Some of our 2019 senior chemistry and biochemistry majors and their research
Sustainable Chemistry Utilizing Terpenes: Synthesis of Anthraquinone Analogs from Myrcene
Dylan J. Babcock Advisor: Dr. Larry French
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 functionalizing, preparing, and exploring reactions involving monoterpenes derived from essential oils. Specifically, myrcene is being used in a multistep synthesis to form more interesting and potentially useful compounds. The current molecule at hand is an anthraquinone analog, which is part of a large group of molecules that have a wide array of uses. The compounds created in this research can be used in further synthesis of chemical products, eliminating the need for some environmentally harmful precursors. Analysis of these compounds is being done through GC/MS as well as C13 and H1 NMR. Future experiments will focus on additional workup and functionalization of the anthraquinone analog at hand.
Design and Fabricat ion of a Homemade T - Jump for Kinetic Characterization of Chemotherapy Drugs
David Bain Advisors: Drs. Samantha Glazier and Catherine Jahncke
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. This heating event disrupts the binding equilibrium, specifically dislodging bound drugs. As the system returns to equilibrate, the fluorescence intensity changes from which binding rates are calculated. Based on a design reported in the literature, we will build an electrically insulated cuvette holder equipped with high voltage electrodes that can be placed in the Fluoromax-3 fluorimeter. The electrodes are connected to a power supply and a capacitor. Part of the project will be optimizing instrument design. For example, a faster capacitor discharge leads to quicker solution heating and increased resolution in relaxation kinetics calculations. The intercalating drug proflavine provides an interesting case study. . 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.
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)
Lillian Devereux Advisor: Dr. Nadia Marano
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 however, it is not an effective measure of amyloid oligomer formation, the smaller building blocks of mature fibers. ANS, Bis-ANS, and DCJV are three alternatives to ThT that are characterized by a multi-ring structure containing a single bond around which the rings are able rotate. However, when these molecules bind to amyloid fibers the rotation is no longer possible, resulting in an increased fluorescence. 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.
Adaptive Python Program for Integrati on of Poorly Resolved 1 H NMR Spectra
Abigail Enders Advisors: Drs. Samuel Tartakoff and Adam Hill
Kinetics studies of the Wagner-Jauregg reaction between para-substituted styrenes and maleic anhydride for determination of the mechanism require reproducible and accurate integration for hundreds of 1H NMR spectra. The existing JEOL software is insufficient for investigating the Wagner-Jauregg reaction and similar heterogeneous reactions. The variability among reactions in addition to the poorly-resolved spectra from the aging NMR can be addressed through a Python-coded program wherein poor spectral results will be repaired through baseline correction and normalization. Additionally, the lack of reproducibility in manual integrations over a collection of spectra will be confronted through the reusable functionality of the program. The Python program will incorporate the integration of multiple peaks, reproducible results, and contain adaptable code necessary for the integration of several spectra. Additional features being developed include a graphical user interface to reduce user interaction with the code. Interactions with the software will include input of desired spectrum file and peaks for integration. The increased functionality compared to the current software will improve utilization of poorly-resolved NMR spectra and provide reproducible results in quantitative analysis of reactions.
Development and Isolation of Stable Fibroblast Growth Factor 2 Proteins
Evan Ketcham Advisor: Dr. Matthew Skeels
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 mutant known as FGFbase (N71E, E78K, K86E, T112E) being more stable under chemical denaturing conditions than the wild type FGF2. Further research is being conducted to probe more mutants for their stabilities.
Isolation and Identification of Amyloid Fibers from Archaeon Haloferax volcanii
Lisa Kozodoy Advisors: Drs. Nadia Marano and Lorraine Olendzinski
Amyloids are proteins that when aggregated form large insoluble fibers characterized by their common cross beta sheet structure. While these proteins are often implicated in human neurodegenerative diseases, such as Alzheimer’s, many organisms make functional amyloids without the toxic side effects. 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 these amyloid fibers from H. volcanii to elucidate more about their composition and structure. Isolation is done through differential centrifugation and sonication, following previous research done by
Heather Raimer ‘17. Sonicating the cells releases the amyloid fibers from the cells and centrifugation allows for the separation of the amyloids from cellular debris. Ultracentrifugation is done to attempt to isolate the larger amyloid fibers from suspension. Lastly, tube SDS-PAGE is used to purify the amyloids from any proteins that are contaminating the sample. The amyloid themselves are detected using Thioflavin T (ThT) assays. ThT will have an emission peak at 487 nm when excited and bound to amyloid fibers. The isolated tube gel sample can be deaggregated into its monomers using formic acid. The monomers are resuspended in urea in order to prevent reaggregation. These can then be sent off to a collaborator for sequencing.
