Chemistry Research Projects 2023

 

Juanita Beenyi

Advisor: Yan Kung

Kinetic and structural characterization of an archaeal mevalonate kinase

The mevalonate pathway, also known as the HMG-CoA reductase pathway, is a metabolic pathway that plays a crucial role in the biosynthesis of steroids such as cholesterol and isoprenoid natural products. The pathway consists of seven different enzymes responsible for synthesizing the precursors to steroids and isoprenoids. One enzyme, mevalonate kinase, catalyzes the first of three consecutive reactions that use ATP. In addition, downstream products of the mevalonate pathway can cause feedback inhibition of mevalonate kinase, but this varies among different organisms. In some archaea, however, MK activity has been found to be unaffected by all downstream products known to cause feedback inhibition. Could MK in archaea be resistant to feedback inhibition? To address this question, I will study the kinetics and structure of MK from Methanocaldococcus jannaschii (MjMK). I will express and purify MjMK and characterize its kinetics to study its activity and possible inhibition. I will also determine its three-dimensional structure by protein X-ray crystallography. The results of this experiment will lead to a better understanding of the structure of MK and how its structure governs its activity and regulation.

Cameron Blair

Advisor: Ashlee Plummer-Medeiros

Purification of YebS Using Different Detergents to Assess Protein Yield

YebS/T is a lipid transport membrane complex found in E. coli and other gram-negative bacteria. These bacteria are composed of a double lipid bilayer, consisting of the inner membrane and the outer membrane, which are separated by the aqueous periplasm. YebS is a transmembrane protein found in the inner membrane, which is thought to be responsible for handing off lipids to YebT in the periplasm, for the purpose of moving lipids from the inner to the outer membrane. This enables cell growth and creates membrane stability. Studying this complex’s structure and function will improve understanding of bacterial membrane stability, which will aid in the development of antibiotics, as bacteria cannot survive when their membranes are unstable or unable to expand. This research aims to express and purify E. coli YebS under varying detergent conditions to test which will yield the largest amount of purified protein. This is important as detergents are useful in solubilizing proteins as their amphipathic nature mimics the lipid bilayer but can also destabilize proteins if too stringent. As such, it is important to choose a detergent in which the protein is stable for use in later experiments. The detergents being used are LMNG, DDM, and Digitonin. Of these, LMNG is predicted to yield the largest amount of protein. The protein will then be used to perform a lipid transport assay with the goal of determining the function of YebS and its role in lipid trafficking. It is important to use the same detergent during protein purification as in the assay. Ascertaining this function will help to understand how YebS passes lipids to YebT, making it a possible protein for future development of antibiotics that target lipid transport in E. coli.

Evelyn Cheng

Advisor: Ashlee Plummer-Medeiros

Studying the lipid transfer function of PqiB and YebT

Bacterial membranes are composed of two phospholipid bilayer membranes: the inner and outer membrane. For bacteria to grow, lipids are continuously shuttled from the inner to the outer membrane. This transfer of lipids is affected by phospholipid interaction with membrane proteins and understanding more about this process can contribute to future designs and more effective antibiotic designs. Instead of studying complex bacterial membranes, vesicles are used as a simplified system for analysis; vesicles are composed of an enclosed phospholipid bilayer, which are used to conduct in vitro lipid transfer assays. Donor vesicles contain fluorescent-tagged lipids and acceptors vesicles have no tagged-lipids, therefore the movement of lipids from donor to acceptor mirrors the shuttling between the inner and outer membranes of bacteria. Assays were conducted to specifically observe how different factors such as changes in time, concentration, temperature, pH, and shaking affect transfer activity. As the bacterial membrane contains lipids with two headgroups PE (Phosphatidyl-Ethanolamine) and PG (Phosphatidyl-Glycerol), both were investigated here. Results showed transport activities were similar at all tested temperature and shaking conditions but differed at variable pH and donor/acceptor ratios. Furthermore, lipid transfer assays will be conducted with the insertion of the proteins PqiB and YebT to study their effect on lipid transport. PqiB and YebT are lipid transport membrane proteins that reside in the periplasm and are suspected to connect two vesicles in the lipid transfer assay, therefore it is hypothesized that the lipid transfer activity will accelerate with the addition of PqiB or YebT.

