Daniel F. ‘65 and Ann H. Sullivan Endowment for Student/Faculty Research
2016 - 2017 Senior Research
Festival of Science and Festival of Scholarship was held April 28th, 2017. Listed are student name, title and abstract of their research.
E. J. Curtis
"The Effect of Structural Differences on Drug Interactions with DNA"
Chemistry Faculty Advisor: Samantha Glazier
In the field of allopathic medicine, when a certain medication is prescribed for an intended therapeutic effect, the efficacy of the drug, as well as the unintended side effects, can be highly varied between patients. A person’s genetic makeup is defined by the sequence of DNA that exists within his or her cells. Recent focuses in cancer biology have found not only that the DNA of cancer differs from the genome of otherwise healthy cells, but also that there exist genetic differences between cells of the same tumor.
Doxorubicin is a highly effective chemotherapy agent that has been used for the better part of a century. Though doxorubicin is well suited to kill many types of cancer, it has some serious side effects in much of the population. Doxorubicin produces harmful free radical oxygen species, that have the potential for both cardiotoxicity and higher order effects.
This study focused on characterizing the interactions of both doxorubicin and a doxorubicin analogue, doxorubicinone, with different types of DNA. Through a combination of spectroscopic techniques, we found that the interactions of the drugs were quite different from one another and the interactions varied greatly between different DNA sequences. These results may help researchers understand the reasons behind differences in drug efficacy between patients.
Vanessa Chilunda "Effects of Growth Stages and Sub-inhibitory Antibiotic Concentrations on Amyloid Protein Formation by Soil Bacteria"
Biochemistry Faculty Advisor: Nadia Marano
Functional amyloids are an important component of biofilms formed by bacterial communities. These amyloids are important due to their hydrophobic nature and other biochemical properties that help bacteria resist harsh conditions such as heat, pH, hosts’ immune systems and even some antibiotics. Studies have shown that subinhibitory antibiotic levels serve as signaling molecules that increase biofilm production and consequently bacterial resistance properties. Thus, I decided to study the effects of subinhibitory tetracycline concentrations on amyloid production in biofilms. Thioflavin T (ThT) spectroscopic assay was used to quantify the amyloid production. I found a statistically significant increase in amyloid production in one of the isolates when treated with 0.2 μg/ml and 0.5 μg/ml tetracycline concentrations. However, at higher tetracycline concentrations, amyloid production decreased. In order to elaborate on these results, I investigated the time taken by the bacteria isolates to produce maximum amounts of amyloid proteins in their biofilms. Bacteria concentration was estimated using absorbance at a wavelength of 600nm on spectrophotometer, and compared to bacteria count under a phase contrast microscope. I found that there was maximum amyloid production around day five when bacteria were cultured in liquid media. However, growing the isolates in liquid media for a longer period of time resulted to less cell-associated amyloid fibers compared to when cultured on agar plates. Currently, I am measuring amyloid production by bacteria isolates grown on agar plates instead of liquid media with different sub-inhibitory tetracycline concentration at different growth periods.