Characterizing the Binding Mechanisms of New Chemotherapy Drugs with Plasmid DNA
So Min (Kate) Park Advisor: Samantha Glazier
Even with the great amount of research currently underway on cancer treatments, development of new chemotherapy drugs remains one of the biggest challenges in the medical field. While there are a few well-known chemotherapy drugs, such as doxorubicin, further understanding of the binding mechanisms of the drug molecules to DNA is essential for the development of new chemotherapy drugs. Our lab previously characterized the binding mode of doxorubicinone, a doxorubicin derivative, to calf thymus DNA using a melting protocol based on UV-Vis spectrometry and observing whether there were changes in the melting temperatures; intercalators increase the melting temperature of DNA and groove binders do not change the melting temperature of DNA. However, as CT DNA has a mixture of both double and single stranded DNA and has unknown DNA fragment sizes, we believe that a more homogenous mixture of DNA could improve our results. In this project, we will develop a protocol to determine the binding mechanisms of new chemotherapy drugs to plasmid DNA. Unlike the CT DNA that was used previously, plasmid DNA only contains circular double stranded DNA that can be easily linearized with restriction enzymes and has a known DNA sequence length of 2686 base pairs. With a more homogenous mixture of DNA fragments, we will be able to compare melting data collected from linearized plasmid to melting data collected from CT DNA. Because testing with millileter volumes can be expensive, we will develop a low volume protocol that detects changes in fluorescence in a PCR (polymerized chain reaction) instrument to collect melting points. As in the UV-Vis based studies, we can determine the binding mechanisms of chemotherapy drugs with plasmid DNA in a more cost effective way. In both techniques, we hope to obtain sharper inflection points in the melting curves and more distinct first derivative curves, so that these protocols can be used to determine the binding mechanisms of future chemotherapy drugs synthesized in Dr. Tartakoff’s lab. Moreover, if we are successful in improving our melting data with these protocols, we will develop another low volume protocol for determining the binding mechanisms of drugs using an agarose gel, as intercalators cause DNA to recoil and recoiled DNA travels further on a gel than DNA bound to drugs that via a minor groove mode.
Chromatographic Analysis of Honey
Sarah Potter Advisor: Dr. Matthew Skeels
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; mono-, di-, and polysaccharides; organic acids; thiamine; minerals; and glycerol. Moisture content will be measured as a percentage of weight after honey is heated to a constant mass. Sugars will be derivatized and analyzed using GC/MS. Organic acids will be analyzed using solid phase extraction followed by HPLC. Thiamine will be oxidized to thiochrome and measured by fluorescence spectroscopy. Samples will be subjected to microwave digestion and minerals will be determined with FAAS. An enzymatic assay will be applied for glycerol determination. Techniques developed will be applied to the study of monofloral honeys to compare their respective nutritional profiles. The nectar and pollen sources of the honeys will be confirmed by an outside laboratory, and comparisons made in and among families of plants. Eventually, this information will be used to create environments with plants that provide appropriate nutrition for pollinators year-round.
Time - Correlated Single - Photon Counting Measurements of Electron Transfer Between Heterobinuclear Units and Bipyridine
Alissa Stone Advisor: Dr. Adam D. Hill
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 singlephoto 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.
Review of the Impact of Road Salting on Soil and Water Chemistry in the Adirondacks; Environmental and Biological Implications and Possible Mitigation Strategies
Dana Tindall Advisor: Dr. Glazier
The application of sodium chloride to roadways to mitigate accidents in the winter has be shown to lead to the accumulation of sodium and chloride in roadside soil and runoff into surface and groundwaters. New York state has the highest road salt application rates in the country and salting rates have shown a dramatic increase in past decades (Kelting et al. 2012). Due to cation exchange, excess sodium replaces biologically necessary metals such as calcium, magnesium, and potassium which negatively affects soil biota (Willmert et al. 2018). The changes in soil chemistry can cause breakdown of “soil aggregates, soil swelling, and reduced soil permeability” which can impair microbial and macrofaunal organisms along with decreasing larger vegetation which can lead to further environmental degradation (Willmert et al. 2018). The chloride ions from the salt are highly water soluble and are carried away from the road sides with snowmelt and rain creating an increase in chloride levels in many lakes (Ke et al. 2013). This study investigates and compiles notable literature on the environmental implications of road salting on soil and water chemistry and the multitude of impacts the salt has on biota in the Adirondacks. It also examines the effectiveness and practicality of various alternatives for winter road management to sodium chloride such as magnesium chloride, calcium chloride, calcium magnesium acetate (CMA), and wetting salts prior to application (Langen et al. 2006).
Synthesis of Anthraquinone Derivatives by Editing Functional Groups Flanking to Anthraquinone for DNA Binding Study
Ryan Jiarui Zhang Advisor: Dr. Larry French
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. Synthesizing such compounds to extend the structural diversity of anthraquinones available for investigation of biological activity, particularly potential DNA binding and cytotoxicity is the aim of this project. It is known that anthraquinone moiety is associated with cytotoxicity with various cell types. Derivatives have appeared in many anticancer chemotherapeutic drugs, such as doxorubicin, mitomycin, and many other biologically active compounds. This synthesis can use to generate multiple derivatives based on the multiplicity and variability of the aldehyde groups; reactivity of the potential derivatives with known DNA intercalator anthraquinone and side functional groups may cause more interactions with DNA backbone or nucleotides for potential pharmaceutical interest.