Lily Desiderio

Advisor: Yan Kung

Understanding the Structure and Inhibition of Homosapien Mevalonate Kinase (HsMK) using X-Ray Crystallography

The mevalonate pathway, also known as the HMG-CoA reductase pathway, is a metabolic pathway responsible for the production of isoprenoid precursors.  Isoprenoids are the largest class of naturally occurring compounds, responsible for various functions throughout the body.  In addition, isoprenoids are vital precursors sex hormones.  In this study, the Homosapien Mevalonate kinase (HsMK) mechanism of action, structure, and inhibition will be observed.  HsMK is the fourth enzyme in the mevalonate pathway in humans.  The active site of mevalonate kinase requires two substrates, mevalonate and ATP, to form 5-phosphomevalonate.  While structural images of MK bound to either mevalonate or ATP have been determined for other organisms, it’s structure has yet to be determined in humans.  In addition, the proposed mechanism of action is not supported by the current structures of MK bound to either mevalonate or ATP.  As such, the structure of HsMK bound to both mevalonate and ATP simultaneously is required.  While studying the structure of MK during reaction, a dilemma occurs.  The speed at which the reaction occurs, is too quick to be analyzed via x-ray crystallography.  Thus, in order to visualize the active site of HsMK bound to mevalonate and ATP, an ATP analog must be utilized.  Similar to ATP, ATP analogs will react with mevalonate and the active site, but prevent the reaction from completing.  Thus, HsMK will be locked in an active site bound to mevalonate and ATP.  By studying the active site of HsMK bound to both mevalonate and ATP, the mechanism of can be better understood.  HsMK is regulated via feedback inhibition.  Thus, downstream products in the mevalonate pathway serve as inhibitors to HsMK.  Considering the inhibitory products in the mevalonate pathway vary in size and structure, how does the active site accommodate all of them?  To answer this question, the kinetic activity and structure of HsMK bound to various inhibitors.  To achieve success, techniques such as molecular cloning and mutagenesis, protein expression and purification, kinetic characterization, and crystallization will be employed.

Rosie Dougherty-Herrmann

Advisor: Bill Malachowski

Development of Chiral Nitrogen Containing Drug Intermediates via Birch-Heck Synthesis

Nitrogen-containing rings such as pyridine and pyrimidine are extremely common components of bioactive drugs and drug candidates due to their aromaticity and the intermolecular forces provided by nitrogen. However, aromatic structures like these often participate in side-reactions, leading to undesired side effects and diminished drug effectiveness. Research has shown that tetrahedral carbons have better results in clinical trials due to improved selectivity over flat aromatic structures. The Malachowski lab uses organic synthesis processes, particularly Birch-Heck process, to create chiral drug intermediates. The process begins with a Birch reduction-alkylation reaction that creates a chiral center from benzoic acid and has been successfully done with several different substituents. Eventually, aza-Heck or aza-Wacker reactions can be used to cyclize the added substituents. My research project in Dr. Malachowski’s lab will aim to create nitrogen-containing drug intermediates with chiral centers. Currently, I am working on adapting the Birch reduction-alkylation to alkylate pyridine. This reaction has been successfully performed, but research into the potential of adding various substituents to pyridine via Birch reduction-alkylation have not been thoroughly explored. Pyridine bears resemblance to important biomolecules such as NADH, and reduced versions could therefore provide potential drug candidates. The eventual goal of successful reduction of pyridine could also include the synthesis of swainsonine, a natural product with potential anti-cancer properties.

Christina Douglas

Advisor: Bill Malachowski

The Functionalization of Cyclohexene Rings Through Hydroamination

Oseltamivir is an antiviral prodrug used in the prevention and treatment of influenza. It is a small molecule, consisting of a cyclohexene ring with four attached groups, including an amino group. In research carried out by the Hartwig group, it was demonstrated that 1,3-dienes could undergo palladium-catalyzed hydroamination, adding an amino group and reducing the diene system to a single alkene. The Birch reduction-alkylation reaction produces 1,4-cyclohexadiene-containing structures that may also be susceptible to this process, and which have the added benefit of chirality, a property that can significantly impact how drugs interact with biological systems. The focus of this research, combining the hydroamination process identified by the Hartwig group with a Birch reduction-alkylation, would allow for the creation of cyclohexenes with a variety of attached groups, which could be used to synthesize derivatives of oseltamivir or other compounds with the potential for bioactivity that contain functionalized cyclohexenes.