Isabel Bogacz "Analysis of Heterobimetallic Materials Through Raman Spectroscopy "
Physics Faculty Advisors: Catherine Jahncke and Adam Hill
Peak oil, global warming, and the modern energy crisis call for new solutions to harnessing and storing solar energy. Harnessing CO2 could provide an alternative fuel source through artificial photosynthesis. Photosynthesis stores energy from light as chemical fuels. In our work we study heterobimetallic materials. When these materials are hit with light, electrons on the outer edge of the molecules are excited to a less stable state. While in this excited state, the material will be able to split carbon dioxide into carbon monoxide and oxygen. Carbon monoxide is already heavily researched as an alternative fuel source. It is the act of obtaining carbon monoxide that eludes scientist. This project uses Raman spectroscopy to study these materials as they change carbon dioxide into carbon monoxide in order to better understand the mechanism involved in this reaction. We present the results of our initial studies where we add various amines or formic acid to our materials and observe the changes in the Raman spectra
Monica Bedford "Doping of Cobalt-Zirconium Heterobimetallic Material with Various Amine Ligands to Enhance Photochemical Carbon Dioxide Reduction "
Chemistry Faculty Advisors: Adam Hill, Catherine Jahncke
Hard-soft metal-metal complexes have been shown to reduce carbon dioxide (CO2). This reaction is one of the first steps in artificial photosynthesis, a way to combat rising levels of carbon dioxide. By inputting photons, an electron transfer (ET) occurs from the cobalt (II) center to the zirconium (IV) center. The excited electrons are then able to react with the carbon dioxide. However, a parallel process known as back-electron transfer (BET), wherein the electron relaxes back to the original energy level within the cobalt 3d orbitals, reduces the availability of excited electrons for further reaction. The energy levels of the cobalt 3d5 orbitals can be adjusted by substituting different amine ligands (triethylamine, diethylamine, diisopropylethylamine, azetidine, pyrrole, piperidine, and N-methylpiperidine) on the cobalt to make BET less energetically favorable. To judge the efficacy of the amine group in reducing BET, time-correlated single-photon counting fluorimetry measurements are considered in conjunction with the Raman spectra for carbon dioxide reduction with the same material.
Kelly Burke "Synthesis and photophysics of lanthanide/transition metal heterobimetallic complexes"
Chemistry Faculty Advisor: Adam Hill
Luminescent lanthanide ions have the potential for applications in the lighting industry, the creation of electroluminescent materials and medical imaging. However, their use is hindered by poor absorption cross sections. In order for luminescence to occur more easily, the lanthanide elements Eu, Tb and La have been paired with first-row transition metals, Co, Ni and Fe to create heterobimetallic compounds using air-free techniques. Hindered Lewis acid/base phosphinoamide ligands support direct electron donation from the transition metal to the lanthanide center, resulting in a significant increase in light absorption. The photophysical properties of the complexes were probed by time-correlated single photon counting phosphorescence spectroscopy, quantifying energy transfer through metal-metal bonds. The results provide an opportunity to explore the relationship between metal identity and excited state lifetime.
Wenyao Zhang "Probing the Reaction Mechanism of the Wagner-Jauregg Reaction: Hammett Study of 4-Substituted Styrenes "
Chemistry Faculty Advisor: Samuel Tartakoff
The mechanism of the Wagner-Jauregg reaction, long considered a double Diels–Alder reaction, was examined through the reaction of maleic anhydride and styrene derivatives. The Wagner-Jauregg reaction was expected to react in the same manner as the classic Diels–Alder reaction, giving a negative correlation in the Hammett plot. However, two other competing mechanisms were also possible. By taking advantage of proton NMR spectroscopy and observing relevant peaks, we could get the relative rate constants for four para-substituted styrenes (p-MeO, p-Me, p-H, p-Cl). The preliminary results suggest that, for the Wagner-Jauregg reaction of para-substituted styrene derivatives, the mechanism might be different for dienes with electron-rich groups (ERG) and with electron-withdrawing groups (EWG). Further experiments will be conducted on another styrene substrate with EWG to complete the Hammett plot and more accurate kinetic data will be collected by high-performance liquid chromatography (HPLC).
Jennifer Finan "Thermodynamic Characterization of a Novel Anthracycline/DNA Binding Mechanism "
Chemistry Faculty Advisor: Samantha Glazier
Anthracyclines are common antitumor antibiotics for cancer treatment characterized by a conjugated four ring system. These drugs bind to the DNA double helix preventing topoisomerase binding, thus halting cellular division. Three binding mechanisms for these drugs are intercalation, groove-binding, and less commonly, threading. A marketed anthracycline, doxorubicin, intercalates into DNA with a positive sugar moiety interacting with the negatively charged phosphate backbone, leading to a strong DNA/drug interaction. We synthesized doxorubicinone, which is an analogue without the sugar moiety, and discovered this change affected the DNA binding significantly. We have previously characterized the thermodynamics of the three binding modes in relation to changes in the DNA hydration layer. To measure these values, the fluorescence of both drugs is utilized in the osmotic stress method to determine the uptake or release of water molecules and the Van’t Hoff method to find the thermodynamic values. We found intercalators have a small disruption in the hydration layer and a small increase in disorder. Unexpectedly, doxorubicinone binding showed a large uptake of water molecules and a large decrease in disorder, meaning it is unique from the three described binding modes. The changes in DNA binding between these two drugs offers areas of study for both binding strength and specificity, which are the two main factors in drug effectiveness.