Taylor Ferreira

Advisor: Jonas Goldsmith

The Synthesis of Bimetallic Catalysts and their Incorporation Into Hydrogen-Based Energy Production

In the United States, hydrogen gas is most commonly derived from harmful fossil fuels through steam methane reforming, which releases carbon dioxide as a byproduct. Sustainably generating and utilizing hydrogen gas as a form of carbon-free energy is the key to a healthier future. This research focuses on the synthesis of a bimetallic molecule which catalyzes the reduction of water by light energy, yielding hydrogen gas. The bimetallic molecule is composed of two transition metal complexes, one of which acts as a photosensitizer (PS) while the other acts as an electron relay (ER). The PS absorbs light energy, becomes excited, and transfers an electron to the ER. The ER then transfers an electron to an acid, ultimately generating hydrogen gas. The excitement of the PS which facilitates this series of electron transfers is brief, therefore it is crucial for the PS and the ER to be in close proximity to one another. A “lock and key” method will be utilized to fix the PS and ER in a position such that they may efficiently react with one another during this limited period of excitement. The two complexes consist of bipyridines bonded to a ruthenium center. The PS and ER can not be connected without the presence of additional functional groups. An amine will be installed to one complex and a carboxylic acid will be installed to another complex allowing them to unite via an amine-carboxylic acid coupling mechanism.

Chelsea Freer

Advisor: Sharon Burgmayer

Synthesis and Optimization of the ligands 2-pivaloyl-6-chloropterin and BMOPP 

Many metals are critical to sustaining life on earth – Iron, Zinc, Copper, etc. One such metal is Molybdenum (Mo). The element forms the catalytic center of a variety of enzymes., which then perform essential reactions such as respiration, protein synthesis, oxidation, and detoxification. This catalytic center, also known as the molybdenum cofactor (Moco), is composed of a Mo atom and a pterin-dithiolene ligand - aka molybdopterin, MPT. The lab focuses on synthetic approaches to create and investigate functions of Moco model compounds. 

I am currently working on the synthesis of 2-pivaloyl-6-chloropterin, in which the pivaloyl group improves the solubility of the system. The next step will utilize a Sonogashira Coupling Reaction to yield 6-(3-butynyl-2-methyl-2-ol)-2-pivaloyl pterin (BMOPP). In addition to synthesizing these materials, I will work on optimizing the procedures to generate a large, pure yield. This goal will be achieved through the mastery of various synthetic techniques including the Schlenk Line and Column Chromatography, as well as analytical techniques such as TLC, NMR, and IR. 

Lana Giha

Advisor: Bill Malachowski

Studies towards the Total Synthesis of Dendrobine via Birch-Heck Sequence

In the Malachowski group there is an interest in using the Birch-Heck sequence to produce bioactive compounds, particularly those inspired by natural products. Using the Birch-Heck sequence we hope to form a bicyclic structure containing a five and six membered ring fused together with a quaternary center forming at one of the bridgehead carbons. This compound is a key intermediate in the synthesis towards dendrobine, a natural product that has exhibited both analgesic and antipyretic effects. Previous syntheses have been developed but all with modest success and relatively low yields. We hope to improve upon these previous efforts.

Hannah Kreider

Advisor: Bill Malachowski

Enantioselective Synthesis of Drug Candidates Through the Birch-Heck Sequence

Recent structural studies of drug candidates have shown that molecules with more tetrahedral, sp3 centers, including chiral ones, are more likely to succeed in clinical trials and eventually become successful drugs. In the Malachowski lab, the goal is to develop a synthetic tool to efficiently create drug candidates with chiral centers in order to reduce these side effects. This summer, I will study the Birch-Heck sequence, a tool that allows the efficient and selective formation of chiral tetrahedral carbons. This sequence begins with the Birch reduction-alkylation reaction to create prochiral sp3 carbons with a variety of substituents from inexpensive benzoic acid and ends with the Heck reaction, which involves an intramolecular desymmetrizing reaction that enantioselectivity creates a cyclic structure with two chiral centers. My research focuses on the addition of alkyl substituents to the bisallylic carbon of the Birch product through radical processes rather than the carboxylic acid, as has been previously done. This allows for the diversification of structures that are able to be made through this Birch-Heck Sequence.

Sydney McDonnough

Advisor: Yan Kung

Investigating the Structure, Kinetics and NADPH/NADH Cofactor Specificity HMG-CoA Reductase of Bordetella petrii

The mevalonate pathway is responsible for making the biosynthetic precursors to all isoprenoids and steroids. In the focused upon mevalonate pathway HMG- CoA reductase (HMGR) is responsible for reducing HMG-CoA to mevalonate and is also the rate determining step in the enzyme pathway. In order to reduce it, it binds the redox cofactors NADPH or NADH and most HMGR enzymes have a cofactor specificity to either of these two cofactors however prior research has shown that Bordetella petrii HMG-CoA reductase (Bp-HMGR) was found to have the ability to use both cofactors and a structure of the enzyme bound to NADH was developed. Thus in my own research I will further investigate the kinetics and catalytic efficiency of Bp-HMGR in the presence of NADH and NADPH to determine which one is favoured by the enzyme as well as using protein crystallisation methods in order to develop a structure of the protein bound to NADPH and determine more specifically which amino acids in the sequence that correlates to the binding region of the cofactor could be contributing to its ability to bind to both NADPH or NADH and how those amino acids could correspond to the enzymes overall preference to either cofactor.

Kirya Miller

Advisor: Patrick Melvin

A Modified Beckmann Rearrangement Using Sulfone Iminium Fluorides (SIFs)

The Melvin group has previously developed a novel class of S(VI) reagent, called sulfone iminium fluorides (SIFs), capable of performing efficient deoxyfluorination reactions in just 60 seconds at room temperature. Work this summer is focused on expanding its use beyond typical deoxyfluorination reactions to mediating a modified Beckmann rearrangement--the rearrangement of an oxime functional group to an amide, often catalyzed by acid--to convert oximes into imidoyl fluorides. My work will expand on this by taking various amidoximes to imidoyl fluorides using SIFs and investigating the reactivity of these intermediates with nitrogen nucleophiles and with water to form guanidine derivatives and urea derivatives, respectively.

Darya Ostapenko

Advisor: Sharon Burgmayer

This abstract outlines a summer research initiative centered on the synthesis and analysis of artificial compounds that emulate Molybdenum cofactor (Moco), a key element in molybdenum-based enzymes. These enzymes are instrumental in a variety of catalytic processes pertinent to oxygen transport, and thus, indispensable to life. My mentor for this venture is Dr. Burgmayer.

A specific focus is on bis(dithiolene)molybdenum complexes, structurally and functionally analogous to Moco in several enzymes such as DMSO reductases. These reductases perform under anaerobic conditions in specific bacteria, crucial for sustaining a balanced human gut microbiome.

This research aims to further ongoing efforts to synthesize a stable bis(dithiolene)molybdenum complex, which is expected to yield critical information regarding the structure, bonding, redox activity, and catalytic mechanisms of molybdenum-dependent enzymes. It is hoped that crafting a steady model will not only advance our research but also offer significant insights to future investigators regarding the functions Moco performs in biological systems.

The implications of this research extend beyond academic curiosity, potentially informing the creation of innovative drugs or treatment approaches for a range of health issues.

Charli Parsons

Advisor: Bill Malachowski

Synthesizing Various Pro-drug Derivatives of Varenicline

The smoking cessation drug varenicline (brand name Chantix) has been in wide use for nearly two decades. As a partial agonist of the nicotinic acetylcholine receptor (nAchR) subtype α4β2, varenicline can reduce withdrawal symptoms in patients discontinuing nicotine use. Large-scale analyses have estimated varenicline treatment’s success rate at between 9% and 20%, with non success frequently due to varenicline’s side effect profile. In order to improve the pharmacokinetics of varenicline, we have synthesized/will synthesize four pro-drug derivatives of varenicline, using various methods of amide coupling along with a wide range of other common synthetic organic reactions. The results of this study show (put results here). With these results in hand, it will be possible to study new smoking cessation treatment plans and finally address the drawbacks of this popular drug.