Kelsey Murphy "Elucidation of the Thermodynamic and Kinetic DNA Binding Mechanism of Anti-Tumor Drug By Fluorescence Spectroscopy"
Chemistry Faculty Advisor: Samantha Glazier
Proflavine is an antitumor antibiotic in the acridine drug family, which effectively works by interacting with DNA. We examine the role of water in the mechanism of binding and investigate the thermodynamic and kinetic profiles of this binding route with respect to the major and minor grooves of DNA. It has been previously thought that the drug enters directly through the minor groove to reach the final intercalated state. Intercalation refers to the insertion of a molecule between the planar purine and pyrimidine base pairs of DNA, perpendicular to the helical axis. Molecular dynamics calculations by Mukerjee et al. showed that the drug may start by pre-binding to the minor groove, but the lowest energy route to intercalation appears to be through the major groove. The major groove of DNA occurs where the backbones are far apart from one another with the nitrogen and oxygen atoms of the base pairs pointing inward toward the helical axis, while the minor groove occurs where they are close together with the oxygen and nitrogen atoms of the base pairs pointing outwards.
Intrigued by the possibility of entry through the major groove, we designed experiments to test thermodynamic and kinetic mechanisms. The thermodynamic binding profile of the drug in calf-thymus (CT) DNA was studied using fluorescence spectroscopy as the principle method of analysis. Water exchange values for the drug show a net uptake of 53.8 water molecules upon binding to CT DNA, and a Van’t Hoff analysis was performed in which the temperature of the titration was varied, resulting in a ΔH value of -44.64 kJ and a TΔS value of -73.91 kJ, suggesting an enthalpy driven process. The extremely unfavorable entropy is consistent with the large uptake of water molecules upon binding. Competitive binding studies were then performed in order to further classify the binding event with respect to how the drug interacts with the major and minor grooves. Hoechst 33258 was used to block the minor groove, resulting in a significantly lower binding constant for proflavine. Methyl green was used to block the major groove, which in turn resulted in a higher binding constant. This suggests the existence of a thermodynamic route and subsequent kinetic route of binding utilized by proflavine. We hypothesize that the blockage of the major groove induces a more stable thermodynamic route through the minor groove, and the blockage of the minor groove results in a less stable but more rapid kinetic route through the major groove. Subsequent kinetic analyses are currently in progress in an effort to further classify and elucidate the binding profile of proflavine in DNA.
Holland Gallup "A Standardization Method for SEM/EDS Determination of Copper, Lead and Mercury Concentrations in Human Teeth "
Chemistry Faculty Advisor: Ning Gao
Four main layers constitute the human tooth; the enamel layer, dentin layer, the pulp and the cementum. Due to the slow and complex formation of teeth, teeth may be subject to the uptake of different elements, including some toxic ones, which may become incorporated into the dentin layer over time. The retention of these elements can serve as a bioindicator to determine the degree of environmental exposure. In particular, heavy metals are of importance due to the large scale exposure from contaminated water sources, diet and potential leeching of mercury from amalgam fillings. Dental treatments or toothpaste may also lead to the incorporation of fluorine into the enamel layer. High levels of exposure to any toxic elements could lead to a wide variety of health complications. By analyzing the concentration levels of heavy metals and fluorine in human teeth, we can draw correlations between age, sex, geographical location and exposure to particularly harmful elements. Building upon the work of previous St. Lawrence University SYE students, we have synthesized dentine standards and created standard curves for different heavy metals (Hg, Sb, Cd, Zn, Pb, Cu) and fluorine using the Scanning Electron Microscope with an X-ray Energy Dispersive Spectrometer. In previous studies, in order to determine the concentration levels of heavy metals in teeth, the teeth were ground up and dissolved in a concentrated acid. However, our method allows us to analyze the elemental composition within different layers of the tooth. In particular, copper, lead and mercury standardized curves were focused upon this year.