Malini Rajbhandari

Advisor: Ashlee Plummer-Medeiros

Mutagenesis Study of PqiA to Study the Effects of Hydrogen Bonding on Protein Function

PqiA is a membrane protein found in gram-negative bacteria which is hypothesized to transport lipids between the inner and the outer membrane of these bacteria. However, the protein is not well studied and developing a better understanding of the function of this protein will help with research on antibacterial resistance, as lipid transport is an essential part of bacterial replication and subsequent survival. The goal of this research is to better understand PqiA function by focusing on the protein-lipid interactions. Previously, all-atom molecular dynamics simulations of PqiA were used to study the protein dynamics of various regions of PqiA. These simulations revealed that Hydrogen Bonding is likely an essential part of the protein function. This raises the hypothesis that the specific hydrogen bonding reactions are required for PqiA-mediated lipid transport. Based on the previous analyses, six Aspartic Acids residues were chosen from PqiA which frequently were identified as amino acids that participate in hydrogen bonding. Site-directed mutagenesis was then used to mutate these individual Aspartic Acid residues to Leucine. The Leucines will be similar in spatial structure and size to the Aspartic Acids but lacks the ability to Hydrogen Bond. Mutated proteins will be expressed in E. Coli and their activity will be tested using a functional assay to see how these mutations impact the rate of lipid transport between liposomes. These results will be combined with MD simulation data to better understand the effects of Aspartate-lipid interactions on PqiA function.

Bernie Schintz

Advisor: Jonas Goldsmith

Synthesizing transition metal complexes using vinyl bypyridine ligands to produce polymers with hydrogen producing abilities

This research focuses on creating transition metal complexes using bidentate chelating bipyridine ligands and ruthenium. Transition metal complexes act as photosynthesizers, absorbing light which causes its electrons to move to a higher energy level. These complexes can also act as electron relays, carrying electrons from one place to another. Vinyl groups are attached to the transition metal complexes so that a polymerization reaction can occur. Layering the synthesized polymer sheets allows the transition metal complexes to have a close proximity enabling electron transfer. Historically, hydrogen gas is created by decomposing methane, releasing pollutants into the atmosphere. The ultimate goal of this research is to synthesize a chemical system where water can be converted into hydrogen gas by harvesting light energy. This process is revolutionary as it could possibly create a thin layer polymer product that can be applied to surfaces and generate hydrogen gas without the use of carbon. Photosystems have not been bonded together in the manner explained above, highlighting the importance of the future success of this research.

Eunjeong (Sal) Shin

Advisor: Patrick Melvin

Fluorine is widely used in pharmaceutical drugs, specifically in drug designs where introduction of fluorine atoms can significantly change the molecule’s lipophilicity, solubility, acidity, and basicity. Despite fluorination’s crucial application, not many methods of installing fluorine atoms into the target molecule are available and they still pose challenges.  Recently, our laboratory has reported the use of sulfone iminium fluorides (SIF), a fluorination reagent that provides high yields of fluorinated products with a significantly shorter reaction times compared to other reagents.  Using this SIF reagent, I will synthesize and characterize different fluorinated intermediates via a modified Beckmann rearrangement process.  We will use amidoximes as our primary substrate with the aim to generate various products that are derivatives of guanidine and urea. 

Tiffany Xue

Advisor: Jonas Goldsmith

Synthesis of bimetallic transition metal macromolecules for photosensitizer and electron relay pre-connection

The transition towards a low-carbon future necessitates the development of environmentally friendly energy sources, including green hydrogen. However, current hydrogen production methods emit substantial amounts of greenhouse gases, rendering them unsustainable. Electrolysis of water offers promise as a green hydrogen production method, but it suffers from high costs and low output. To address these challenges, our laboratory aims to enhance the photocatalytic water reduction system, thereby increasing hydrogen output. In this system, a light-trapping Photosensitizer (PS) rapidly excites and transfers electrons to an Electron Relay (ER). To optimize this system, a crucial aspect is establishing effective connections between PS and ER within a limited timeframe. The objective of our research is to establish pre-connections between PSs and ERs through amide coupling, accomplished by synthesizing transition metal macromolecules, specifically ruthenium, with carboxylic acid one carbon away on either PS or ER and amine on the other. This research has the potential to make a significant impact on the energy sector and the environment by reducing greenhouse gas emissions and contributing to global climate goals. Additionally, the mechanism employed to attach groups onto the original molecule to establish connections holds further potential for application in other synthesis cases.