Stephanie Johnnson "Factors Controlling the Wagner-Jauregg Reaction "
Chemistry Faculty Advisor: Samuel Tartakoff
While Lewis acids are widely used to accelerate Diels–Alder reactions, none have been investigated in relation to the Wagner–Jauregg reaction. The Wagner-Jauregg reaction is a double Diels Alder reaction which breaks the aromaticity of the reactants to form a bis-adduct with four new carbon-carbon bonds and two rings. The intermolecular Wagner-Jauregg reaction of maleic anhydride and trans-anethole generally proceeds over 3 hours at 80°C in the presence of dimethylaniline under solvent-free conditions, and produces a 60% yield of the
bis-adduct. Using Lewis acids, which coordinate to the dienophile to lower its LUMO energy, the reaction can be accelerated, allowing it to proceed at lower temperatures or in solution. A variety of Lewis acids were tested at different temperatures in an attempt to run the reaction at room temperature. Cerium (III) chloride catalyzed the reaction, with the best conversion produced in the presence of a Lewis base. The combination of CeCl3 and dimethylaniline allowed the reaction to proceed at room temperature and in 3 M solution.
George Misiewicz Using Chemical Oscillations to Determine the Antioxidant Strength of Wine: Development of an Undergraduate Laboratory"
Chemistry Faculty Advisor: Samantha Glazier
Oscillating reactions are a field in chemistry that encompass the foundations of numerical modeling, non-linear dynamics, and the theories behind organic interactions. Varying parameters such as the temperature, concentration, type of catalyst, and organic substrate, have profound physical effects on the reaction, such as changing the period of oscillation, the induction time, and colour of the reaction. Due to all of these variances, extensive research has gone into exactly how these reactions occur, and how they can help us learn more about our world. One such way is through close analysis of how certain compounds quench the reaction. Products containing phenols and chlorides are known to completely inhibit the oscillations through the prevention of the oxidative step that resets the entire reaction.
In this study we look at determining the antioxidant strength of different wines due to their phenolic content through the controlled quenching of a variant of the Belousov-Zhabotinskii reaction. The reported antioxidant strengths are relative to that of gallic acid, a known phenol present in all wines. It was found that red wines had a considerably larger antioxidant strength than that of white wines. It was also noted that the addition of wine significantly affected the reaction on a whole, such as a decrease in both oscillation period and amplitude. Additionally, we looked at how change in body temperature (hyperthermia, physiological, and high fever) alters the antioxidant effects of the wines through relative analyses and Arrhenius calculations. From this we are able to see that the higher the temperature, the greater the relative antioxidant value. This work was additionally designed as a possible undergraduate laboratory for physical chemistry students, focusing on kinetics, Arrhenius calculations, and phenol chemistry, and was successfully carried out with a class of seventeen students over a two-week period.
Kirsten Padalis "Optimizing a Thioflavin-T Assay using the Microplate Reader"
Biochemistry Faculty Advisor: Nadia Marano
Amyloids are proteins that are characterized by fibrils that have beta sheets which are perpendicular to the fiber axis. There are functional amyloid fibers that can be produced by bacteria as part of their biofilms. An assay optimized by Jordan Koloski using Thioflavin T, a
dye that fluoresces when bound to amyloid fibrils, was used to measure the growth of amyloids of different tetracycline resistant soil bacteria. This assay has been done by measuring the fluorescence using the Horiba FluoroMax Spectrofluorimeter. I created a new assay to be able to read and analyze the fluorescence on the Synergy HTX Multi-Mode Microplate reader. This plate reader can measure fluorescence on a 96 well plate so that more samples can be analyzed at once, which will help cut down time for gathering data allowing more trials to be performed. The assay was first optimized using aggregated insulin, which gives consistently high fluorescence then further studied with bacteria grown on plates. I concluded that the plate reader is not as sensitive at reading fluorescence as the fluorimeter. Therefore, the plate reader is a good way to check a lot of samples at once and to see which ones should be studied further using the fluorimeter.
Avery Peck "Exploring the Tandem Wagner-Jauregg/Diels–Alder Reactions "
Chemistry Faculty Advisor: Samuel Tartakoff
In this day and age, our ability to create begins at the molecular level. Chemical synthesis is a fundamental aspect of global industry ranging from materials science and manufacturing to pharmaceuticals. This makes the exploration of new methods and the development of old methodology a vibrant field as we look for new ways to create. In the early 20th century, a rapid and robust reaction was discovered that provides a way to build highly functionalized molecules with little waste. Many versions of this Diels–Alder reaction exist, with one in particular, the Wagner-Jauregg, being the focus of this study. My work is expanding on what is known about this reaction and its facility in chemical synthesis, which can be applied to numerous industrial applications. A number of reaction conditions have been manipulated to achieve the first intermolecular and subsequent intramolecular reaction. Modifications to the original diene and dienophile used in Wagner-Jauregg’s work, combined with various reaction temperatures, solvents and/or Lewis acid catalysis, has shown progress towards the formation of the desired adduct. Refining the ideal reaction conditions with the right substrates has revealed the sensitivity of the reaction with respect to each condition. This type of double Diels–Alder reaction further establishes the efficiency if this reaction in a wide range of applications.
Jamie Wilson "Analyzing the Levels of Mercury (Hg) in Rice and Rice Products Consumed by Residents and Students in St. Lawrence County, NY "
Chemistry Faculty Advisor: Ning Gao
It is common knowledge that a major source of mercury (Hg) in humans comes from the consumption of marine and freshwater fish. However, it is not widely known that Hg has also been found in terrestrial plants, and recent studies have proven that this may be another significant cause of Hg exposure for humans. This study pays particular attention to the levels of Hg found in rice and rice products consumed by students and residents of St. Lawrence County, NY. The AMA254 Mercury Analyzer, which uses direct combustion to analyze
samples, determined the concentrations of Hg in a variety of raw rice grains, cooked grains, and rice-based products. Initial studies showed that the Hg levels diminished significantly in cooked rice products. In order to compare the raw rice to the cooked rice, the amount of mercury per grain was calculated. The average mercury content in the two types of raw rice grains was found to be 0.635 ng/grain and 0.786 ng/grain for the white rice and brown rice respectively. For the cooked rice, the average level came out to be 0.295 ng/grain and 0.046 ng/grain for white rice and brown rice respectively. Understanding that significant concentrations of heavy metals like Hg are found in commonly consumed plant materials, such as rice, will hopefully spark new studies on how the intake of these metals can be inhibited in plants.
Brenda Winn "Philatelic Table of the Elements as a Teaching Tool: Topics in Health and Medicine "
Chemistry Faculty Advisor: Larry G. French
A philatelic table of the elements has previously been assembled with each element portrayed by a postage stamp. 73 different stamp-issuing entities are represented. This table provides a platform to discuss the rich histories and applications of 114 elements. Four elements – nitrogen, silicon, arsenic, and chlorine – are represented by stamps celebrating their drug, human health, and medical fields. A French stamp celebrates the anniversary of the isolation of quinine (nitrogen), an anti-malarial medication, while a Cambodian stamp highlights the importance of malaria control via the use of DDT (chlorine). A Swazi stamp delves into the history of the Havelock Asbestos Mine (silicon) where lasting effects of lung cancer and mesothelioma present concern. A stamp issued by Germany celebrates the birthday of Ehrlich, who introduced the Salvarsan magic bullet (arsenic), the start of targeted chemotherapy. Narratives behind these four stamps will be developed to provide a new teaching tool for secondary or non-major science classes.