Zahraa Zamir

Advisor: Yan Kung

Structural modification of cofactor specificity in HMG-CoA reductase

The mevalonate pathway is an important pathway in multiple biological systems and consists of seven enzymes that act as precursors to the production of steroids and isoprenoids, which are used in many industries. The enzyme HMG-CoA reductase (HMGR), the third enzyme in the mevalonate pathway, binds to either NADPH or NADH to reduce HMG-CoA to mevalonate. Previous research has indicated that a “cofactor helix” near the NAD(P)H binding site is responsible for this NAD(P)H preference, and we have recently shown that switching the entire cofactor helix results in a switching of NAD(P)H preference. Here, I will study the effects of multiple mutations of the cofactor helix to determine exactly which amino acid or combination of amino acids cause the change in cofactor preference. Specifically, I will study D146Y, V148S, and L152R mutations in HMGR from Delftia acidovorans (DaHMGR), and the corresponding inverse mutations in HMGR from Streptococcus pneumoniae (SpHMGR). This will be done through expressing and purifying these mutant proteins, then testing their activities via kinetics studies. If we find that any of our mutants have interesting activities, we will continue protein crystallization to allow us a clearer understanding of the mutant HMGR structure. Through this study, I wish to contribute to a better understanding of the importance of structural basis for HMGR in relation to cofactor specificity.

Ruolin Zhang

Advisor: Jonas Goldsmith

Synthesis of vinyl bipyridine thin films to optimize the photocatalytic water reduction system


In recent years, the demand for clean and renewable energy has increased. A famous example of green energy, hydrogen could be produced from water and light using transitional metal complexes as electron carriers and catalysts. This new method is called a photocatalytic water reduction system. Transition metal complexes would perform as photosensitizers (PS) and electron relays (ER) in electron transfer to help the water splitting. Sacrificial reductant donates an electron to the PS to excite the electron to a higher energy level using light energy. ER acts as the intermediate to carry the energy from the excited electron to reduce water. The interaction between PS and ER is considered a workable application to increase the efficiency of the overall electron transfer and water-splitting reaction. Our research generally aims to form transition metal complexes with vinyl groups attacking and polymerized complexes using electropolymerization. Then, the polymers will be pre-positioned in thin films to achieve the interaction. The proximity of the PS and ER helps to increase the electron transfer rate and reduce energy consumption. Currently, the research focuses on producing and increasing the yield of complex precursors: 4-methoxy ethyl-4'-methyl-2,2’-bipyridine and 4-methyl-4’-vinyl-2,2’-bipyridine, and making a connection between the vinyl precursor and transition metal- Rhodium.

Chiara Zuccoli

Advisor: Sharon Burgmayer

Synthesis of Tungsten Enzyme Models

Molybdenum and tungsten enzymes play a vital role as catalysts in a wide range of organisms, including humans. Studying their chemical properties helps us to better understand their role in important biochemical reactions. The Burgmayer lab’s primary focus is the synthesis and study of molecules containing the catalytic site known as Moco, which includes a molybdenum atom and the pterin-dithiolene ligand. Similar complexes can be synthesized using tungsten in the place of molybdenum with several key considerations to keep in mind, such as its preference to create a trisulfide structure as opposed to molybdenum’s tetrasulfide compound. The synthesis pathway for tungsten models has several obstacles that need to be considered at each step in order to efficiently characterize each synthetic intermediate. An unidentified side product (-704)  that appears at the first synthetic step (tungsten tricarbonyl) might be interfering greatly with yield, so its identification could be useful moving forward. The tendency of the trisulfide-BMOPP complex to spontaneously move to its oxo form makes characterization of the target complex difficult. My goal is to tackle these issues, in addition to several others associated with the synthetic pathway, using techniques requiring a Schlenk line and glove box, as well as analytical tools such as mass spectrometry, IR spectroscopy, and NMR.